WorldWideScience

Sample records for aminopeptidases

  1. Aminopeptidase C of Aspergillus niger is a Novel Phenylalanine Aminopeptidase

    NARCIS (Netherlands)

    Basten, E.J.W.; Dekker, P.J.T.; Schaap, P.J.

    2003-01-01

    A novel enzyme with a specific phenylalanine aminopeptidase activity (ApsC) from Aspergillus niger (CBS 120.49) has been characterized. The derived amino acid sequence is not similar to any previously characterized aminopeptidase sequence but does share similarity with some mammalian acyl-peptide hy

  2. [Endocellular aminopeptidase from Astasia longa].

    Science.gov (United States)

    Rudenskaia, Iu A; Aseev, V V; Rudensaia, G N

    2008-01-01

    A new aminopeptidase was isolated from the biomass of the flagellate Astasia longa by precipitation with ammonium sulfate, gel filtration, and affinity chromatography on Arginine-Silochrome in 41% yield and with purification degree 490. The enzyme is irreversible inhibited by mercury chloride, EDTA, o-phenanthroline and, partially, bestatin and zinc chloride. It has an optimum pH 8.5 toward the hydrolysis of a synthetic chromogenic substrate Ala-pNA. The enzyme molecular mass is 45 kDa, isoelectric point 5.5, and temperature optimum 45 degrees C. The enzyme most effectively hydrolyzes p-nitroanilides of alanine, arginine, and leucine; it is classified as metalloaminopeptidase. PMID:18672681

  3. Arginine specific aminopeptidase from Lactobacillus brevis

    Directory of Open Access Journals (Sweden)

    Arya Nandan

    2010-12-01

    Full Text Available The proteolytic system of lactic acid bacteria contribute to the development of flavor during the ripening of cheese through the generation of short peptides and free amino acids, which directly or indirectly act as flavor precursors. Newly isolated lactic acid bacteria (LAB as well as those procured from culture collection centers were screened for the production of various substrate specific aminopeptidases. Among all the strains screened, L. brevis (NRRL B-1836 was found to produce quantifiable amount of intracellular arginine specific aminopeptidase (EC 3.4.11.6. The productivity of arginine aminopeptidase in 5 L fermentor was 36 IU/L/h. The Luedeking and Piret model was tested for intracellular production of aminopeptidase and the data seemed to fit well, as the correlation coefficient was 0.9964 for MRS. The αAP and βAP was 0.4865 and 0.0046, respectively in MRS medium indicating that the yield was predominantly depended on growth. The culture produced lactic acid and also tolerated pH 2.0-3.0 and 0.3-0.5% bile salts, the most important probiotic features.

  4. Characterisation of Aspergillus niger prolyl aminopeptidase

    NARCIS (Netherlands)

    Basten, E.J.W.; Moers, A.P.H.A.; Ooyen, van A.J.J.; Schaap, P.J.

    2005-01-01

    We have cloned a gene (papA) that encodes a prolyl aminopeptidase from Aspergillus niger. Homologous genes are present in the genomes of the Eurotiales A. nidulans, A. fumigatus and Talaromyces emersonii, but the gene is not present in the genome of the yeast Saccharomyces cerevisiae. Cell extracts

  5. Novel selective inhibitors of aminopeptidases that generate antigenic peptides.

    Science.gov (United States)

    Papakyriakou, Athanasios; Zervoudi, Efthalia; Theodorakis, Emmanuel A; Saveanu, Loredana; Stratikos, Efstratios; Vourloumis, Dionisios

    2013-09-01

    Endoplasmic reticulum aminopeptidases, ERAP1 and ERAP2, as well as Insulin regulated aminopeptidase (IRAP) play key roles in antigen processing, and have recently emerged as biologically important targets for manipulation of antigen presentation. Taking advantage of the available structural and substrate-selectivity data for these enzymes, we have rationally designed a new series of inhibitors that display low micromolar activity. The selectivity profile for these three highly homologous aminopeptidases provides a promising avenue for modulating intracellular antigen processing. PMID:23916253

  6. Aminothiols: synthesis and effect on chicken brain aminopeptidases.

    Science.gov (United States)

    Weiss, B; Hui, K S; Hui, M; Lajtha, A

    1988-10-01

    An amino acid derivative, leucinethiol, was reported to be a strong inhibitor of aminopeptidase activity. In order to obtain selective inhibitors of various brain aminopeptidases, we tested the inhibition by amino acid analogs of brain aminopeptidase activity. In particular, we synthesized the trifluoroacetyl derivatives of phenylalaninol, tyrosinol, and leucinol; leucinethiol; and phenylalaninethiol and measured their effect on soluble puromycin-sensitive aminopeptidase S1 and SII purified in our laboratory. Two of the compounds, L-bis (1-thio-2-amino-4-methylpentane) dihydrochloride (TAMP) and L-bis (1-thio-2-amino-3-phenylpropane) dihydrochloride (TAPP), caused significant inhibition. PMID:3205972

  7. Aminopeptidase activity from germinated jojoba cotyledons.

    Science.gov (United States)

    Johnson, R; Storey, R

    1985-11-01

    One major and two minor aminopeptidase activities from germinated jojoba (Simmondsia chinensis) cotyledon extracts were separated by ammonium sulfate precipitation and chromatofocusing. None of the activities were inhibited by 1,10 phenanthroline.The major aminopeptidase, purified 260-fold, showed a pH optimum of 6.9 with leucine-p-nitroanilide as substrate, a molecular weight estimated at 14,200 by electrophoretic analysis, and an isoelectric point of 4.5 according to the chromatofocusing pattern. Activity was inhibited by p-chloromercuribenzoate, slightly stimulated by 1,10 phenanthroline and 2-mercaptoethanol, and not influenced by Mg(2+) or diethyl pyrocarbonate. Inhibition by p-chloromercuribenzoate was prevented by the presence of cysteine in the assay. Leucine-p-nitroanilide and leucine-beta-naphthylamide were the most rapidly hydrolyzed of 11 carboxy-terminal end blocked synthetic substrates tested. No activity on endopeptidase or carboxypeptidase specific substrates was detected. The major aminopeptidase showed activity on a saline soluble, jojoba seed protein preparation and we suggest a possible physiological role for the enzyme in the concerted degradation of globulin reserve proteins during cotyledon senescence.

  8. Purification and characterization of an immunogenic aminopeptidase of Brucella melitensis.

    Science.gov (United States)

    Contreras-Rodriguez, Araceli; Ramirez-Zavala, Bernardo; Contreras, Andrea; Schurig, Gerhardt G; Sriranganathan, Nammalwar; Lopez-Merino, Ahide

    2003-09-01

    An immunogenic aminopeptidase was purified from Brucella melitensis strain VTRM1. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps. This procedure resulted in a yield of 29% and a 144-fold increase in specific activity. The aminopeptidase appeared to be a monomeric enzyme with a molecular mass of 96 kDa and an isoelectric point of 4.8. Its activity was optimal at pH 7.0 at 40 degrees C. The enzyme was strongly inhibited by EDTA, 1,10-phenathroline, and divalent cations (Zn(2+) and Hg(2+)), suggesting that this protein was a metalloaminopeptidase. The enzyme showed preference for alanine at the N termini of aminoacyl derivatives. The K(m) values for L-alanine-p-nitroanilide (Ala-pNA) and Lys-pNA were 0.35 and 0.18 mM, respectively. The N-terminal sequence of aminopeptidase was used for a homologous search in the genomes of B. melitensis 16M and Brucella suis 1330. The analysis revealed an exact match of the probe sequence (36 bp) with an open reading frame of 2,652 bp encoding a protein predicted to be alanyl aminopeptidase (aminopeptidase N). Collectively, these data suggest designation of the B. melitensis enzyme as an aminopeptidase N. The aminopeptidase was recognized by sera from patients with acute and chronic brucellosis, suggesting that the enzyme may have important diagnostic implications. PMID:12933870

  9. Characterization of aspartyl aminopeptidase from Toxoplasma gondii

    Science.gov (United States)

    Zheng, Jun; Cheng, Ziying; Jia, Honglin; Zheng, Yonghui

    2016-01-01

    Aminopeptidases have emerged as new promising drug targets for the development of novel anti-parasitic drugs. An aspartyl aminopeptidase-like gene has been identified in the Toxoplasma gondii genome (TgAAP), although its function remains unknown. In this study, we characterized TgAAP and performed functional analysis of the gene product. Firstly, we expressed a functional recombinant TgAAP (rTgAAP) protein in Escherichia coli, and found that it required metal ions for activity and showed a substrate preference for N-terminal acidic amino acids Glu and Asp. Then, we evaluated the function and drug target potential of TgAAP using the CRISPR/Cas9 knockout system. Western blotting demonstrated the deletion of TgAAP in the knockout strain. Indirect immunofluorescence analysis showed that TgAAP was localized in the cytoplasm of the wild-type parasite, but was not expressed in the knockout strain. Phenotype analysis revealed that TgAAP knockout inhibited the attachment/invasion, replication, and substrate-specific activity in T. gondii. Finally, the activity of drug CID 23724194, previously described as targeting Plasmodium and malarial parasite AAP, was tested against rTgAAP and the parasite. Overall, TgAAP knockout affected the growth of T. gondii but did not completely abolish parasite replication and growth. Therefore, TgAAP may comprise a useful adjunct drug target of T. gondii. PMID:27678060

  10. Identification and Characterization of Novel Inhibitors of Mammalian Aspartyl Aminopeptidase

    OpenAIRE

    Chen, Yuanyuan; Tang, Hong; Seibel, William; Papoian, Ruben; Oh, Ki; Li, Xiaoyu; Zhang, Jianye; Golczak, Marcin; Palczewski, Krzysztof; Kiser, Philip D.

    2014-01-01

    Aspartyl aminopeptidase (DNPEP) has been implicated in the control of angiotensin signaling and endosome trafficking, but its precise biologic roles remain incompletely defined. We performed a high-throughput screen of ∼25,000 small molecules to identify inhibitors of DNPEP for use as tools to study its biologic functions. Twenty-three confirmed hits inhibited DNPEP-catalyzed hydrolysis of angiotensin II with micromolar potency. A counter screen against glutamyl aminopeptidase (ENPEP), an enz...

  11. Transcytosis of Aminopeptidase N in caco-2 cells is mediated by a Non-cytoplasmic Signal

    DEFF Research Database (Denmark)

    Vogel, L K; Norén, Ove; Sjöström, H

    1995-01-01

    transmembrane or cytoplasmic domain of aminopeptidase N for transport of aminopeptidase N by the indirect pathway by analysis of mutated forms of aminopeptidase N recombinantly expressed in Caco-2 cells. A tail-less and two secretory forms of aminopeptidase N, all deprived of the cytoplasmic tail, were...... transported to the basolateral plasma membrane in proportions equivalent to the wild type enzyme. This shows that no cytoplasmic basolateral sorting signal is involved in directing aminopeptidase N to the basolateral plasma membrane. Both the wild type and the tail-less aminopeptidase N were transcytosed from...

  12. Probing the S1 specificity pocket of the aminopeptidases that generate antigenic peptides

    OpenAIRE

    Zervoudi, Efthalia; Papakyriakou, Athanasios; Georgiadou, Dimitra; Evnouchidou, Irini; Gajda, Anna; Poreba, Marcin; Salvesen, Guy S.; Drag, Marcin; Hattori, Akira; Swevers, Luc; Vourloumis, Dionisios; Stratikos, Efstratios

    2011-01-01

    Abstract ER aminopeptidase 1 (ERAP1), ER aminopeptidase 2 (ERAP2) and Insulin Regulated aminopeptidase (IRAP) are three homologous enzymes that play critical roles in the generation of antigenic peptides. These aminopeptidases excise amino acids from N-terminally extended precursors of antigenic peptides in order to generate the correct length epitopes for binding onto MHC class I molecules. The specificity of these peptidases can affect antigenic peptide selection, but has not yet...

  13. Identification and characterization of Paragonimus westermani leucine aminopeptidase.

    Science.gov (United States)

    Song, Su-Min; Park, Joon-Hyung; Kim, Jin; Kim, Suk-Il; Hong, Yeon-Chul; Kong, Hyun-Hee; Chung, Dong-Il

    2008-09-01

    Paragonimus westermani is a tissue-invading trematode parasite that causes inflammatory lung disease as well as systemic infections including cerebral invasion in carnivorous mammals. While aminopeptidases play important roles in trematodes in the catabolism of host hemoglobin, an essential source of nutrient for the parasite, little is known about aminopeptidase in Paragonimus. Presently, we isolated a cDNA encoding a 58 kDa P. westermani leucine aminopeptidase (PwLAP). Deduced amino acid sequence of PwLAP exhibited significant sequence homology with LAP from Schistosoma spp. and Fasciola hepatica. Biochemical analysis of the recombinant PwLAP protein demonstrated preferential substrate specificity for Leu-NHMec and inhibition by EDTA, 1,10-phenanthroline, and bestatin, which are conserved characteristics of the M17 family of leucine aminopeptidase. PwLAP exhibited relatively higher enzyme activity in the presence of Mn2+ compared to Schistosoma mansoni LAP. Based on the biochemical properties and immunohistochemical analysis, PwLAP is concluded to represent a leucine aminopeptidase. The enzyme is most likely responsible for the catabolism of host hemoglobin, and, hence, represents a potential target of Paragonimus chemotherapy.

  14. Placental Leucine Aminopeptidase- and Aminopeptidase A- Deficient Mice Offer Insight concerning the Mechanisms Underlying Preterm Labor and Preeclampsia

    Directory of Open Access Journals (Sweden)

    Shigehiko Mizutani

    2011-01-01

    Full Text Available Preeclampsia and preterm delivery are important potential complications in pregnancy and represent the leading causes for maternal and perinatal morbidity and mortality. The mechanisms underlying both diseases remain unknown, thus available treatments (beta2-stimulants and magnesium sulfate are essentially symptomatic. Both molecules have molecular weights less than 5–8 kDa, cross the placental barrier, and thus exert their effects on the fetus. The fetus produces peptides that are highly vasoactive and uterotonic and increase in response to maternal stress and with continued development. Fetal peptides are also small molecules that inevitably leak across into the maternal circulation. Aminopeptidases such as placental leucine aminopeptidase (P-LAP and aminopeptidase A (APA are large molecules that do not cross the placental barrier. We have shown that APA acts as an antihypertensive agent in the pregnant spontaneously hypertensive rat by degrading vasoactive peptides and as a result returns the animal to a normotensive state. P-LAP also acts as an antiuterotonic agent by degrading uterotonic peptides and thus prolongs gestation in the pregnant mouse. Given the ever increasing worldwide incidences of preeclampsia and preterm labor, it is imperative that new agents be developed to safely prolong gestation. We believe that the use of aminopeptidases hold promise in this regard.

  15. Genetic associations and functional characterization of M1 aminopeptidases and immune-mediated diseases.

    Science.gov (United States)

    Agrawal, N; Brown, M A

    2014-12-01

    Endosplasmic reticulum aminopeptidase 1 (ERAP1), endoplasmic reticulum aminopeptidase 2 (ERAP2) and puromycin-sensitive aminopeptidase (NPEPPS) are key zinc metallopeptidases that belong to the oxytocinase subfamily of M1 aminopeptidase family. NPEPPS catalyzes the processing of proteosome-derived peptide repertoire followed by trimming of antigenic peptides by ERAP1 and ERAP2 for presentation on major histocompatibility complex (MHC) Class I molecules. A series of genome-wide association studies have demonstrated associations of these aminopeptidases with a range of immune-mediated diseases such as ankylosing spondylitis, psoriasis, Behçet's disease, inflammatory bowel disease and type I diabetes, and significantly, genetic interaction between some aminopeptidases and HLA Class I loci with which these diseases are strongly associated. In this review, we highlight the current state of understanding of the genetic associations of this class of genes, their functional role in disease, and potential as therapeutic targets. PMID:25142031

  16. Genetic characterization of a polymorphic dipeptidyl aminopeptidase of Apis mellifera

    OpenAIRE

    Marco Antonio Del Lama,; Boraschi, Daniele; Ademilson Espencer Egea Soares,; Duran, Ximena

    2004-01-01

    International audience Dipeptidyl aminopeptidase (DAP) activity towards L-leucylglycine-$\\beta$-naphthylamide (Leu-Gly NA) was characterized in pupae and adult extracts of Apis mellifera. Enzyme activity was more conspicuous in pupae than in adult extracts and it seemed to be concentrated in digestive tract tissues. Two genetically determined electrophoretic variants were observed in honeybee samples from the USA and Chile; in Brazilian Africanized bees, two additional variants were observ...

  17. Immunogold labelling is a quantitative method as demonstrated by studies on aminopeptidase N in microvillar membrane vesicles

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Wetterberg, L L; Sjöström, H;

    1992-01-01

    Microvillar membrane vesicle preparations with varying content of aminopeptidase N were prepared from enterocytes of the pig small intestine. Postembedding immunogold labelling of aminopeptidase N was performed on these glutaraldehyde/paraformaldehyde-fixed, osmium tetroxide-treated and Epon...

  18. The M1 family of vertebrate aminopeptidases: role of evolutionarily conserved tyrosines in the enzymatic mechanism of aminopeptidase B.

    Science.gov (United States)

    Cadel, Sandrine; Darmon, Cécile; Pernier, Julien; Hervé, Guy; Foulon, Thierry

    2015-02-01

    Aminopeptidase B (Ap-B), a member of the M1 family of Zn(2+)-aminopeptidases, removes basic residues at the NH2-terminus of peptides and is involved in the in vivo proteolytic processing of miniglucagon and cholecystokinin-8. M1 enzymes hydrolyze numerous different peptides and are implicated in many physiological functions. As these enzymes have similar catalytic mechanisms, their respective substrate specificity and/or catalytic efficiency must be based on subtle structural differences at or near the catalytic site. This leads to the hypothesis that each primary structure contains a consensus structural template, strictly necessary for aminopeptidase activity, and a specific amino acid environment localized in or outside the catalytic pocket that finely tunes the substrate specificity and catalytic efficiency of each enzyme. A multiple sequence alignment of M1 peptidases from vertebrates allowed to identify conserved tyrosine amino acids, which are members of this catalytic backbone. In the present work, site-directed mutagenesis and 3D molecular modeling of Ap-B were used to specify the role of four fully (Y281, Y229, Y414, and Y441) and one partially (Y409) conserved residues. Tyrosine to phenylalanine mutations allowed confirming the influence of the hydroxyl groups on the enzyme activity. These groups are implicated in the reaction mechanism (Y414), in substrate specificity and/or catalytic efficiency (Y409), in stabilization of essential amino acids of the active site (Y229, Y409) and potentially in the maintenance of its structural integrity (Y281, Y441). The importance of hydrogen bonds is verified by the Y229H substitution, which preserves the enzyme activity. These data provide new insights into the catalytic mechanism of Ap-B in the M1 family of aminopeptidases. PMID:25530263

  19. The M1 family of vertebrate aminopeptidases: role of evolutionarily conserved tyrosines in the enzymatic mechanism of aminopeptidase B.

    Science.gov (United States)

    Cadel, Sandrine; Darmon, Cécile; Pernier, Julien; Hervé, Guy; Foulon, Thierry

    2015-02-01

    Aminopeptidase B (Ap-B), a member of the M1 family of Zn(2+)-aminopeptidases, removes basic residues at the NH2-terminus of peptides and is involved in the in vivo proteolytic processing of miniglucagon and cholecystokinin-8. M1 enzymes hydrolyze numerous different peptides and are implicated in many physiological functions. As these enzymes have similar catalytic mechanisms, their respective substrate specificity and/or catalytic efficiency must be based on subtle structural differences at or near the catalytic site. This leads to the hypothesis that each primary structure contains a consensus structural template, strictly necessary for aminopeptidase activity, and a specific amino acid environment localized in or outside the catalytic pocket that finely tunes the substrate specificity and catalytic efficiency of each enzyme. A multiple sequence alignment of M1 peptidases from vertebrates allowed to identify conserved tyrosine amino acids, which are members of this catalytic backbone. In the present work, site-directed mutagenesis and 3D molecular modeling of Ap-B were used to specify the role of four fully (Y281, Y229, Y414, and Y441) and one partially (Y409) conserved residues. Tyrosine to phenylalanine mutations allowed confirming the influence of the hydroxyl groups on the enzyme activity. These groups are implicated in the reaction mechanism (Y414), in substrate specificity and/or catalytic efficiency (Y409), in stabilization of essential amino acids of the active site (Y229, Y409) and potentially in the maintenance of its structural integrity (Y281, Y441). The importance of hydrogen bonds is verified by the Y229H substitution, which preserves the enzyme activity. These data provide new insights into the catalytic mechanism of Ap-B in the M1 family of aminopeptidases.

  20. Biosynthesis of intestinal microvillar proteins. Expression of aminopeptidase N along the crypt-villus axis

    DEFF Research Database (Denmark)

    Danielsen, E M

    1984-01-01

    The expression of pig small-intestinal aminopeptidase N (EC 3.4.11.2) along the crypt-villus axis was studied in tangential sections of [35S]-methionine-labelled, organ-cultured explants. The only detectable molecular forms of aminopeptidase N along the crypt-villus axis were polypeptides of Mr 140...

  1. Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA

    DEFF Research Database (Denmark)

    Cowell, G M; Kønigshøfer, E; Danielsen, E M;

    1988-01-01

    The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250-555 p...

  2. Zinc Regulation of Aminopeptidase B Involved in Neuropeptide Production

    OpenAIRE

    Hwang, Shin-Rong; Hook, Vivian

    2008-01-01

    Aminopeptidase B (AP-B) is a metallopeptidase that removes basic residues from the N-termini of neuropeptide substrates in secretory vesicles. This study assessed zinc regulation of AP-B activity, since secretory vesicles contain endogenous zinc. AP-B was inhibited by zinc at concentrations typically present in secretory vesicles. Zinc effects were dependent on concentration, incubation time, and the molar ratio of zinc to enzyme. AP-B activity was recovered upon removal of zinc. AP-B with zi...

  3. Functional characterization of X-prolyl aminopeptidase from Toxoplasma gondii.

    Science.gov (United States)

    Yang, Mingfa; Zheng, Jun; Jia, Honglin; Song, Mingxin

    2016-09-01

    In the present study, a recombinant aminopeptidase P (rTgAPP) from Toxoplasma gondii was expressed in Escherichia coli to evaluate its enzyme parameters. The rTgAPP showed strong activity against a synthetic substrate for aminopeptidase P at pH 8·0 with a K m value of 0·255 µ m and a k cat value of 35·6 s-1. The overall catalytic efficiency (k cat/K m) of the rTgAPP was 139·6 × 105 M-1 s-1. The activity of rTgAPP was enhanced by the addition of divalent cations and inhibited by bestatin. Deletion of TgAPP gene in the parasite through a CRISPR/Cas9 system resulted in inhibition of growth indicating the importance of TgAPP. Thus our findings reveal that TgAPP is an active enzyme in T. gondii and provide an insight into the function of TgAPP. PMID:27220680

  4. Investigation of the metal binding site in methionine aminopeptidase by density functional theory

    DEFF Research Database (Denmark)

    Jørgensen, Anne Techau; Norrby, Per-Ola; Liljefors, Tommy

    2002-01-01

    All methionine aminopeptidases exhibit the same conserved metal binding site. The structure of this site with either Co2+ ions or Zn2+ ions was investigated using density functional theory. The calculations showed that the structure of the site was not influenced by the identity of the metal ions....... This was the case for both of the systems studied; one based on the X-ray structure of the human methionine aminopeptidase type 2 (hMetAP-2) and the other based on the X-ray structure of the E. coli methionine aminopeptidase type 1 (eMetAP-1). Another important structural issue is the identity of the bridging...

  5. The Aminopeptidase Inhibitor CHR-2863 Is an Orally Bioavailable Inhibitor of Murine Malaria

    OpenAIRE

    Skinner-Adams, Tina S.; Peatey, Christopher L.; Anderson, Karen; Trenholme, Katharine R.; Krige, David; Christopher L. Brown; Stack, Colin; Nsangou, Desire M. M.; Mathews, Rency T.; Thivierge, Karine; Dalton, John P; GARDINER, DONALD L.

    2012-01-01

    Malaria remains a significant risk in many areas of the world, with resistance to the current antimalarial pharmacopeia an ever-increasing problem. The M1 alanine aminopeptidase (PfM1AAP) and M17 leucine aminopeptidase (PfM17LAP) are believed to play a role in the terminal stages of digestion of host hemoglobin and thereby generate a pool of free amino acids that are essential for parasite growth and development. Here, we show that an orally bioavailable aminopeptidase inhibitor, CHR-2863, is...

  6. Influence of chronic ethanol intake on mouse synaptosomal aspartyl aminopeptidase and aminopeptidase A: relationship with oxidative stress indicators.

    Science.gov (United States)

    Mayas, María Dolores; Ramírez-Expósito, María Jesús; García, María Jesús; Carrera, María Pilar; Martínez-Martos, José Manuel

    2012-08-01

    Aminopeptidase A (APA) and aspartyl aminopeptidase (ASAP) not only act as neuromodulators in the regional brain renin-angiotensin system, but also release N-terminal acidic amino acids (glutamate and aspartate). The hyperexcitability of amino acid neurotransmitters is responsible for several neurodegenerative processes affecting the central nervous system. The purpose of the present work was to study the influence of chronic ethanol intake, a well known neurotoxic compound, on APA and ASAP activity under resting and K(+)-stimulated conditions at the synapse level. APA and ASAP activity were determined against glutamate- and aspartate-β-naphthylamide respectively in mouse frontal cortex synaptosomes and in their incubation supernatant in a Ca(2+)-containing or Ca(2+)-free artificial cerebrospinal fluid. The neurotoxic effects were analyzed by determining free radical generation, peroxidation of membrane lipids and the oxidation of synaptosomal proteins. In addition, the bioenergetic behavior of synaptosomes was analyzed under different experimental protocols. We obtained several modifications in oxidative stress parameters and a preferential inhibitor effect of chronic ethanol intake on APA and ASAP activities. Although previous in vitro studies failed to show signs of neurodegeneration, these in vivo modifications in oxidative stress parameters do not seem to be related to changes in APA and ASAP, invalidating the idea that an excess of free acidic amino acids released by APA and ASAP induces neurodegeneration.

  7. HISTOCHEMICAL DISTRIBUTION OF LEUCINE AMINOPEPTIDASE (LAP-ase)IN THE YOUNG RABBIT INTESTINE

    OpenAIRE

    Sabatakou, O.; Xylouri-Frangiadaki, E.; Paraskevakou, E.

    2000-01-01

    Abstract not available. Sabatakou, O.; Xylouri-Frangiadaki, E.; Paraskevakou, E. (2000). HISTOCHEMICAL DISTRIBUTION OF LEUCINE AMINOPEPTIDASE (LAP-ase)IN THE YOUNG RABBIT INTESTINE. http://hdl.handle.net/10251/10176.

  8. Development of cognitive enhancers based on inhibition of insulin-regulated aminopeptidase

    OpenAIRE

    Chai, Siew Yeen; Yeatman, Holly R; Parker, Michael W.; Ascher, David B.; Thompson, Philip E.; Mulvey, Hayley T; Albiston, Anthony L.

    2008-01-01

    The peptides angiotensin IV and LVV-hemorphin 7 were found to enhance memory in a number of memory tasks and reverse the performance deficits in animals with experimentally induced memory loss. These peptides bound specifically to the enzyme insulin-regulated aminopeptidase (IRAP), which is proposed to be the site in the brain that mediates the memory effects of these peptides. However, the mechanism of action is still unknown but may involve inhibition of the aminopeptidase activity of IRAP,...

  9. Antigenic peptide trimming by ER aminopeptidases – insights from structural studies

    OpenAIRE

    Stratikos, Efstratios; Stern, Lawrence J.

    2013-01-01

    Generation and destruction of antigenic peptides by ER resident aminopeptidases ERAP1 and ERAP2 have been shown in the last few years to be important for the correct functioning and regulation of the adaptive immune response. These two highly homologous aminopeptidases appear to have evolved complex mechanisms well suited for their biological role in antigen presentation. Furthermore, polymorphic variability in these enzymes appears to affect their function and predispose individuals to disea...

  10. Endoplasmic reticulum aminopeptidases in the pathogenesis of ankylosing spondylitis.

    Science.gov (United States)

    Kenna, Tony J; Robinson, Philip C; Haroon, Nigil

    2015-09-01

    There has been significant progress in our understanding of the pathogenesis of AS. The advent of genome-wide association studies has increased the known loci associated with AS to more than 40. The endoplasmic reticulum resident aminopeptidases (ERAP) 1 and 2 were identified in this manner and are of particular interest. There appears to be a genetic as well as a functional interaction of ERAP1 and 2 with HLA-B27 based on the known functions of these molecules. Recent studies on the structure, immunological effects and the peptide-trimming properties of ERAP 1 and 2 have helped to provide insight into their pathogenic potential in AS. In this review, we explore the role of ERAP 1 and 2 in the pathogenesis of AS. PMID:26070942

  11. Insights into the molecular interactions between aminopeptidase and amyloid beta peptide using molecular modeling techniques.

    Science.gov (United States)

    Dhanavade, Maruti J; Sonawane, Kailas D

    2014-08-01

    Amyloid beta (Aβ) peptides play a central role in the pathogenesis of Alzheimer's disease. The accumulation of Aβ peptides in AD brain was caused due to overproduction or insufficient clearance and defects in the proteolytic degradation of Aβ peptides. Hence, Aβ peptide degradation could be a promising therapeutic approach in AD treatment. Recent experimental report suggests that aminopeptidase from Streptomyces griseus KK565 (SGAK) can degrade Aβ peptides but the interactive residues are yet to be known in detail at the atomic level. Hence, we developed the three-dimensional model of aminopeptidase (SGAK) using SWISS-MODEL, Geno3D and MODELLER. Model built by MODELLER was used for further studies. Molecular docking was performed between aminopeptidase (SGAK) with wild-type and mutated Aβ peptides. The docked complex of aminopeptidase (SGAK) and wild-type Aβ peptide (1IYT.pdb) shows more stability than the other complexes. Molecular docking and MD simulation results revealed that the residues His93, Asp105, Glu139, Glu140, Asp168 and His255 are involved in the hydrogen bonding with Aβ peptide and zinc ions. The interactions between carboxyl oxygen atoms of Glu139 of aminopeptidase (SGAK) with water molecule suggest that the Glu139 may be involved in the nucleophilic attack on Ala2-Glu3 peptide bond of Aβ peptide. Hence, amino acid Glu139 of aminopeptidase (SGAK) might play an important role to degrade Aβ peptides, a causative agent of Alzheimer's disease.

  12. Substrate-dependent nitric oxide synthesis by secreted endoplasmic reticulum aminopeptidase 1 in macrophages.

    Science.gov (United States)

    Goto, Yoshikuni; Ogawa, Kenji; Nakamura, Takahiro J; Hattori, Akira; Tsujimoto, Masafumi

    2015-06-01

    In this study, we examined the role of aminopeptidases with reference to endoplasmic reticulum aminopeptidase 1 (ERAP1) in nitric oxide (NO) synthesis employing murine macrophage cell line RAW264.7 cells activated by lipopolysaccharide (LPS) and interferon (IFN)-γ and LPS-activated peritoneal macrophages derived from ERAP1 knockout mouse. When NO synthesis was measured in the presence of peptides having N-terminal Arg, comparative NO synthesis was seen with that measured in the presence of Arg. In the presence of an aminopeptidase inhibitor amastatin, NO synthesis in activated RAW264.7 cells was significantly decreased. These results suggest that aminopeptidases are involved in the NO synthesis in activated RAW264.7 cells. Subsequently, significant reduction of NO synthesis was observed in ERAP1 knockdown cells compared with wild-type cells. This reduction was rescued by exogenously added ERAP1. Furthermore, when peritoneal macrophages prepared from ERAP1 knockout mouse were employed, reduction of NO synthesis in knockout mouse macrophages was also attributable to ERAP1. In the presence of amastatin, further reduction was observed in knockout mouse-derived macrophages. Taken together, these results suggest that several aminopeptidases play important roles in the maximum synthesis of NO in activated macrophages in a substrate peptide-dependent manner and ERAP1 is one of the aminopeptidases involved in the NO synthesis. PMID:25577645

  13. Probing the S1 specificity pocket of the aminopeptidases that generate antigenic peptides: S1 specificity of ERAP1, ERAP2 and IRAP

    OpenAIRE

    Zervoudi, Efthalia; Papakyriakou, Athanasios; Georgiadou, Dimitra; Evnouchidou, Irini; Gajda, Anna; Poreba, Marcin; Salvesen, Guy S.; Drag, Marcin; Hattori, Akira; Swevers, Luc; Vourloumis, Dionisios; Stratikos, Efstratios

    2011-01-01

    ER aminopeptidase 1 (ERAP1), ER aminopeptidase 2 (ERAP2) and Insulin Regulated aminopeptidase (IRAP) are three homologous enzymes that play critical roles in the generation of antigenic peptides. These aminopeptidases excise amino acids from N-terminally extended precursors of antigenic peptides in order to generate the correct length epitopes for binding onto MHC class I molecules. The specificity of these peptidases can affect antigenic peptide selection, but has not yet been investigated i...

  14. Proteins of the kidney microvillar membrane. Aspartate aminopeptidase: purification by immunoadsorbent chromatography and properties of the detergent- and proteinase-solubilized forms

    DEFF Research Database (Denmark)

    Danielsen, Erik Michael; Norén, O; Sjöström, H;

    1980-01-01

    Aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) was purified 2000-fold from pig kidney cortex. The essential step in the purification was chromatography on an immunoadsorbent column prepared from a rabbit antiserum raised against pig intestinal aminopeptidase A. Glutamyl and aspartyl...

  15. Characterisation of the aminopeptidase from non-germinated winter rape (Brassica napus L.) seeds.

    Science.gov (United States)

    Kania, Joanna; Gillner, Danuta M

    2016-09-15

    Rapeseed plays a crucial role in food and fuel industry. Since aminopeptidases take part in many physiological processes in all organisms, it is important to learn their role and characteristics in economically relevant plants. Extracts of non-germinated winter rape seeds were screened for aminopeptidase activity. Substrate specificity, the influence of pH and temperature, as well as effect of protease inhibitors and chosen metal ions on the aminopeptidase activity were determined. The approximate molecular weight estimated by NATIVE-PAGE and SDS-PAGE electrophoresis was ∼60 kDa. The partially purified enzyme as well as the aminopeptidases present in crude extract cleaved preferentially Phe-pNA. The activity profiles toward several substrates were also determined. Maximum activity was observed at pH 6.5 and temperature of 40 °C for Phe-pNA as a substrate. Two visible picks in the pH profile toward Phe-pNA, together with other results (IEF) suggest the presence of more than one aminopeptidase, having similar molecular mass. Much lower activity and broad pH profiles were observed for Leu- and Ala-pNA as substrates. PMID:27080895

  16. Biosynthesis of intestinal microvillar proteins. Processing of aminopeptidase N by microsomal membranes

    DEFF Research Database (Denmark)

    Danielsen, E M; Norén, Ove; Sjöström, H

    1983-01-01

    The biosynthesis of small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in a cell-free translation system derived from rabbit reticulocytes. When dog pancreatic microsomal fractions were present during translation, most of the aminopeptidase N synthesized was found in a membrane-bound rat......The biosynthesis of small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in a cell-free translation system derived from rabbit reticulocytes. When dog pancreatic microsomal fractions were present during translation, most of the aminopeptidase N synthesized was found in a membrane......-bound rather than a soluble form, indicating that synthesis of the enzyme takes place on ribosomes attached to the rough endoplasmic reticulum. The microsomal fractions process the Mr-115 000 polypeptide, which is the primary translation product of aminopeptidase N, to a polypeptide of Mr 140 000...... of microsomal fractions was found to be similar to that on one of the forms of the enzyme obtained from tunicamycin-treated organ-cultured intestinal explants....

  17. Alterations in the Helicoverpa armigera midgut digestive physiology after ingestion of pigeon pea inducible leucine aminopeptidase.

    Directory of Open Access Journals (Sweden)

    Purushottam R Lomate

    Full Text Available Jasmonate inducible plant leucine aminopeptidase (LAP is proposed to serve as direct defense in the insect midgut. However, exact functions of inducible plant LAPs in the insect midgut remain to be estimated. In the present investigation, we report the direct defensive role of pigeon pea inducible LAP in the midgut of Helicoverpa armigera (Lepidoptera: Noctuidae and responses of midgut soluble aminopeptidases and serine proteinases upon LAP ingestion. Larval growth and survival was significantly reduced on the diets supplemented with pigeon pea LAP. Aminopeptidase activities in larvae remain unaltered in presence or absence of inducible LAP in the diet. On the contrary, serine proteinase activities were significantly decreased in the larvae reared on pigeon pea LAP containing diet as compared to larvae fed on diet without LAP. Our data suggest that pigeon pea inducible LAP is responsible for the degradation of midgut serine proteinases upon ingestion. Reduction in the aminopeptidase activity with LpNA in the H. armigera larvae was compensated with an induction of aminopeptidase activity with ApNA. Our findings could be helpful to further dissect the roles of plant inducible LAPs in the direct plant defense against herbivory.

  18. Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV

    DEFF Research Database (Denmark)

    Danielsen, E M; Sjöström, H; Norén, Ove

    1983-01-01

    The biogenesis of three intestinal microvillar enzymes, maltase-glucoamylase (EC 3.2.1.20), aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) and dipeptidyl peptidase IV (EC 3.4.14.5), was studied by pulse-chase labelling of pig small-intestinal explants kept in organ culture. The earliest...

  19. Debittering of Protein Hydrolysates by Lactobacillus LBL-4 Aminopeptidase

    Directory of Open Access Journals (Sweden)

    Bozhidar Tchorbanov

    2011-01-01

    Full Text Available Yoghurt strain Lactobacillus LBL-4 cultivated for 8–10 h at pH ~6.0 was investigated as a considerable food-grade source of intracellular aminopeptidase. Cell-free extract manifesting >200 AP U/l was obtained from cells harvested from 1 L culture media. Subtilisin-induced hydrolysates of casein, soybean isolate, and Scenedesmus cell protein with degree of hydrolysis 20–22% incubated at 45∘C for 10 h by 10 AP U/g peptides caused an enlarging of DH up to 40–42%, 46–48%, and 38–40% respectively. The DH increased rapidly during the first 4 h, but gel chromatography studies on BioGel P-2 showed significant changes occurred during 4–10 h of enzyme action when the DH increased gradually. After the digestion, the remained AP activity can be recovered by ultrafiltration (yield 40–50%. Scenedesmus protein hydrolysate with DH 20% was inoculated by Lactobacillus LBL-4 cells, and after 72 h cultivation the DH reached 32%. The protein hydrolysates (DH above 40% obtained from casein and soybean isolate (high Q value demonstrated a negligible bitterness while Scenedesmus protein hydrolysates (low Q value after both treatments were free of bitterness.

  20. Partial purification and characterization of an aminopeptidase from Eimeria tenella.

    Science.gov (United States)

    Fetterer, R H; Miska, K B; Barfield, R C

    2005-12-01

    Our previous investigation demonstrated the expression in Eimeria tenella sporulated oocysts of an aminopeptidase (AP) with strong homology to AP N. To further understand the role of proteases during development, we investigated the molecular and biochemical properties of E. tenella AP. Greater than 95% AP activity was present in a soluble extract during sporulation of oocysts with highest activity in fully sporulated oocysts. The AP activity was inhibited by the AP inhibitors bestatin and 1,6-phenanthroline, but not by serine protease inhibitors. The AP had specificity for synthetic endopeptidase substrates that contain arginine, alanine, or glycine at the N terminus. Partial purification of the enzyme yielded a major protein band with an Mr of about 106 kDa and an isoelectric point (Ip) of 5.1. Reverse transcription-polymerase chain reaction indicated that the gene for AP is expressed during sporulation, but expression is absent or greatly reduced in the sporozoites and merozoites. On the basis of the deduced gene structure, the predicted Mr is 110 kDa with a pI of 5.59. Database search indicates that the E. tenella AP shares significant homology with the AP from Apicomplexan taxa: Toxoplasma gondii, Cryptosporidium parvum, and Cryptosporidium hominis. Together, these results confirm the presence of a cytosolic AP related to AP N, which is expressed and active during sporulation of E. tenella oocysts. PMID:16539006

  1. Altered glutamyl-aminopeptidase activity and expression in renal neoplasms

    International Nuclear Information System (INIS)

    Advances in the knowledge of renal neoplasms have demonstrated the implication of several proteases in their genesis, growth and dissemination. Glutamyl-aminopeptidase (GAP) (EC. 3.4.11.7) is a zinc metallopeptidase with angiotensinase activity highly expressed in kidney tissues and its expression and activity have been associated wtih tumour development. In this prospective study, GAP spectrofluorometric activity and immunohistochemical expression were analysed in clear-cell (CCRCC), papillary (PRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytoma (RO). Data obtained in tumour tissue were compared with those from the surrounding uninvolved kidney tissue. In CCRCC, classic pathological parameters such as grade, stage and tumour size were stratified following GAP data and analyzed for 5-year survival. GAP activity in both the membrane-bound and soluble fractions was sharply decreased and its immunohistochemical expression showed mild staining in the four histological types of renal tumours. Soluble and membrane-bound GAP activities correlated with tumour grade and size in CCRCCs. This study suggests a role for GAP in the neoplastic development of renal tumours and provides additional data for considering the activity and expression of this enzyme of interest in the diagnosis and prognosis of renal neoplasms

  2. Co-and post-translational events in the biogenesis of pig small intestinal aminopeptidase N

    DEFF Research Database (Denmark)

    Danielsen, Erik Michael; Norén, O; Sjöström, H

    1982-01-01

    ,000. When translation was performed in the presence of dog pancreatic microsomes, a Mr 140,000 polypeptide was also observed. A polypeptide of Mr 115,000 was seen for the enzyme, purified from tunicamycin exposed explants. This result suggests that aminopeptidase N is co-translationally inserted...... into the membrane without cleavage of the signal. Pulse-chase labelling of explants gave the following results: 1. Immediately after a 10 min pulse with [35S] methionine, aminopeptidase N was detected in the Ca2+-precipitated membrane fraction. 2. The earliest detectable form of the enzyme, a polypeptide of Mr 140...... mannose to complex glycosylation and the appearance of the enzyme in the microvillar membrane, indicating a role of the Golgi complex in these processes. Colchicine prevented aminopeptidase N from reaching the microvillar membrane, suggesting the involvement of microtubules in the transport....

  3. Thiolation of polycarbophil enhances its inhibition of intestinal brush border membrane bound aminopeptidase N.

    Science.gov (United States)

    Bernkop-Schnürch, A; Zarti, H; Walker, G F

    2001-11-01

    The purpose of this study was to evaluate the potential of polycarbophil-cysteine conjugates (PCP-Cys) as an oral excipient to protect leucine enkephalin (leu-enkp) from enzymatic degradation by the intestinal mucosa. Cysteine was covalently linked to polycarbophil by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC). Inhibitory activity was tested towards isolated aminopeptidase N and excised intact pig intestinal mucosa, with native mucus. Aminopeptidase N activity was assayed spectrophotometrically using L-leucine p-nitroanilide (leu-pNA) as a synthetic substrate and against the model peptide drug leu-enkp, by high-performance liquid chromatography (HPLC). Free cysteine at 6.3 and 63 microM (pH 6) significantly (p < 0.05) inhibited aminopeptidase N activity, and PCP-Cys (0.25% w/v, pH 6) had a significantly (p < 0.05) greater inhibitory effect than PCP on the aminopeptidase N activity towards both substrates. PCP-Cys completely protected leu-enkp against aminopeptidase N activity over a 2-h incubation period, whereas 83 +/- 4 and 60 +/- 7% remained stable in the presence of PCP and buffer only, respectively. Leu-enkp in the absence and presence of PCP (0.25% w/v) at pH 6 was completely digested by the intact intestinal mucosa at the 60- and 90-min incubation time points, respectively, whereas in the presence of PCP-Cys (0.25% w/v, pH 6) 11 +/- 3.5% of leu-enkp remained at the 120-min time point. Thiolation of PCP increased the stability of leu-enkp against the enzymatic degradation by aminopeptidase N and the intact intestinal mucosa, identifying a promising new excipient for peroral delivery of peptides.

  4. Antigenic peptide trimming by ER aminopeptidases--insights from structural studies.

    Science.gov (United States)

    Stratikos, Efstratios; Stern, Lawrence J

    2013-10-01

    Generation and destruction of antigenic peptides by ER resident aminopeptidases ERAP1 and ERAP2 have been shown in the last few years to be important for the correct functioning and regulation of the adaptive immune response. These two highly homologous aminopeptidases appear to have evolved complex mechanisms well suited for their biological role in antigen presentation. Furthermore, polymorphic variability in these enzymes appears to affect their function and predispose individuals to disease. This review discusses our current understanding of the molecular mechanisms behind ERAP1/2 function as suggested by several recently determined crystallographic structures of these enzymes. PMID:23545452

  5. Glycylproline dipeptidyl aminopeptidase isoenzyme in diagnosis of primary hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Run-Zhou Ni; Jie-Fei Huang; Ming-Bing Xiao; Mei Li; Xian-Yong Meng

    2003-01-01

    AIM: To investigate the role of glycylproline dipeptidyl aminopeptidase (GPDA) isoenzyme in the diagnosis of primary hepatocellular carcinoma (PHC), especially in patients with negative alpha-fetoprotein (AFP).METHODS: A stage gradient polyacrylamide gel discontinuous electrophoresis system was developed to separate serum GPDA isoenzymes, which were determined in 102 patients with PHC, 45 cases with liver cirrhosis, 24cases with chronic hepatitis, 35 cases with benign liver spaceoccupying lesions, 20 cases with metastatic liver cancer and 50 cases with extra-hepatic cancer, as well as 80 healthy subjects. The relationships between GPDA isoenzymes and AFP, the sizes of tumors, as well as alanine aminotransferase (ALT) were also analyzed.RESULTS: Serum GPDA was separated into two isoenzymes,GPDA-S and GPDA-F. The former was positive in all subjects,while the latter was found mainly in majority of PHC (85.3 %)and a few cases with liver cirrhosis (11.1%), chronic hepatitis (33.3 %), metastatic liver cancer (15.0 %) and non-hepatic cancer (16.0 %). GPDA-F was negative in all healthy subjects and patients with benign liver space-occupying lesions,including abscess, cysts and angioma. There was no correlation between GPDA-F and AFP concentration or tumor size. GPDA-F was consistently positive and not correlated with ALT in PHC, but GPDA-F often converted to negative as decline of ALT in benign liver diseases. The electrophoretic migration of GPDA-F became sluggish after the treatment of neuraminidase.CONCLUSION: GPDA-F is a new useful serum marker for PHC. Measurement of serum GPDA-F is helpful in diagnosis of PHC, especially in patients with negative AFP. GPDA-F is one kind of glycoproteins rich in sialic acid.

  6. Effect of X-Prolyl Dipeptidyl Aminopeptidase Deficiency on Lactococcus lactis

    NARCIS (Netherlands)

    Mayo, Baltasar; Kok, Jan; Bockelmann, Wilhelm; Haandrikman, Alfred; Leenhouts, Kees J.; Venema, Gerhardus

    1993-01-01

    The genetic determinant (pepXP) of an X-prolyl dipeptidyl aminopeptidase (PepXP) has recently been cloned and sequenced from both Lactococcus lactis subsp. cremoris (B. Mayo, J. Kok, K. Venema, W. Bockelmann, M. Teuber, H. Reinke, and G. Venema, Appl. Environ. Microbiol. 57:38-44, 1991) and L. lacti

  7. Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase.

    Science.gov (United States)

    McGowan, Sheena; Porter, Corrine J; Lowther, Jonathan; Stack, Colin M; Golding, Sarah J; Skinner-Adams, Tina S; Trenholme, Katharine R; Teuscher, Franka; Donnelly, Sheila M; Grembecka, Jolanta; Mucha, Artur; Kafarski, Pawel; Degori, Ross; Buckle, Ashley M; Gardiner, Donald L; Whisstock, James C; Dalton, John P

    2009-02-24

    Plasmodium falciparum parasites are responsible for the major global disease malaria, which results in >2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase, PfA-M1, a validated drug target. The X-ray crystal structure of PfA-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH(2)]Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite.

  8. Biosynthesis of intestinal microvillar proteins. Forskolin reduces surface expression of aminopeptidase N

    DEFF Research Database (Denmark)

    Danielsen, E M; Hansen, Gert Helge; Cowell, G M

    1987-01-01

    The effect of forskolin on the biosynthesis and intracellular transport of pig intestinal aminopeptidase N (EC 3.4.11.2) was studied in organ cultured mucosal explants. The drug which activates adenylate cyclase and hence the cAMP-dependent glycogenolytic pathway did not affect the explant content...

  9. Tyrosine sulphation is not required for microvillar expression of intestinal aminopeptidase N

    DEFF Research Database (Denmark)

    Danielsen, E M

    1988-01-01

    The effect of 2,6-dichloro-4-nitrophenol (DCNP), an inhibitor of phenol sulphotransferases (EC 2.8.2.-), on the biosynthesis of aminopeptidase N (EC 3.4.11.2) was studied in organ-cultured pig intestinal mucosal explants. At 50 microM DCNP did not affect protein synthesis but it decreased...

  10. Characterization and cDNA cloning of aminopeptidase A from the venom of Gloydius blomhoffi brevicaudus.

    Science.gov (United States)

    Ogawa, Yuko; Murayama, Nobuhiro; Fujita, Yoshiaki; Yanoshita, Ryohei

    2007-06-15

    The aminopeptidase activities of snake venoms from Gloydius blomhoffi brevicaudus, Gloydius halys blomhoffii, Trimeresurus flavoviridis, Bothrops jararaca and Crotalus atrox were investigated. Aminopeptidase A (APA), aminopeptidase B and aminopeptidase N activities were present in all snake venoms. The strongest APA activity was found in venom from G. blomhoffi brevicaudus. The susceptibility to metallopeptidase inhibitors and the pH optimum of the partially purified enzyme from G. blomhoffi brevicaudus venom were similar to those of known APAs from mammals. A G. blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on highly conserved amino acid sequences in known APAs. Molecular cloning of APA from G. blomhoffi brevicaudus venom predicted that it was a type II integral membrane protein containing 958 amino acid residues with 17 potential N-linked glycosylation sites. It possessed a His-Glu-Xaa-Xaa-His-(Xaa)(18)-Glu zinc binding motif that allowed the classification of this protein as a member of the M1 family of zinc-metallopeptidases, or gluzincins. The deduced amino acid sequence shows approximately 60% sequence identity to mammalian APA sequences. This is the first study to report the primary structure of APA from a reptile. PMID:17383704

  11. Characterization and overexpression of the Lactococcus lactis pepN gene and localization of its product, aminopeptidase N.

    OpenAIRE

    van Alen-Boerrigter, I J; Baankreis, R; de Vos, W M

    1991-01-01

    The chromosomal pepN gene encoding lysyl-aminopeptidase activity in Lactococcus lactis has been identified in a lambda EMBL3 library in Escherichia coli by using an immunological screening with antiserum against a purified aminopeptidase fraction. The pepN gene was localized and subcloned in E. coli on the basis of its expression and hybridization to a mixed-oligonucleotide probe for the previously determine N-terminal amino acid sequence of lysyl-aminopeptidase (P. S. T. Tan and W. N. Koning...

  12. Mammary renin-angiotensin system-regulating aminopeptidase activities are modified in rats with breast cancer.

    Science.gov (United States)

    del Pilar Carrera, Maria; Ramírez-Expósito, Maria Jesus; Mayas, Maria Dolores; García, Maria Jesus; Martínez-Martos, Jose Manuel

    2010-12-01

    Angiotensin II in particular and/or the local renin-angiotensin system in general could have an important role in epithelial tissue growth and modelling; therefore, it is possible that it may be involved in breast cancer. In this sense, previous works of our group showed a predominating role of angiotensin II in tumoral tissue obtained from women with breast cancer. However, although classically angiotensin II has been considered the main effector peptide of the renin-angiotensin system cascade, several of its catabolism products such as angiotensin III and angiotensin IV also possess biological functions. These peptides are formed through the activity of several proteolytic regulatory enzymes of the aminopeptidase type, also called angiotensinases. The aim of this work was to analyse several specific angiotensinase activities involved in the renin-angiotensin system cascade in mammary tissue from control rats and from rats with mammary tumours induced by N-methyl-nitrosourea (NMU), which may reflect the functional status of their target peptides under the specific conditions brought about by the tumoural process. The results show that soluble and membrane-bound specific aspartyl aminopeptidase activities and membrane-bound glutamyl aminopeptidase activity increased in mammary tissue from NMU-treated animals and soluble aminopeptidase N and aminopeptidase B activities significantly decreased in mammary tissue from NMU-treated rats. These changes support the existence of a local mammary renin-angiotensin system and that this system and its putative functions in breast tissue could be altered by the tumour process, in which we suggest a predominant role of angiotensin III. All described data about the renin-angiotensin system in mammary tissue support the idea that it must be involved in normal breast tissue functions, and its disruption could be involved in one or more steps of the carcinogenesis process.

  13. Transcriptional regulation of cytosol and membrane alanyl-aminopeptidase in human T cell subsets.

    Science.gov (United States)

    Bukowska, Alicja; Tadje, Janine; Arndt, Marco; Wolke, Carmen; Kähne, Thilo; Bartsch, Jaqueline; Faust, Jürgen; Neubert, Klaus; Hashimoto, Yuichi; Lendeckel, Uwe

    2003-04-01

    Aminopeptidase inhibitors strongly affect the proliferation and function of immune cells in man and animals and are promising agents for the pharmacological treatment of inflammatory or autoimmune diseases. Membrane alanyl-aminopeptidase (mAAP) has been considered as the major target of these anti-inflammatory aminopeptidase inhibitors. Recent evidence also points to a role of the cytosol alanyl-aminopeptidase (cAAP) in the immune response. In this study we used quantitative RT-PCR to determine the mRNA expression of both cAAP and mAAP in resting and activated peripheral T cells and also in CD4+, CD8+, Th1, Th2 and Treg (CD4+ CD25+) subpopulations. Both mAAP and cAAP mRNAs were expressed in all cell types investigated, and in response to activation their expression appeared to be upregulated in CD8+ cells, but downregulated in Treg cells. In CD4+ cells, mAAP and cAAP mRNAs were affected in opposite ways in response to activation. The cAAP-specific inhibitor, PAQ-22, did not affect either cAAP or mAAP expression in activated CD4+ or CD8+ cells, whereas in activated Treg cells it markedly upregulated the mRNA levels of both aminopeptidases. The non-discriminatory inhibitor, phebestin, significantly increased the amount of mAAP and cAAP mRNA in CD4+ and that of cAAP in Treg cells.

  14. The Thermophilic, Homohexameric Aminopeptidase of Borrelia burgdorferi Is a Member of the M29 Family of Metallopeptidases

    OpenAIRE

    Bertin, Patrícia B.; Silene P Lozzi; Howell, Jerrilyn K.; Restrepo-Cadavid, Glória; Neves, David; Teixeira, Antonio R. L.; de Sousa, Marcelo V.; Norris, Steven J; Santana, Jaime M.

    2005-01-01

    Proteases are implicated in several aspects of the physiology of microorganisms, as well as in host-pathogen interactions. Aminopeptidases are also emerging as novel drug targets in infectious agents. In this study, we have characterized an aminopeptidase from the spirochete Borrelia burgdorferi, the causative agent of Lyme disease. The aminopeptidolytic activity was identified in cell extracts from B. burgdorferi by using the substrate leucine-7-amido-4-methylcoumarin. A protein displaying t...

  15. Onset of transcription of the aminopeptidase N (leukemia antigen CD 13) gene at the crypt/villus transition zone during rabbit enterocyte differentiation

    DEFF Research Database (Denmark)

    Norén, O; Dabelsteen, E; Høyer, P E;

    1989-01-01

    The sequence of a cDNA clone (2.82 kbp) of rabbit intestinal aminopeptidase N (CD 13) is reported. Using the corresponding anti-sense RNA probe, the distribution of aminopeptidase N mRNA along the crypt/villus axis of the rabbit small intestine was studied by in situ hybridization. The aminopepti......The sequence of a cDNA clone (2.82 kbp) of rabbit intestinal aminopeptidase N (CD 13) is reported. Using the corresponding anti-sense RNA probe, the distribution of aminopeptidase N mRNA along the crypt/villus axis of the rabbit small intestine was studied by in situ hybridization...

  16. N-Terminal methionine processing by the zinc-activated Plasmodium falciparum methionine aminopeptidase 1b.

    Science.gov (United States)

    Calcagno, Sarah; Klein, Christian D

    2016-08-01

    The methionine aminopeptidase 1b from Plasmodium falciparum (PfMetAP 1b) was cloned, expressed in Escherichia coli and characterized. Surprisingly, and in contrast to other methionine aminopeptidases (MetAPs) that require heavy-metal cofactors such as cobalt, the enzyme is reliably activated by zinc ions. Immobilization of the enzyme is possible by His-tag metal chelation to iminodiacetic acid-agarose and by covalent binding to chloroacetamido-hexyl-agarose. The covalently immobilized enzyme shows long-term stability, allowing a continuous, heterogenous processing of N-terminal methionines, for example, in recombinant proteins. Activation by zinc, instead of cobalt as for other MetAPs, avoids the introduction of heavy metals with toxicological liabilities and oxidative potential into biotechnological processes. The PfMetAP 1b therefore represents a useful tool for the enzymatic, posttranslational processing of recombinant proteins. PMID:27023914

  17. Biosynthesis of intestinal microvillar proteins. Dimerization of aminopeptidase N and lactase-phlorizin hydrolase

    DEFF Research Database (Denmark)

    Danielsen, E M

    1990-01-01

    explants. For aminopeptidase N, dimerization did not begin until 5-10 min after synthesis, and maximal dimerization by cross-linking of the transient form of the enzyme required 1 h, whereas the mature form of aminopeptidase N cross-linked with unchanged efficiency from 45 min to 3 h of labeling. Formation...... of dimers of this enzyme therefore occurs prior to the Golgi-associated processing, and the slow rate of dimerization may be the rate-limiting step in the transport from the endoplasmic reticulum to the Golgi complex. For lactase-phlorizin hydrolase, the posttranslational processing includes a...... proteolytic cleavage of its high molecular weight precursor. Since only the mature form and not the precursor of this enzyme could be cross-linked, formation of tightly associated dimers only takes place after transport out of the endoplasmic reticulum. Dimerization of the two brush border enzymes therefore...

  18. Crystal structure of human insulin-regulated aminopeptidase with specificity for cyclic peptides.

    Science.gov (United States)

    Hermans, Stefan J; Ascher, David B; Hancock, Nancy C; Holien, Jessica K; Michell, Belinda J; Chai, Siew Yeen; Morton, Craig J; Parker, Michael W

    2015-02-01

    Insulin-regulated aminopeptidase (IRAP or oxytocinase) is a membrane-bound zinc-metallopeptidase that cleaves neuroactive peptides in the brain and produces memory enhancing effects when inhibited. We have determined the crystal structure of human IRAP revealing a closed, four domain arrangement with a large, mostly buried cavity abutting the active site. The structure reveals that the GAMEN exopeptidase loop adopts a very different conformation from other aminopeptidases, thus explaining IRAP's unique specificity for cyclic peptides such as oxytocin and vasopressin. Computational docking of a series of IRAP-specific cognitive enhancers into the crystal structure provides a molecular basis for their structure-activity relationships and demonstrates that the structure will be a powerful tool in the development of new classes of cognitive enhancers for treating a variety of memory disorders such as Alzheimer's disease. PMID:25408552

  19. Localization and biosynthesis of aminopeptidase N in pig fetal small intestine

    DEFF Research Database (Denmark)

    Danielsen, E M; Niels-Christiansen, L L; Hansen, Gert Helge

    1995-01-01

    BACKGROUND & AIMS: Little is known about the expression of brush border enzymes in fetal enterocytes. The aim of this study was to describe the localization and biosynthesis of porcine fetal aminopeptidase N. METHODS: This study was performed using histochemistry and immunoelectron microscopy...... and [35S]methionine labeling of cultured mucosal explants. RESULTS: Enzyme activity was present in the brush border membrane and extended into the apical cytoplasm. The protein was colocalized with cationized ferritin at the surface of endocytic structures including coated pits, vesicles, tubules......, and large vacuoles in the apical cytoplasm. The transient high mannose-glycosylated form of fetal aminopeptidase N was processed to the mature complex-glycosylated form at a markedly slower rate than the enzyme in adult intestine. Likewise, dimerization occurred slowly compared with the adult form...

  20. Crystal structure of human insulin-regulated aminopeptidase with specificity for cyclic peptides.

    Science.gov (United States)

    Hermans, Stefan J; Ascher, David B; Hancock, Nancy C; Holien, Jessica K; Michell, Belinda J; Chai, Siew Yeen; Morton, Craig J; Parker, Michael W

    2015-02-01

    Insulin-regulated aminopeptidase (IRAP or oxytocinase) is a membrane-bound zinc-metallopeptidase that cleaves neuroactive peptides in the brain and produces memory enhancing effects when inhibited. We have determined the crystal structure of human IRAP revealing a closed, four domain arrangement with a large, mostly buried cavity abutting the active site. The structure reveals that the GAMEN exopeptidase loop adopts a very different conformation from other aminopeptidases, thus explaining IRAP's unique specificity for cyclic peptides such as oxytocin and vasopressin. Computational docking of a series of IRAP-specific cognitive enhancers into the crystal structure provides a molecular basis for their structure-activity relationships and demonstrates that the structure will be a powerful tool in the development of new classes of cognitive enhancers for treating a variety of memory disorders such as Alzheimer's disease.

  1. Role of Endoplasmic Reticulum Aminopeptidases in Health and Disease: from Infection to Cancer

    OpenAIRE

    Doriana Fruci; Franco Locatelli; Paolo Romania; Silvia Lorenzi; Loredana Cifaldi

    2012-01-01

    Endoplasmic reticulum (ER) aminopeptidases ERAP1 and ERAP2 (ERAPs) are essential for the maturation of a wide spectrum of proteins involved in various biological processes. In the ER, these enzymes work in concert to trim peptides for presentation on MHC class I molecules. Loss of ERAPs function substantially alters the repertoire of peptides presented by MHC class I molecules, critically affecting recognition of both NK and CD8+ T cells. In addition, these enzymes are ...

  2. Endoplasmic Reticulum Aminopeptidase-1 Functions Regulate Key Aspects of the Innate Immune Response

    OpenAIRE

    Aldhamen, Yasser A; Seregin, Sergey S.; Rastall, David P. W.; Charles F Aylsworth; Pepelyayeva, Yuliya; Busuito, Christopher J.; Godbehere-Roosa, Sarah; Kim, Sungjin; Amalfitano, Andrea

    2013-01-01

    Endoplasmic reticulum aminopeptidase-1 (ERAP1) is a multifunctional, ubiquitously expressed enzyme whose peptide-trimming role during antigen processing for presentation by MHC I molecules is well established, however, a role for ERAP1 in modulating global innate immune responses has not been described to date. Here we demonstrate that, relative to wild type mice, mice lacking ERAP1 exhibit exaggerated innate immune responses early during pathogen recognition, as characterized by increased ac...

  3. Structural insights into the molecular ruler mechanism of the endoplasmic reticulum aminopeptidase ERAP1

    OpenAIRE

    Amit Gandhi; Damodharan Lakshminarasimhan; Yixin Sun; Hwai-Chen Guo

    2011-01-01

    Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an essential component of the immune system, because it trims peptide precursors and generates the N--restricted epitopes. To examine ERAP1's unique properties of length- and sequence-dependent processing of antigen precursors, we report a 2.3 Å resolution complex structure of the ERAP1 regulatory domain. Our study reveals a binding conformation of ERAP1 to the carboxyl terminus of a peptide, and thus provides direct evidence for the molecular...

  4. The two authentic methionine aminopeptidase genes are differentially expressed in Bacillus subtilis

    Directory of Open Access Journals (Sweden)

    Wang YiPing

    2005-10-01

    Full Text Available Abstract Background Two putative methionine aminopeptidase genes, map (essential and yflG (non-essential, were identified in the genome sequence of Bacillus subtilis. We investigated whether they can function as methionine aminopeptidases and further explored possible reasons for their essentiality or dispensability in B. subtilis. Results In silico analysis of MAP evolution uncovered a coordinated pattern of MAP and deformylase that did not correlate with the pattern of 16S RNA evolution. Biochemical assays showed that both MAP (MAP_Bs and YflG (YflG_Bs from B. subtilis overproduced in Escherichia coli and obtained as pure proteins exhibited a methionine aminopeptidase activity in vitro. Compared with MAP_Bs, YflG_Bs was approximately two orders of magnitude more efficient when assayed on synthetic peptide substrates. Both map and yflG genes expressed in multi-copy plasmids could complement the function of a defective map gene in the chromosomes of both E. coli and B. subtilis. In contrast, lacZ gene transcriptional fusions showed that the promoter activity of map was 50 to 100-fold higher than that of yflG. Primer extension analysis detected the transcription start site of the yflG promoter. Further work identified that YvoA acted as a possible weak repressor of yflG expression in B. subtilis in vivo. Conclusion Both MAP_Bs and YflG_Bs are functional methionine aminopeptidases in vitro and in vivo. The high expression level of map and low expression level of yflG may account for their essentiality and dispensality in B. subtilis, respectively, when cells are grown under laboratory conditions. Their difference in activity on synthetic substrates suggests that they have different protein targets in vivo.

  5. Bioluminogenic Imaging of AminopeptidaseN In Vitro and In Vivo.

    Science.gov (United States)

    Wu, Wenxiao; Chen, Laizhong; Li, Jing; Du, Lupei; Li, Minyong

    2016-01-01

    Bioluminescence is a process that converts biochemical energy to visible light. Bioluminescence-based imaging technology has been widely applicable in the imaging of process envisioned in life sciences. As one of the most popular bioluminescence system, the firefly luciferin-luciferase system is exceptionally suitable for deep tissue imaging in living animals owing to its long wavelength emission light. Herein, we report the experimental detail of bioluminogenic imaging of aminopeptidase N activity both in vitro and in vivo. PMID:27424897

  6. Purification and functional characterisation of rhiminopeptidase A, a novel aminopeptidase from the venom of Bitis gabonica rhinoceros.

    Directory of Open Access Journals (Sweden)

    Sakthivel Vaiyapuri

    Full Text Available BACKGROUND: Snake bite is a major neglected public health issue within poor communities living in the rural areas of several countries throughout the world. An estimated 2.5 million people are bitten by snakes each year and the cost and lack of efficacy of current anti-venom therapy, together with the lack of detailed knowledge about toxic components of venom and their modes of action, and the unavailability of treatments in rural areas mean that annually there are around 125,000 deaths worldwide. In order to develop cheaper and more effective therapeutics, the toxic components of snake venom and their modes of action need to be clearly understood. One particularly poorly understood component of snake venom is aminopeptidases. These are exo-metalloproteases, which, in mammals, are involved in important physiological functions such as the maintenance of blood pressure and brain function. Although aminopeptidase activities have been reported in some snake venoms, no detailed analysis of any individual snake venom aminopeptidases has been performed so far. As is the case for mammals, snake venom aminopeptidases may also play important roles in altering the physiological functions of victims during envenomation. In order to further understand this important group of snake venom enzymes we have isolated, functionally characterised and analysed the sequence-structure relationships of an aminopeptidase from the venom of the large, highly venomous West African gaboon viper, Bitis gabonica rhinoceros. METHODOLOGY AND PRINCIPAL FINDINGS: The venom of B. g. rhinoceros was fractionated by size exclusion chromatography and fractions with aminopeptidase activities were isolated. Fractions with aminopeptidase activities showed a pure protein with a molecular weight of 150 kDa on SDS-PAGE. In the absence of calcium, this purified protein had broad aminopeptidase activities against acidic, basic and neutral amino acids but in the presence of calcium, it had only

  7. Effects of tannic acid on trypsin and leucine aminopeptidase activities in gypsy moth larval midgut

    Directory of Open Access Journals (Sweden)

    Mrdaković Marija

    2013-01-01

    Full Text Available The effects of allelochemical stress on genetic variations in the specific activities of gypsy moth digestive enzymes (trypsin and leucine aminopeptidase and relative midgut mass (indirect measure of food consumption, as well as variability in their plasticity, were investigated in fifth instar gypsy moths originating from two populations with different trophic adaptations (oak and locust-tree forests. Thirty-two full-sib families from the Quercus population and twenty-six full-sib families from the Robinia population were reared on an artificial diet with or without supplementation with tannic acid. Between population differences were observed as higher average specific activity of trypsin and relative midgut mass in larvae from the Robinia population. Significant broad-sense heritabilities were observed for the specific activity of trypsin in the control state, and for specific activity of leucine aminopeptidase in a stressful environment. Significantly lower heritability for relative midgut mass was recorded in larvae from the Robinia population reared under stressful conditions. Significant variability of trypsin plasticity in larvae from both populations and significant variability of leucine aminopeptidase plasticity in larvae from the Robinia population point to the potential for the evolution of enzyme adaptive plastic responses to the presence of stressor. Non-significant across-environment genetic correlations do not represent a constraint for the evolution of enzyme plasticity. [Projekat Ministarstva nauke Republike Srbije, br. 173027

  8. A Plasmodium falciparum S33 proline aminopeptidase is associated with changes in erythrocyte deformability.

    Science.gov (United States)

    da Silva, Fabio L; Dixon, Matthew W A; Stack, Colin M; Teuscher, Franka; Taran, Elena; Jones, Malcolm K; Lovas, Erica; Tilley, Leann; Brown, Christopher L; Trenholme, Katharine R; Dalton, John P; Gardiner, Donald L; Skinner-Adams, Tina S

    2016-10-01

    Infection with the apicomplexan parasite Plasmodium falciparum is a major cause of morbidity and mortality worldwide. One of the striking features of this parasite is its ability to remodel and decrease the deformability of host red blood cells, a process that contributes to disease. To further understand the virulence of Pf we investigated the biochemistry and function of a putative Pf S33 proline aminopeptidase (PfPAP). Unlike other P. falciparum aminopeptidases, PfPAP contains a predicted protein export element that is non-syntenic with other human infecting Plasmodium species. Characterization of PfPAP demonstrated that it is exported into the host red blood cell and that it is a prolyl aminopeptidase with a preference for N-terminal proline substrates. In addition genetic deletion of this exopeptidase was shown to lead to an increase in the deformability of parasite-infected red cells and in reduced adherence to the endothelial cell receptor CD36 under flow conditions. Our studies suggest that PfPAP plays a role in the rigidification and adhesion of infected red blood cells to endothelial surface receptors, a role that may make this protein a novel target for anti-disease interventions strategies.

  9. Crystallization and preliminary X-ray diffraction analysis of human endoplasmic reticulum aminopeptidase 2

    International Nuclear Information System (INIS)

    The luminal domain of human endoplasmic reticulum aminopeptidase 2 has been expressed, purified and crystallized. The crystals belonged to the orthorhombic space group P21212 and diffracted anisotropically to 3.3 Å resolution in the best direction on an in-house source. Endoplasmic reticulum aminopeptidase 2 (ERAP2) is a critical enzyme involved in the final processing of MHC class I antigens. Peptide trimming by ERAP2 and the other members of the oxytocinase subfamily is essential to customize longer precursor peptides in order to fit them to the correct length required for presentation on major histocompatibility complex class I molecules. While recent structures of ERAP1 have provided an understanding of the ‘molecular-ruler’ mechanism of substrate selection, little is known about the complementary activities of its homologue ERAP2 despite their sharing 49% sequence identity. In order to gain insights into the structure–function relationship of the oxytocinase subfamily, and in particular ERAP2, the luminal region of human ERAP2 has been crystallized in the presence of the inhibitor bestatin. The crystals belonged to an orthorhombic space group and diffracted anisotropically to 3.3 Å resolution in the best direction on an in-house X-ray source. A molecular-replacement solution suggested that the enzyme has adopted the closed state as has been observed in other inhibitor-bound aminopeptidase structures

  10. Substrate specificity screening of oat (Avena sativa) seeds aminopeptidase demonstrate unusually broad tolerance in S1 pocket.

    Science.gov (United States)

    Gajda, Anna D; Pawełczak, Małgorzata; Drag, Marcin

    2012-05-01

    Aminopeptidases are proteolytic enzymes that remove one amino acid at a time from N-terminus of peptidic substrates. In plants, inhibitors of aminopeptidases can find potential applications in agriculture as herbicides. In this report we have used a library of fluorogenic derivatives of natural and unnatural amino acids for substrate specificity profiling of oat (Avena sativa) aminopeptidase. Interestingly, we have found that this enzyme recognizes effectively among the natural amino acids basic residues like Arg and Lys, hydrophobic Phe, Leu and Met, but also to some extent acidic residues Asp and Glu. In the case of unnatural amino acids hydrophobic residues (hPhe and hCha) and basic hArg were preferentially recognized.

  11. cDNAs of aminopeptidase-like protein genes from Plodia interpunctella strains with different susceptibilities to Bacillus thuringiensis toxins.

    Science.gov (United States)

    Zhu, Y C; Kramer, K J; Oppert, B; Dowdy, A K

    2000-03-01

    Aminopeptidase N has been reported to be a Bacillus thuringiensis (Bt) Cry1A toxin-binding protein in several lepidopteran insects. cDNAs of aminopeptidase-like proteins from both Bt-susceptible RC688s and Bt-resistant HD198r strains of the Indianmeal moth, Plodia interpunctella, were cloned and sequenced. They contain 3345 and 3358 nucleotides, respectively, and each has a 3048 bp open reading frame that encodes 1016 amino acids. Putative protein sequences include 10 potential glycosylation sites and a zinc metal binding site motif of HEXXH, which is typical of the active site of zinc-dependent metallopeptidases. Sequence analysis indicated that the deduced protein sequences are most similar to an aminopeptidase from Heliothis virescens with 62% sequence identity and highly similar to three other lepidopteran aminopeptidases from Plutella xylostella, Manduca sexta, Bombyx mori with sequence identities of 51-52%. Four nucleotide differences were observed in the open reading frames that translated into two amino acid differences in the putative protein sequences. Polymerase chain reaction (PCR) confirmed an aminopeptidase gene coding difference between RC688s and HD198r strains of P. interpunctella in the PCR amplification of a specific allele (PASA) using preferential primers designed from a single base substitution. The gene mutation for Asp185-->Glu185 was also confirmed in two additional Bt-resistant P. interpunctella strains. This mutation is located within a region homologous to the conserved Cry1Aa toxin binding regions from Bombyx mori and Plutella xylostella. The aminopeptidase-like mRNA expression levels in the Bt-resistant strain were slightly higher than those in the Bt-susceptible strain. The sequences reported in this paper have been deposited in the GenBank database (accession numbers AF034483 for susceptible strain RC688s and AF034484 for resistant strain HD198r).

  12. Structural Basis for Antigenic Peptide Recognition and Processing by Endoplasmic Reticulum (ER) Aminopeptidase 2.

    Science.gov (United States)

    Mpakali, Anastasia; Giastas, Petros; Mathioudakis, Nikolas; Mavridis, Irene M; Saridakis, Emmanuel; Stratikos, Efstratios

    2015-10-23

    Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5 and 2.7 Å, respectively, and compared them to the apo-form structure determined to 3.0 Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center toward the distal end of the internal cavity, making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product, although decreasing electron density toward its C terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias. PMID:26381406

  13. Hypertension and Angiotensin II Hypersensitivity in Aminopeptidase A–deficient Mice

    OpenAIRE

    MITSUI, Takashi; NOMURA, Seiji; Okada, Mayumi; Ohno, Yasumasa; Kobayashi, Honami; Nakashima,Yutaka; Murata, Yasutaka; Takeuchi, Mikihito; Kuno, Naohiko; Nagasaka, Tetsuo; O-Wang, Jiyang; Cooper, Max D.; Mizutani, Shigehiko

    2003-01-01

    Local concentrations of the vasopressor peptide, angiotensin II (AngII), depend upon the balance between synthesis and degradation. Previous studies of blood pressure (BP) regulation have focused primarily on the generation of AngII and its receptors, and less attention has been devoted to angiotensin degradation. Aminopeptidase A (APA, EC 3.4.11.7) is responsible for the N-terminal cleavage of AngII, a hydrolytic event that serves as a rate-limiting step in angiotensin degradation. To evalua...

  14. Biosynthesis of intestinal microvillar proteins. Effect of castanospermine on cell-free synthesis of aminopeptidase N

    DEFF Research Database (Denmark)

    Danielsen, E M; Olsen, Jørgen

    1988-01-01

    the blocked removal of glucose residues. In contrast to its reduced expression in a mucosal explant system [(1986) Biochem. J. 240, 777-782], this molecular form of aminopeptidase N was at least as abundant in cell-free translation as its normal high mannose-glycosylated counterpart, ruling out degradation...... taking place in the rough endoplasmic reticulum. Degradation of newly produced, malprocessed enzyme must therefore occur at a later stage during intracellular transport, presumably in the sarcoplasmic reticulum or in transitional elements between this organelle and the Golgi complex....

  15. Aminopeptidase N/CD13 targeting fluorescent probes: synthesis and application to tumor cell imaging.

    Science.gov (United States)

    Zhang, Zhouen; Harada, Hiroshi; Tanabe, Kazuhito; Hatta, Hiroshi; Hiraoka, Masahiro; Nishimoto, Sei-ichi

    2005-11-01

    A family of fluorescein-peptide conjugates (CNP1-3) for aminopeptidase N (APN/CD13) targeting fluorescent probes were designed and synthesized. Among the three conjugates, CNP1 bearing tumor-homing cyclic peptide CNGRC, could selectively label APN/CD13 over-expressing on the surface of tumor cells of HT-1080, as identified by means of fluorescent microscopic cell imaging. CNP1 was shown to be a promising fluorescent probe applicable to tumor-targeting molecular imaging. PMID:15885853

  16. Biosynthesis of intestinal microvillar proteins. Surface expression of aminopeptidase N is not affected by chloroquine

    DEFF Research Database (Denmark)

    Danielsen, E M; Sjöström, H; Norén, Ove

    1985-01-01

    The effect of chloroquine on the biosynthesis of pig intestinal aminopeptidase N (EC 3.4.11.2) was studied by labelling with [35S]methionine in organ cultured mucosal explants. The lysosomotropic agent did not alter the molecular size of either the transient or the mature form of the enzyme and d...... no consequence on the intracellular transport of the newly synthesized microvillar enzyme. This suggests that the acidic compartments are not involved in the post-Golgi transport and that this, in turn, probably occurs via a constitutive rather than a regulated pathway....

  17. Purification, Characterization, Gene Cloning, Sequencing, and Overexpression of Aminopeptidase N from Streptococcus thermophilus A

    OpenAIRE

    Chavagnat, Frederic; Casey, Michael G.; Meyer, Jacques

    1999-01-01

    The general aminopeptidase PepN from Streptococcus thermophilus A was purified to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtration chromatographies. The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity on N-Lys–7-amino-4-methylcoumarin at pH 7 and 37°C. It was strongly inhibited by metal chelating agents, suggesting that it is a metallopeptidase. The activity was greatly restored by the bivalent cations Co2+, Zn2+, and Mn2+. Except for prolin...

  18. Potent macrocyclic inhibitors of insulin-regulated aminopeptidase (IRAP) by olefin ring-closing metathesis.

    Science.gov (United States)

    Andersson, Hanna; Demaegdt, Heidi; Johnsson, Anders; Vauquelin, Georges; Lindeberg, Gunnar; Hallberg, Mathias; Erdelyi, Mate; Karlen, Anders; Hallberg, Anders

    2011-06-01

    Macrocyclic analogues of angiotensin IV (Ang IV, Val(1)-Tyr(2)-Ile(3)-His(4)-Pro(5)-Phe(6)) targeting the insulin-regulated aminopeptidase (IRAP) have been designed, synthesized, and evaluated biologically. Replacement of His(4)-Pro(5)-Phe(6) by a 2-(aminomethyl)phenylacetic acid (AMPAA) moiety and of Val(1) and Ile(3) by amino acids bearing olefinic side chains followed by macrocyclization provided potent IRAP inhibitors. The impact of the ring size and the type (saturated versus unsaturated), configuration, and position of the carbon-carbon bridge was assessed. The ring size generally affects the potency more than the carbon-carbon bond characteristics. Replacing Tyr(2) by β(3)hTyr or Phe is accepted, while N-methylation of Tyr(2) is deleterious for activity. Removal of the carboxyl group in the C-terminal slightly reduced the potency. Inhibitors 7 (K(i) = 4.1 nM) and 19 (K(i) = 1.8 nM), both encompassing 14-membered ring systems connected to AMPAA, are 10-fold more potent than Ang IV and are also more selective over aminopeptidase N (AP-N). Both compounds displayed high stability against proteolysis by metallopeptidases.

  19. Unexpected Diversity of pepA Genes Encoding Leucine Aminopeptidases in Sediments from a Freshwater Lake

    Science.gov (United States)

    Tsuboi, Shun; Yamamura, Shigeki; Imai, Akio; Iwasaki, Kazuhiro

    2016-01-01

    We herein designed novel PCR primers for universal detection of the pepA gene, which encodes the representative leucine aminopeptidase gene, and investigated the genetic characteristics and diversity of pepA genes in sediments of hypereutrophic Lake Kasumigaura, Japan. Most of the amino acid sequences deduced from the obtained clones (369 out of 370) were related to PepA-like protein sequences in the M17 family of proteins. The developed primers broadly detected pepA-like clones associated with diverse bacterial phyla—Alpha-, Beta-, Gamma-, and Deltaproteobacteria, Acidobacteria, Actinobacteria, Aquificae, Chlamydiae, Chloroflexi, Cyanobacteria, Firmicutes, Nitrospirae, Planctomycetes, and Spirochetes as well as the archaeal phylum Thaumarchaeota, indicating that prokaryotes in aquatic environments possessing leucine aminopeptidase are more diverse than previously reported. Moreover, prokaryotes related to the obtained pepA-like clones appeared to be r- and K-strategists, which was in contrast to our previous findings showing that the neutral metalloprotease gene clones obtained were related to the r-strategist genus Bacillus. Our results suggest that an unprecedented diversity of prokaryotes with a combination of different proteases participate in sedimentary proteolysis. PMID:26936797

  20. Renin angiotensin system-regulating aminopeptidase activities in serum of pre- and postmenopausal women with breast cancer.

    Science.gov (United States)

    Martínez-Martos, José Manuel; del Pilar Carrera-González, María; Dueñas, Basilio; Mayas, María Dolores; García, María Jesús; Ramírez-Expósito, María Jesús

    2011-10-01

    Angiotensin peptides regulate vascular tone and natriohydric balance through the renin angiotensin system (RAS) and are related with the angiogenesis which plays an important role in the metastatic pathway. Estrogen influences the aminopeptidases (APs) involved in the metabolism of bioactive peptides of RAS through several pathways. We analyze RAS-regulating AP activities in serum of pre- and postmenopausal women with breast cancer to evaluate the putative value of these activities as biological markers of the development of breast cancer. We observed an increase in aminopeptidase N (APN) and aminopeptidase B (APB) activities in women with breast cancer; however, a decrease in aspartyl-aminopeptidase (AspAP) activity in premenopausal women. These results suggest a slow metabolism of angiotensin II (Ang II) to angiotensin III (Ang III) in premenopausal women and a rapid metabolism of Ang III to angiotensin IV (Ang IV) in pre- and postmenopausal women with breast cancer. An imbalance in the signals activated by Ang II may produce abnormal vascular growth with different response between pre- and postmenopausal women depending on the hormonal profile and the development of the disease.

  1. Crystal Structure of the Leucine Aminopeptidase from Pseudomonas putida Reveals the Molecular Basis for its Enantioselectivity and Broad Substrate Specificity

    NARCIS (Netherlands)

    Kale, Avinash; Pijning, Tjaard; Sonke, Theo; Dijkstra, Bauke W.; Thunnissen, Andy-Mark W. H.; Guss, M.

    2010-01-01

    The zinc-dependent leucine aminopeptidase from Pseudomonas putida (ppLAP) is an important enzyme for the industrial production of enantiomerically pure amino acids. To provide a better understanding of its structure function relationships, the enzyme was studied by X-ray crystallography. Crystal str

  2. Changes in vasopressin-converting aminopeptidase activity in the rat pineal gland during summer : Relationship to vasopressin contents

    NARCIS (Netherlands)

    Liu, B.; Burbach, J.P.H.

    1988-01-01

    Vasopressin (VP)-converting aminopeptidase (VP-AP) activity and VP contents were measured in single rat pineal glands during the summer of two successive years. The peptidase activity decreased significantly in August. The lowest activity (±SEM) of 0.18±0.02 pmol·hour−1 was recorded on August 14, co

  3. Bacillus thuringiensis Cry1Ca-resistant Spodoptera exigua lacks expression of one of four Aminopeptidase N genes

    NARCIS (Netherlands)

    Herrero, S.; Gechev, T.; Bakker, P.L.; Moar, W.; Maagd, de R.A.

    2005-01-01

    BACKGROUND: Insecticidal toxins from Bacillus thuringiensis bind to receptors on midgut epithelial cells of susceptible insect larvae. Aminopeptidases N (APNs) from several insect species have been shown to be putative receptors for these toxins. Here we report the cloning and expression analysis of

  4. Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on aminopeptidase N and sucrase-isomaltase

    DEFF Research Database (Denmark)

    Danielsen, Erik Michael

    1982-01-01

    The biogenesis of two microvillar enzymes, aminopeptidase N (EC 3.4.11.2) and sucrase (EC 3.2.1.48)-isomaltase (EC 3.2.1.10), was studied by pulse-chase labelling of pig small-intestinal explants kept in organ culture. Both enzymes became inserted into the membrane during or immediately after...

  5. Biosynthesis of intestinal microvillar proteins. The effect of swainsonine on post-translational processing of aminopeptidase N

    DEFF Research Database (Denmark)

    Danielsen, E M; Cowell, G M; Norén, Ove;

    1983-01-01

    The post-translational processing of pig small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in organ-cultured mucosal explants. Exposure of the explants to swainsonine, an inhibitor of Golgi mannosidase II, resulted in the formation of a Mr-160000 polypeptide, still sensitive to endo...

  6. Molecular Cloning and Sequence Analysis of the X-Prolyl Dipeptidyl Aminopeptidase Gene From Lactococcus lactis subsp. cremoris

    NARCIS (Netherlands)

    Mayo, Baltasar; Kok, Jan; Venema, Konraad; Bockelmann, Wilhelm; Teuber, Michael; Reinke, Heinz; Venema, Gerhardus

    1991-01-01

    Lactococcus lactis subsp. cremoris P8-2-47 contains an X-prolyl dipeptidyl aminopeptidase (X-PDAP; EC 3.4.14.5). A mixed-oligonucleotide probe prepared on the basis of the N-terminal amino acid sequence of the purified protein was made and used to screen a partial chromosomal DNA bank in Escherichia

  7. Endoplasmic reticulum aminopeptidase 2 is highly expressed in papillary thyroid microcarcinoma with cervical lymph node metastasis

    Directory of Open Access Journals (Sweden)

    Woo Young Kim

    2015-01-01

    Full Text Available Background: The cervical lymph node metastasis (CLNM of papillary thyroid microcarcinoma (PTMC is not uncommon. However, prophylactic cervical lymph node dissection in all PTMC is debatable. Molecular markers of predicting CLNM would help to decide to either do or not do cervical lymph node dissection which might increase morbidities. Aims: We aimed to characterize gene expression profiles and molecular markers of CLNM in PTMC. Settings and Design: The thyroid frozen tissues were obtained with from six PTMC patients, who underwent total thyroidectomy. Methods: We performed oligonucleotide microarray analysis with three PTMCs with CLNM and three without CLNM. Real-time quantitative reverse transcription-polymerase chain reaction was used to validate the gene. Statistical Analysis Used: We used linear models for microarray data. Results: We identified 12 differentially expressed gene, and most one is endoplasmic reticulum aminopeptidase 2 (ERAP2. Conclusion: ERAP2 might be associated with CLNM in PTMC.

  8. Bacillus thuringiensis Cry1Aa toxin-binding region of Bombyx mori aminopeptidase N.

    Science.gov (United States)

    Yaoi, K; Nakanishi, K; Kadotani, T; Imamura, M; Koizumi, N; Iwahana, H; Sato, R

    1999-12-17

    The Bacillus thuringiensis Cry1Aa toxin-binding region of Bombyx mori aminopeptidase N (APN) was analyzed, to better understand the molecular mechanism of susceptibility to the toxin and the development of resistance in insects. APN was digested with lysylendopeptidase and the ability of the resulting fragments to bind to Cry1Aa and 1Ac toxins was examined. The binding abilities of the two toxins to these fragments were different. The Cry1Aa toxin bound to the fragment containing 40-Asp to 313-Lys, suggesting that the Cry1Aa toxin-binding site is located in the region between 40-Asp and 313-Lys, while Cry1Ac toxin bound exclusively to mature APN. Next, recombinant APN of various lengths was expressed in Escherichia coli cells and its ability to bind to Cry1Aa toxin was examined. The results localized the Cry1Aa toxin binding to the region between 135-Ile and 198-Pro. PMID:10606725

  9. Cloning of aminopeptidase Npromoter and its activity in hematopoietic cell and different tumor cell lines

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Aminopeptidase N (APN) promoter region was cloned and sequenced from peripheral blood mononuclear cells. The recombinant reporter construct containing the promoter and luciferase gene, designated pXP1-APNLuc, was introduced into myeloblastic cell line, T lymphocyte cell line and various tumor cell lines. Luciferase assay showed that APN upstream promoter is myeloid-specific for high expression in myeloblastic cell line and much lower expres sion in T lymphocyte cell line. The promoter activity was relatively high in lung adenoma cell line compared with other tumor cell lines including hepatoma cell line, tong cancer cell line and esophageal cancer cell line in which the promoter activity significantly diminished or was almost undetectable. The characteristics of APN promoter may pro vide a new strategy for specific myeloprotection while tumor patients are being treated with chemotherapy and/or radio therapy.

  10. Feline aminopeptidase N is not a functional receptor for avian infectious bronchitis virus

    Directory of Open Access Journals (Sweden)

    Harbison Carole E

    2007-02-01

    Full Text Available Abstract Background Coronaviruses are an important cause of infectious diseases in humans, including severe acute respiratory syndrome (SARS, and have the continued potential for emergence from animal species. A major factor in the host range of a coronavirus is its receptor utilization on host cells. In many cases, coronavirus-receptor interactions are well understood. However, a notable exception is the receptor utilization by group 3 coronaviruses, including avian infectious bronchitis virus (IBV. Feline aminopeptidase N (fAPN serves as a functional receptor for most group 1 coronaviruses including feline infectious peritonitis virus (FIPV, canine coronavirus, transmissible gastroenteritis virus (TGEV, and human coronavirus 229E (HCoV-229E. A recent report has also suggested a role for fAPN during IBV entry (Miguel B, Pharr GT, Wang C: The role of feline aminopeptidase N as a receptor for infectious bronchitis virus. Brief review. Arch Virol 2002, 147:2047–2056. Results Here we show that, whereas both transient transfection and constitutive expression of fAPN on BHK-21 cells can rescue FIPV and TGEV infection in non-permissive BHK cells, fAPN expression does not rescue infection by the prototype IBV strain Mass41. To account for the previous suggestion that fAPN could serve as an IBV receptor, we show that feline cells can be infected with the prototype strain of IBV (Mass 41, but with low susceptibility compared to primary chick kidney cells. We also show that BHK-21 cells are slightly susceptible to certain IBV strains, including Ark99, Ark_DPI, CA99, and Iowa97 ( Conclusion We conclude that fAPN is not a functional receptor for IBV, the identity of which is currently under investigation.

  11. Expression levels of aminopeptidase-N genes in the lightbrown apple moth,Epiphyas postvittana

    Institute of Scientific and Technical Information of China (English)

    Robert M.Simpson; Joanne Poulton; Ngaire P.Markwick

    2008-01-01

    Five aminopeptidase-N genes(EpAPNl-5)of the tortricid moth Epiphyas postvittana have been isolated from a midgut expressed sequence tag(EST)library.Relative RNA expression of these genes was measured by quantitative reverse transcription poly-merase chain reaction using actin as a reference gene.Measurements were made on various tissues of fifth instar larvac.over all stages of the life cycle and under differing dietary conditions:different protein sources and levels,and in presence of trypsin and metalloproteatm ininbitors.Gene expression for all five genes Was the greatest in midgut tissue,but Was also detected in the hindgut,fat body and Malpighian tubules.EpAPN4 was consistently the highest expressed,with EpAPN3 at about half that level;EpAPN5 Was the least expressed.During larval stages expression Was high,generally increasing OVer the instars,after an early Deak as neonates or first instars.Expression in other life stages Was much lower.Males and females showed differing expression in the pupal and adult stages:female expression was higher in the pupa,this reversed in tlle adult.Gene expression levels and ratios both changed with diet.A natural apple leaf diet depressed levels.Diets with the most impaired amino acid balance induced the greatest change;generally EpAPN1 increased by the greatest proportion.The addition of proteinase inhibitor also increased gene expression,and it Was noteworthy that the trypsin inhibitor addition,which has been shown to double aminopeptidase activity,also doubled levels of gene expression.

  12. Cloning and Sequencing of a Gene Encoding Aminopeptidase N in the Midgut of Helicoverpa armigera (Hübner)

    Institute of Scientific and Technical Information of China (English)

    WANG Gui-rong; LIANG Ge-mei; WU Kong-ming; GUO Yu-yuan

    2003-01-01

    A cDNA encoding aminopeptidase N was cloned by degenerated PCR combined with RACEtechnique in this paper. The full-length of APN-Harm is 3 043 bp. Open reading frame is 2 856 bp inlength, encoding 951 amino acid residues. Its predicted molecular weight and isoelectric point are 108.3kDa and 5.29, respectively. This deduced amino acid sequence shares some common structural features withaminopeptidase N from several moth species, including the consensus zinc-binding motif HEXXHX18 E and the GAMEN motif common to gluzincin aminopeptidases. The first 20 amino acid residues at N-termini ishydrophobic transmembrane helix. The sequence of APN-Harm was deposited in GenBank and the accessionnumber is AY181026.

  13. Identification of a Bacillus thuringiensis Cry11Ba toxin-binding aminopeptidase from the mosquito, Anopheles quadrimaculatus

    Directory of Open Access Journals (Sweden)

    Abdullah Mohd

    2006-05-01

    Full Text Available Abstract Background Aminopeptidase N (APN type proteins isolated from several species of lepidopteran insects have been implicated as Bacillus thuringiensis (Bt toxin-binding proteins (receptors for Cry toxins. We examined brush border membrane vesicle (BBMV proteins from the mosquito Anopheles quadrimaculatus to determine if APNs from this organism would bind mosquitocidal Cry toxins that are active to it. Results A 100-kDa protein with APN activity (APNAnq 100 was isolated from the brush border membrane of Anopheles quadrimaculatus. Native state binding analysis by surface plasmon resonance shows that APNAnq 100 forms tight binding to a mosquitocidal Bt toxin, Cry11Ba, but not to Cry2Aa, Cry4Ba or Cry11Aa. Conclusion An aminopeptidase from Anopheles quadrimaculatus mosquitoes is a specific binding protein for Bacillus thuringiensis Cry11Ba.

  14. Roles of the Peptide Transport Systems and Aminopeptidase PepA in Peptide Assimilation by Helicobacter pylori.

    Science.gov (United States)

    Ki, Mi Ran; Lee, Ji Hyun; Yun, Soon Kyu; Choi, Kyung Min; Hwang, Se Young

    2015-10-01

    Peptide assimilation in Helicobacter pylori necessitates a coordinated working of the peptide transport systems (PepTs) and aminopeptidase (PepA). We found that H. pylori hydrolyzes two detector peptides, L-phenylalanyl-L-3-thiaphenylalanine (PSP) and L-phenylalanyl-L-2- sulfanilylglycine (PSG), primarily before intake and excludes their antibacterial effects, whereas Escherichia coli readily transports them with resultant growth inhibition. PSP assimilation by H. pylori was inhibited by aminopeptidase inhibitor bestatin, but not by dialanine or cyanide-m-chlorophenylhydrazone, contrary to that of E. coli. RT- and qRT-PCR analyses showed that H. pylori may express first the PepTs (e.g., DppA and DppB) and then PepA. In addition, western blot analysis of PepA suggested that the bacterium secretes PepA in response to specific inducers.

  15. 3’-RACE Amplification of Aminopeptidase N Gene from Anopheles stephensi Applicable in Transmission Blocking Vaccines

    OpenAIRE

    Bokharaei, Hanieh; Raz, Abbasali; Zakeri, Sedigheh; Djadid, Navid Dinparast

    2012-01-01

    Background Because of the lack of an effective and economical control strategy against malaria (the most devastating infectious disease in developing countries) Transmission-Blocking Vaccines (TBVs) concept has been raised in recent years, promising a more efficient way to malaria control. TBVs aim at interfering and/or blocking pathogen development within the vector, halting transmission to non-infected vertebrate host. Aminopeptidase N (APN) is one of the most potent proteins in parasite de...

  16. A novel virulence strategy for Pseudomonas aeruginosa mediated by an autotransporter with arginine-specific aminopeptidase activity

    OpenAIRE

    Luckett, Jeni C. A.; Owen Darch; Chase Watters; Manal Abuoun; Victoria Wright; Esteban Paredes-Osses; Jenny Ward; Hana Goto; Stephan Heeb; Stéphanie Pommier; Rumbaugh, Kendra P.; Miguel Cámara; Hardie, Kim R.

    2012-01-01

    Author Summary We present a new Pseudomonas aeruginosa virulence factor that promotes chronic skin wound infections. We propose the name AaaA for this cell-surface tethered autotransporter. This arginine-specific aminopeptidase confers a growth advantage upon P. aeruginosa, providing a fitness advantage by creating a supply of arginine in chronic wounds where oxygen availability is limited and biofilm formation is involved. To our knowledge, this is the first mechanistic evidence linking the ...

  17. A common SNP in ER aminopeptidase 2 induces a specificity switch that leads to altered antigen processing

    OpenAIRE

    Evnouchidou, Irini; Birtley, James; Seregin, Sergey; Papakyriakou, Athanasios; Zervoudi, Efthalia; Samiotaki, Martina; Panayotou, George; Giastas, Petros; Petrakis, Olivia; Georgiadis, Dimitris; Amalfitano, Andrea; Saridakis, Emmanuel; Mavridis, Irene M.; Stratikos, Efstratios

    2012-01-01

    ER aminopeptidases 1 and 2 (ERAP1 and ERAP2) cooperate to trim antigenic peptide precursors for loading onto MHC class I molecules and help regulate the adaptive immune response. Common coding single nucleotide polymorphisms (SNPs) in ERAP1 and ERAP2 have been linked with predisposition to human diseases ranging from viral and bacterial infections to autoimmunity and cancer. It has been hypothesized that altered antigen processing by these enzymes is a causal link to disease etiology but the ...

  18. A Role for Naturally Occurring Alleles of Endoplasmic Reticulum Aminopeptidases in Tumor Immunity and Cancer Pre-Disposition

    OpenAIRE

    Stratikos, Efstratios; Stamogiannos, Athanasios; Zervoudi, Efthalia; Fruci, Doriana

    2014-01-01

    Endoplasmic reticulum aminopeptidase 1 and 2 (ERAP1 and ERAP2) are key components on the pathway that generates antigenic epitopes for presentation to cytotoxic T-lymphocytes (CTLs). Coding single nucleotide polymorphisms (SNPs) in these enzymes have been associated with pre-disposition to several major human diseases including inflammatory diseases with autoimmune etiology, viral infections, and virally induced cancer. The function of these enzymes has been demonstrated to affect CTL and nat...

  19. Secretion of Endoplasmic Reticulum Aminopeptidase 1 Is Involved in the Activation of Macrophages Induced by Lipopolysaccharide and Interferon-γ*

    OpenAIRE

    Goto, Yoshikuni; Ogawa, Kenji; Hattori, Akira; Tsujimoto, Masafumi

    2011-01-01

    Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme with an important role in processing antigenic peptides presented to class I major histocompatibility complex in the endoplasmic reticulum. In this study, we found that endoplasmic reticulum-retained ERAP1 was secreted from macrophages in response to activation by treatment with lipopolysaccharide (LPS) and interferon (IFN)-γ and enhanced their phagocytic activity. Enhancement of the phagocytic activity of murine macro...

  20. Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming

    OpenAIRE

    Kochan, Grazyna; Krojer, Tobias; Harvey, David; Fischer, Roman; Chen, Liye; Vollmar, Melanie; von Delft, Frank; Kavanagh, Kathryn L.; Brown, Matthew A.; Bowness, Paul; Wordsworth, Paul; Kessler, Benedikt M.; Oppermann, Udo

    2011-01-01

    Endoplasmatic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in trimming of peptides to an optimal length for presentation by major histocompatibility complex (MHC) class I molecules. Polymorphisms in ERAP1 have been associated with chronic inflammatory diseases, including ankylosing spondylitis (AS) and psoriasis, and subsequent in vitro enzyme studies suggest distinct catalytic properties of ERAP1 variants. To understand structure-activity relationships of this enzy...

  1. Concerted In Vitro Trimming of Viral HLA-B27-Restricted Ligands by Human ERAP1 and ERAP2 Aminopeptidases

    OpenAIRE

    Lorente, Elena; Barriga, Alejandro; Johnstone, Carolina; Mir, Carmen; Jiménez, Mercedes; López, Daniel

    2013-01-01

    In the classical human leukocyte antigen (HLA) class I antigen processing and presentation pathway, the antigenic peptides are generated from viral proteins by multiple proteolytic cleavages of the proteasome (and in some cases other cytosolic proteases) and transported to the endoplasmic reticulum (ER) lumen where they are exposed to aminopeptidase activity. In human cells, two different ER-resident enzymes, ERAP1 and ERAP2, can trim the N-terminally extended residues of peptide precursors. ...

  2. Molecular cloning and heterologous expression in Pichia pastoris of X-prolyl-dipeptidyl aminopeptidase from basidiomycete Ustilago maydis.

    Science.gov (United States)

    Juárez-Montiel, Margarita; Ibarra, J Antonio; Chávez-Camarillo, Griselda; Hernández-Rodríguez, César; Villa-Tanaca, Lourdes

    2014-03-01

    Dipeptidyl aminopeptidases are enzymes involved in the posttranslational control of bioactive peptides. Here we identified the gene dapUm in Ustilago maydis by homology with other fungal dipeptidyl aminopeptidases. Analysis of the dapUm-deduced amino acid sequence indicated that it encodes for membrane-type serine protease with a characteristic prolyl oligopeptidase catalytic motif triad: Ser, Asp, His. In order to overexpress the DapUm, the gene encoding for it was cloned and transformed into Pichia. Using this system, we observed a ∼ 125-kDa recombinant protein with an optimal enzymatic activity at pH 6.0 and at 40 °C for the Ala-Pro-p-nitroanilide substrate and an experimental pH of 6.9. U. maydis DapUm was specifically inhibited by phenylmethylsulfonyl fluoride and Pefabloc, confirming the presence of a serine residue in the active site. To our knowledge, this study is the first report on the cloning and expression of a DPP IV dipeptidyl aminopeptidase from a basidiomycete organism. Moreover, the use of recombinant DapUm will allow us to further study and characterize this enzyme, in addition to testing chemical compounds for pharmaceutical purposes.

  3. Plasma renin-angiotensin system-regulating aminopeptidase activities are modified in early stage Alzheimer's disease and show gender differences but are not related to apolipoprotein E genotype.

    Science.gov (United States)

    Puertas, María Del Carmen; Martínez-Martos, José Manuel; Cobo, Manuela; Lorite, Pedro; Sandalio, Rosa María; Palomeque, Teresa; Torres, María Isabel; Carrera-González, María Pilar; Mayas, María Dolores; Ramírez-Expósito, María Jesús

    2013-06-01

    Alterations in blood pressure and components of the renin-angiotensin system (RAS) contribute to the development and progression of Alzheimer's disease (AD), resulting in changes that can lead or contribute to cognitive decline. Aspartyl aminopeptidase (ASAP), aminopeptidase A (APA), aminopeptidase N (APN) and aminopeptidase B (APB) catabolise circulating angiotensins, whereas insulin-regulated aminopeptidase (IRAP) has been described as the AT4 receptor. We have found in AD patients a significant decrease of APA activity in men but not in women, and of APN, APB and IRAP in both genders, when compared with control subjects. No changes were found in ASAP activity. Also, APN, APB and IRAP but not APA correlated with the Mini-Mental test, but no relationship with APOE genotype was found. We conclude that several components of the RAS are modified in AD patients, with gender differences. Furthermore, ROC analysis indicates that APN, APB and IRAP activities could be useful non-invasive biomarkers of AD from the earliest stages.

  4. Activity of leucine aminopeptidase of Telchin licus licus: an important insect pest of sugarcane.

    Science.gov (United States)

    Valencia, Jorge W Arboleda; de Sá, Maria Fátima Grossi; Jiménez, Arnubio Valencia

    2014-06-01

    The enzymatic activity of leucine aminopeptidase (EC 3.4.11.1) from the intestinal tract of sugarcane giant borer (Telchin licus licus) was assayed by using a simple and sensitive spectrophotometric assay that uses L-leucyl-2- naphthylamide as substrate. In this assay, L-leucyl-2-naphthylamide is hydrolyzed to produce 2-naphthylamine and Lleucine. The product 2-naphthylamine reacts with Fast Black K and can be monitored using a continuous spectrophotometric measurement at 590 nm. The data on the kinetic parameters indicates that the Km for the L-leucyl-2- naphthylamide at pH 7.0 was found to be lower than those found for other LAP substrates. The Km and Vmax for the LAP were determined to be 84.03 µM and 357.14 enzymatic units mg(-1), respectively. A noticeable difference of LAP activity between the two insect orders tested was observed. This method could be used to screen for natural LAP inhibitors. PMID:24410745

  5. Aminopeptidase activity in rat brain synaptosomes - 2-mercaptoethanol stimulation and Arg-vasopressin degradation

    Energy Technology Data Exchange (ETDEWEB)

    Simmons, W.H.; Orawski, A.T.

    1986-03-05

    Rat brain synaptic plasma membranes contain an amastatin-inhibited aminopeptidase activity which degrades Arg-vaso-pressin (AVP). The pH optimum for AVP cleavage was found to be 6.8, similar to that reported for oxytocin. The ability of other peptides and arylamides such as oxytocin, Tyr-Phe-Met-Arg-Phe-NH/sub 2/ and Arg-Arg-..beta..NA to inhibit cleavage of (/sup 3/H-Tyr/sup 2/)-AVP suggests that the enzyme may not be specific for AVP. The AVP-cleaving activity has been solubilized and partially characterized. Synaptosomes were lysed with hypotonic buffer, washed, and extracted with 1% Nonidet P-40 detergent. The solubilized protein was chromatographed by gel filtration HPLC on Superose 6. A single peak of activity was found with a M.W. = 117,000 which could hydrolyze 1mM Ala-..beta..NA, Arg-..beta..NA, Arg-Arg-..beta..NA, Phe-Met and Phe-Arg as well as slowly cleave AVP with the ultimate release of /sup 3/H-Tyr. 2-Mercaptoethanol (3.9mM) (ME) stimulated activity 3.6 to 6.6-fold for arylamide and dipeptide substrates, but 35-fold for labelled AVP, possibly owing to reduction of the AVP disulfide bond. All activities in the presence of ME were completely inhibited by 0.2mM amastatin.

  6. Recombinant methionine aminopeptidase protein of Babesia microti: immunobiochemical characterization as a vaccine candidate against human babesiosis.

    Science.gov (United States)

    Munkhjargal, Tserendorj; Yokoyama, Naoaki; Igarashi, Ikuo

    2016-09-01

    Human babesiosis is the most important zoonotic protozoan infection in the world. This is the first report of the cloning, expression, purification, and immunobiochemical characterization of a methionine aminopeptidase 1 (MetAP1) protein from Babesia microti (B. microti). The gene encodes a MetAP1 protein of B. microti (BmMetAP1) of approximately 66.8 kDa that includes glutathione S-transferase (GST) tag and shows MetAP activity. BmMetAP1 was detected in a lysate of B. microti and further localized in cytoplasm of the B. microti merozoite. rBmMetAP1 was found to be immunogenic, eliciting a high antibody titer in mice. Moreover, rBmMetAP1 stimulated the production of IFN-γ and IL-12 but not IL-4. Finally, rBmMetAP1 was able to provide considerable protection to mice against a B. microti challenge infection based on a reduction in peak parasitemia levels and earlier clearance of the parasite as compared with control mice. Taken together, these results suggest that rBmMetAP1 confers significant protection against experimental B. microti infection and might be considered a potential vaccine target against human babesiosis. PMID:27306898

  7. Type I methionine aminopeptidase from Saccharomyces cerevisiae is a potential target for antifungal drug screening

    Institute of Scientific and Technical Information of China (English)

    Ling-ling CHEN; Jia LI; Jing-ya LI; Qun-li LUO; Wei-feng MAO; Qiang SHEN; Fa-jun NAN; Qi-zhuang YE

    2004-01-01

    AIM: To screen antifungal drug candidates using in vitro and in vivo assays based on type I methionine aminopeptidase from Saccharomyces cerevisiae (ScMetAP1). METHODS: A colorimetric assay suitable for high throughput screening (HTS) using recombinant ScMetAP1 protein expressed in Escherichia coli was established for antifungal lead discovery. A series of pyridine-2-carboxylic acid derivatives were characterized and a chemical library of 12 800 pure organic compounds was screened with the in vitro ScMetAP1 assay. Active compounds from the in vitro assay were further evaluated by a growth inhibition assay on yeast strain with deletion of ScMetAP1 gene mapl in comparison with the wild-type yeast strain and the yeast strain with deletion of type II enzyme (ScMetAP2)gene map2. RESULTS: Active ScMetAP1 inhibitors were identified from HTS. Some of the pyridine-2-carboxylic acid derivatives (compound 2 and 3) had selective inhibition of the growth of map2 deletion yeast and weak inhibition on wild-type yeast growth, while no inhibition on mapl deletion yeast. CONCLUSION: ScMetAP1 is a novel potential target for developing antifungal drugs. The in vitro and in vivo ScMetAP1 assays can serve as tools in discovering antifungal drug candidates.

  8. In ovo Administration of Ghrelin and Subsequent Intestinal Leucine aminopeptidase (LAP Activity in Broiler Chickens

    Directory of Open Access Journals (Sweden)

    J. Ghiasi Ghaleh-kandi,

    2011-02-01

    Full Text Available Aim of this study was to investigation on effect of in ovo administration of ghrelin on subsequent Leucine Aminopeptidase (LAP activity in broiler chickens. In this experiment 250 fertilized eggs were collected from commercial breeder flock. The eggs were divided into five experimental groups; control T1 (without injection, group T2 (in ovo injected with solution, group T3 (in ovo injected with 50 μg/egg ghrelin, group T4 (in ovo injected with 100 μg/egg ghrelin and group T5 (in ovo injected with 150 μg/egg ghrelin. All of groups were incubated. In ovo injection was done at day 7 of incub ation. in ovo administration of 150 μg/egg ghrelin in embryonic period, could stimulate LAP activity at 21-day- old chicks in 10, 30 and 50% of intestine with 3520.4, 266.9, 4595.6 IU/g protein, also in ovo injected 50 and 150 μg/egg ghrelin could stimulate LAP activity in 1, 50 and 70% of intestine with 3071.4, 4779.3 and 5013.4 IU/g. In 42-day-old chicks, in ovo injected 50 μg/egg ghrelin could stimulate LAP activity in 1, 10, 30, 40, 70, and 90% percent of intestine. These findings demonstrated stimulatory effects of ghrelin in low doses (50 μg in chicken intestine LAP activity.

  9. Structure of a microsporidian methionine aminopeptidase type 2 complexed with fumagillin and TNP-470

    Energy Technology Data Exchange (ETDEWEB)

    Alvarado, J.; Nemkal, A; Sauder, J; Russell, M; Akiyoshi, D; Shi, W; Almo, S; Weiss, L

    2009-01-01

    Microsporidia are protists that have been reported to cause infections in both vertebrates and invertebrates. They have emerged as human pathogens particularly in patients that are immunosuppressed and cases of gastrointestinal infection, encephalitis, keratitis, sinusitis, myositis and disseminated infection are well described in the literature. While benzimidazoles are active against many species of microsporidia, these drugs do not have significant activity against Enterocytozoon bieneusi. Fumagillin and its analogues have been demonstrated to have activity in vitro and in animal models of microsporidiosis and human infections due to E. bieneusi. Fumagillin and its analogues inhibit methionine aminopeptidase type 2. Encephalitozoon cuniculi MetAP2 (EcMetAP2) was cloned and expressed as an active enzyme using a baculovirus system. The crystal structure of EcMetAP2 was determined with and without the bound inhibitors fumagillin and TNP-470. This structure classifies EcMetAP2 as a member of the MetAP2c family. The EcMetAP2 structure was used to generate a homology model of the E. bieneusi MetAP2. Comparison of microsporidian MetAP2 structures with human MetAP2 provides insights into the design of inhibitors that might exhibit specificity for microsporidian MetAP2.

  10. Biochemical Properties and Potential Applications of Recombinant Leucine Aminopeptidase from Bacillus kaustophilus CCRC 11223

    Directory of Open Access Journals (Sweden)

    Yonghua Wang

    2011-11-01

    Full Text Available Experiments were carried out to investigate the effects of various factors on the activity and conformation of recombinant leucine aminopeptidase of Bacillus kaustophilus CCRC 11223 (BkLAP and potential utilization of BkLAP in the hydrolysis of anchovy protein. Optimal temperature and pH of BkLAP were 70 °C and 8.0 in potassium-phosphate buffer, respectively, and the activity was strongly stimulated by Ni2+, followed by Mn2+ and Co2+. Conformational studies via circular dichroism spectroscopy indicated that various factors could influence the secondary structure of BkLAP to different extents and further induce the changes in enzymatic activity. The secondary structure of BkLAP was slightly modified by Ni2+ at the concentration of 1×10−4 M, however, significant changes on the secondary structures of the enzyme were observed when Hg2+ was added to the concentration of 1×10−4 M. The potential application of BkLAP was evaluated through combination with the commercial or endogenous enzyme to hydrolysis the anchovy protein. Results showed that combining the BkLAP with other enzymes could significantly increase the degree of hydrolysis and amino acid component of hydrolysate. In this regard, BkLAP is a potential enzyme that can be used in the protein hydrolysate industry.

  11. TLR-mediated secretion of endoplasmic reticulum aminopeptidase 1 from macrophages.

    Science.gov (United States)

    Goto, Yoshikuni; Ogawa, Kenji; Nakamura, Takahiro J; Hattori, Akira; Tsujimoto, Masafumi

    2014-05-01

    Macrophages play an important role in host defense under several immunological, inflammatory, and/or infectious conditions. In our previous work, we demonstrated that endoplasmic reticulum aminopeptidase 1 (ERAP1) was secreted from macrophages in response to LPS and IFN-γ, and it enhanced their phagocytic activity. In this study, we analyzed the mechanism of LPS/IFN-γ-induced ERAP1 secretion. LPS/IFN-γ-induced secretion of the enzyme from the murine macrophage cell line RAW264.7 was suppressed by polymyxin B. Several agonists of TLRs, such as Pam3CSK4, FSL-1, and ODN1826, induced its secretion. In contrast, neutralizing Abs to IFN-β and TNF-α receptor type 1 suppressed its secretion. Using murine peritoneal macrophages derived from TNF-α and type 1 IFNR knockout mice, we confirmed the involvement of these two cytokines in ERAP1 secretion. In addition, secretion of ERAP1 from both RAW264.7 cells and murine peritoneal macrophages was induced by A23187 and thapsigargin and inhibited by BAPTA-AM and the calmodulin inhibitor W7. These results suggest that LPS/IFN-γ-induced secretion of ERAP1 is mediated by TLRs via induction of intermediate cytokines such as IFN-β and TNF-α, which in turn lead to enhanced cytosolic Ca(2+) levels and calmodulin activation. PMID:24688025

  12. Structural Basis For Antigenic Peptide Precursor Processing by the Endoplasmic Reticulum Aminopeptidase ERAP1

    Energy Technology Data Exchange (ETDEWEB)

    T Nguyen; S Chang; I Evnouchidou; I York; C Zikos; K Rock; A Goldberg; E Stratikos; L Stern

    2011-12-31

    ERAP1 trims antigen precursors to fit into MHC class I proteins. To fulfill this function, ERAP1 has unique substrate preferences, trimming long peptides but sparing shorter ones. To identify the structural basis for ERAP1's unusual properties, we determined the X-ray crystal structure of human ERAP1 bound to bestatin. The structure reveals an open conformation with a large interior compartment. An extended groove originating from the enzyme's catalytic center can accommodate long peptides and has features that explain ERAP1's broad specificity for antigenic peptide precursors. Structural and biochemical analyses suggest a mechanism for ERAP1's length-dependent trimming activity, whereby binding of long rather than short substrates induces a conformational change with reorientation of a key catalytic residue toward the active site. ERAP1's unique structural elements suggest how a generic aminopeptidase structure has been adapted for the specialized function of trimming antigenic precursors.

  13. A role for naturally occurring alleles of Endoplasmic Reticulum Aminopeptidases in tumor immunity and cancer predisposition

    Directory of Open Access Journals (Sweden)

    Efstratios eStratikos

    2014-12-01

    Full Text Available Endoplasmic Reticulum aminopeptidase 1 and 2 (ERAP1 and ERAP2 are key components on the pathway that generates antigenic epitopes for presentation to cytotoxic T-lymphocytes (CTLs. Coding single nucleotide polymorphisms (SNPs in these enzymes have been associated with predisposition to several major human diseases including inflammatory diseases with autoimmune aetiology, viral infections and virally induced cancer. The function of these enzymes has been demonstrated to affect cytotoxic T-lymphocyte (CTL and natural killer (NK cell responses towards healthy and malignant cells as well as the production of inflammatory cytokines. Recent studies have demonstrated that SNPs in ERAP1 and ERAP2 can affect their ability to generate or destroy antigenic epitopes and define the immunopeptidome. In this review we examine the potential role of these enzymes and their polymorphic states on the generation of cytotoxic responses towards malignantly transformed cells. Given the current state-of-the-art, it is possible that polymorphic variation in these enzymes may contribute to the individual’s predisposition to cancer through altered generation or destruction of tumor antigens that can facilitate tumor immune evasion.

  14. Specific myeloprotection via multidrug resistance 1 gene controlled by aminopeptidase N myeloid promoter

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    In the treatment of tumor patients introduction of multidrug resistance genes into hematopoietic cells has been reported as an approach for reducing myelotoxicity created by antitumor drugs. However, the nonspecific expression of the genes can also increase the chemoresistance of the tumor cells invaded into bone marrow, which influences seriously the effectiveness of chemotherapy. In this study, a new strategy is described for specific myeloprotection. The recombinant retroviral vector containing multidrug resistance 1 (MDR1) gene regulated by aminopeptidase N (APN) myeloid promoter was constructed and then introduced into myeloblastic cells KG1a and tumor cell line BEL7402. The specific transcript of MDR1 was detected in KG1a cells transduced with MDR1 gene and rhodamine 123 was effectively extruded by Pgp, the protein of MDR1 gene. The resistance elevated markedly by 10.6, 10.4, 11.2, 4.2 and 14.2 folds in MDR1 gene-transduced KG1a cells to chemotherapeutic drugs such as cochicine, VP-16, vincristine, doxorubi- cin and paclitaxel, respectively. In contrast, the chemoresistance had no significant changes in BEL7402 cells transduced with MDR1 gene. Expression of MDR1 directed by APN myeloid promoter resulted in myelospecific protection during the killing of tumor cells treated with antitumor drugs. The study would provide a new mean for circumventing myelosuppression of tumor patients undergoing chemotherapy.

  15. Inhibition of the methionine aminopeptidase 2 enzyme for the treatment of obesity

    Directory of Open Access Journals (Sweden)

    Joharapurkar AA

    2014-02-01

    Full Text Available Amit A Joharapurkar, Nirav A Dhanesha, Mukul R Jain Department of Pharmacology and Toxicology, Zydus Research Centre, Cadila Healthcare Limited, Ahmedabad, India Abstract: Worldwide prevalence of obesity has nearly doubled since 1980. Obesity is the result of interactions among the environmental factors, genetic predisposition, and human behavior. Even modest weight reduction in obese patients provides beneficial health outcomes. For effective weight reduction, a drug should either increase energy expenditure or decrease energy intake without causing serious adverse effects. To overcome lack of efficacy and central nervous system related side effects, exploitation of the peripheral mechanism of anti-obesity action is needed. Inhibition of pathological angiogenesis in adipose tissue is one such peripheral mechanism that has attracted the attention of researchers in this area. Although originally developed as anti-cancer agents, methionine aminopeptidase (MetAP2 inhibitors induce significant and sustained weight reduction. Here, we review preclinical and clinical pharmacology of MetAP2 inhibitors. Beloranib is a prototype MetAP2 inhibitor, and currently in advanced clinical trials for the treatment of obesity. Clinical data of beloranib indicate that MetAP2 inhibitors could be a future treatment option for weight reduction without serious adverse effects. Further clinical data from Phase III trials will add to our growing knowledge of MetAP2 inhibitor potential for anti-obesity therapy. Keywords: angiogenesis, beloranib, body weight, MetAP2, anti-obesity

  16. Tumor suppression by a rationally designed reversible inhibitor of methionine aminopeptidase-2.

    Science.gov (United States)

    Wang, Jieyi; Sheppard, George S; Lou, Pingping; Kawai, Megumi; BaMaung, Nwe; Erickson, Scott A; Tucker-Garcia, Lora; Park, Chang; Bouska, Jennifer; Wang, Yi-Chun; Frost, David; Tapang, Paul; Albert, Daniel H; Morgan, Sherry J; Morowitz, Michael; Shusterman, Suzanne; Maris, John M; Lesniewski, Rick; Henkin, Jack

    2003-11-15

    Methionine aminopeptidase (MetAP)-2 has been suggested as a novel target for cancer therapy because the anticancer agent TNP-470 irreversibly inactivates the catalytic activity of this enzyme. However, the importance of MetAP2 in cell growth and tumor progression was uncertain because previous data were based on the chemically reactive TNP-470. Here we show that a rationally designed reversible MetAP2 inhibitor, A-357300, suppresses tumor growth preclinically without the toxicities observed with TNP-470. We have synthesized this bestatin-type MetAP2 inhibitor with the aid of crystal structures of the enzyme-inhibitor complexes and parallel synthesis. A-357300 induces cytostasis by cell cycle arrest at the G(1) phase selectively in endothelial cells and in a subset of tumor cells, but not in most primary cells of nonendothelial type. A-357300 inhibits angiogenesis both in vitro and in vivo and shows potent antitumor efficacy in carcinoma, sarcoma, and neuroblastoma murine models. These data affirm that MetAP2 plays a pivotal role in cell growth and establish that reversible MetAP2 inhibitors are promising novel cancer therapeutic agents.

  17. LJNK, an indoline-2,3-dione-based aminopeptidase N inhibitor with promising antitumor potency.

    Science.gov (United States)

    Hou, Jinning; Jin, Kang; Li, Jin; Jiang, Yuqi; Li, Xiaoyang; Wang, Xuejian; Huang, Yongxue; Zhang, Yingjie; Xu, Wenfang

    2016-07-01

    In our previous study, we found that LJNK showed potent aminopeptidase N (APN)-inhibitory activity. In the current study, we further evaluated the antitumor effects of LJNK both in vitro and in vivo. Enzyme experiments showed that LJNK showed better inhibitory activity than bestatin against APN both from human carcinoma cells' surface and from porcine kidney microsomes. In addition, LJNK could suppress rat aortic ring microvessel growth and HUVEC tubular structure formation, which showed its stronger antiangiogenesis effects than bestatin. [(3-[4,5-Dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide)] assay and clonogenic assay showed that LJNK suppressed cancer cell growth both in the short and the long term. Mice bearing H22 transplantation tumor proved its antitumor effects in vivo. Annexin V-fluorescein isothiocyanate/propidium iodide assay showed that LJNK could induce 28.1% PLC/PRF/5 cell apoptosis and the apoptotic pathway was probably identified by western blot. The above-mentioned results suggested that LJNK inhibited cell proliferation and angiogenesis, and induced apoptosis by decreasing APN activity. PMID:26872309

  18. Role of Endoplasmic Reticulum Aminopeptidases in Health and Disease: from Infection to Cancer

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    Doriana Fruci

    2012-07-01

    Full Text Available Endoplasmic reticulum (ER aminopeptidases ERAP1 and ERAP2 (ERAPs are essential for the maturation of a wide spectrum of proteins involved in various biological processes. In the ER, these enzymes work in concert to trim peptides for presentation on MHC class I molecules. Loss of ERAPs function substantially alters the repertoire of peptides presented by MHC class I molecules, critically affecting recognition of both NK and CD8+ T cells. In addition, these enzymes are involved in the modulation of inflammatory responses by promoting the shedding of several cytokine receptors, and in the regulation of both blood pressure and angiogenesis. Recent genome-wide association studies have identified common variants of ERAP1 and ERAP2 linked to several human diseases, ranging from viral infections to autoimmunity and cancer. More recently, inhibition of ER peptide trimming has been shown to play a key role in stimulating innate and adaptive anti-tumor immune responses, suggesting that inhibition of ERAPs might be exploited for the establishment of innovative therapeutic approaches against cancer. This review summarizes data currently available for ERAP enzymes in ER peptide trimming and in other immunological and non-immunological functions, paying attention to the emerging role played by these enzymes in human diseases.

  19. Aminopeptidase N (CD13 Is Involved in Phagocytic Processes in Human Dendritic Cells and Macrophages

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    Mónica I. Villaseñor-Cardoso

    2013-01-01

    Full Text Available Aminopeptidase N (APN or CD13 is a membrane ectopeptidase expressed by many cell types, including myelomonocytic lineage cells: monocytes, macrophages, and dendritic cells. CD13 is known to regulate the biological activity of various peptides by proteolysis, and it has been proposed that CD13 also participates in several functions such as angiogenesis, cell adhesion, metastasis, and tumor invasion. We had previously reported that, in human monocytes and macrophages, CD13 modulates the phagocytosis mediated by receptors for the Fc portion of IgG antibodies (FcγRs. In this work, we analyzed the possible interaction of CD13 with other phagocytic receptors. We found out that the cross-linking of CD13 positively modulates the phagocytosis mediated by receptors of the innate immune system, since a significant increase in the phagocytosis of zymosan particles or heat-killed E. coli was observed when CD13 was cross-linked using anti-CD13 antibodies, in both macrophages and dendritic cells. Also, we observed that, during the phagocytosis of zymosan, CD13 redistributes and is internalized into the phagosome. These findings suggest that, besides its known functions, CD13 participates in phagocytic processes in dendritic cells and macrophages.

  20. Novel adipocyte aminopeptidases are selectively upregulated by insulin in healthy and obese rats.

    Science.gov (United States)

    Alponti, Rafaela Fadoni; Alves, Patricia Lucio; Silveira, Paulo Flavio

    2016-02-01

    The lack of a complete assembly of the sensitivity of subcellular aminopeptidase (AP) activities to insulin in different pathophysiological conditions has hampered the complete view of the adipocyte metabolic pathways and its implications in these conditions. Here we investigated the influence of insulin on basic AP (APB), neutral puromycin-sensitive AP (PSA), and neutral puromycin-insensitive AP (APM) in high and low density microsomal and plasma membrane fractions from adipocytes of healthy and obese rats. Catalytic activities of these enzymes were fluorometrically monitoring in these fractions with or without insulin stimulus. Canonical traffic such as insulin-regulated AP was not detected for these novel adipocyte APs in healthy and obese rats. However, insulin increased APM in low density microsomal and plasma membrane fractions from healthy rats, APB in high density microsomal fraction from obese rats and PSA in plasma membrane fraction from healthy rats. A new concept of intracellular compartment-dependent upregulation of AP enzyme activities by insulin emerges from these data. This relatively selective regulation has pathophysiological significance, since these enzymes are well known to act as catalysts and receptor of peptides directly related to energy metabolism. Overall, the regulation of each one of these enzyme activities reflects certain dysfunction in obese individuals.

  1. Expression of Caenorhabditis elegans-expressed Trans-HPS, partial aminopeptidase H11 from Haemonchus contortus.

    Science.gov (United States)

    Zhou, Qian-Jin; Yang, Yi; Guo, Xiao-Lu; Duan, Li-Jun; Chen, Xue-Qiu; Yan, Bao-Long; Zhang, Hong-Li; Du, Ai-Fang

    2014-10-01

    Aminopeptidase H11 present in the surface of intestine microvilli in Haemonchus contortus was identified as the most effective antigen candidate. However, its recombinant forms produced in Escherichiacoli, insect cells and yeast could not provide promising protection against H. contortus challenge, probably due to the inappropriate glycosylation and/or conformational folding. Herein, partial H11 containing the potential zinc-binding domain and two predicted glycosylation sites (nt 1 bp-1710 bp, Trans-HPS) was subcloned downstream of 5' flanking region of Caenorhabditis elegans cpr-1 gene in pPD95.77 vector, with the deletion of GFP gene. The recombinant was expressed in C. elegans and verified by blotting with anti-H11 and anti-Trans-HPS rabbit polyclonal antibodies and anti-His monoclonal antibody. Stably inherited Trans-HPS in worm descendants was achieved by integration using UV irradiation. Immunization with the crude Trans-HPS extracted from transgenic worms resulted in 37.71% reduction in faecal egg counts (FEC) (Pgoats. These results suggested an apparent delay against H. contortus egg-laying in goats, which differed from that with bacteria-origin form of partial H11 (nt 670 bp-1710 bp, HPS) (26.04% reduction in FEC and 18.46% reduction in worm burden). These findings indicate the feasibility of sufficient C. elegans-expressed H11 for the immunological research and vaccine development. PMID:25128369

  2. Hormonal status modifies renin-angiotensin system-regulating aminopeptidases and vasopressin-degrading activity in the hypothalamus-pituitary-adrenal axis of female mice.

    Science.gov (United States)

    García, María Jesús; Martínez-Martos, José Manuel; Mayas, María Dolores; Carrera, María Pilar; De la Chica, Susana; Cortés, Pedro; Ramírez-Expósito, María Jesús

    2008-07-01

    The hypothalamus-pituitary-adrenal axis (HPA) participates in the maintenance of cardiovascular functions and in the control of blood pressure. By other hand, it is known that blood pressure regulation and HPA activity are affected by sex hormones. The aim of the present work is to analyze the influence of estradiol and progesterone on renin-angiotensin system (RAS)-regulating aminopeptidase A, aminopeptidase B and aminopeptidase N activities and vasopressin-degrading activity in the HPA axis of ovariectomized mice and ovariectomized mice treated subscutaneously with different doses of estradiol and progesterone. Our data suggest that in female mice, estradiol and progesterone influence RAS-regulating and vasopressin-degrading activities at different levels of the HPA axis.

  3. Silencing or inhibition of endoplasmic reticulum aminopeptidase 1 (ERAP1) suppresses free heavy chain expression and Th17 responses in ankylosing spondylitis

    OpenAIRE

    Chen, Liye; Ridley, Anna; Hammitzsch, Ariane; Al-Mossawi, Mohammad Hussein; Bunting, Helen; Georgiadis, Dimitris; Chan, Antoni; Kollnberger, Simon; Bowness, Paul

    2015-01-01

    Objective Human leucocyte antigen (HLA)-B27 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly associated with ankylosing spondylitis (AS). ERAP1 is a key aminopeptidase in HLA class I presentation and can potentially alter surface expression of HLA-B27 free heavy chains (FHCs). We studied the effects of ERAP1 silencing/inhibition/variations on HLA-B27 FHC expression and Th17 responses in AS. Methods Flow cytometry was used to measure surface expression of HLA class I in peripher...

  4. The ER aminopeptidase, ERAP1, trims precursors to lengths of MHC class I peptides by a “molecular ruler” mechanism

    OpenAIRE

    Chang, Shih-Chung; Momburg, Frank; Bhutani, Nidhi; Goldberg, Alfred L.

    2005-01-01

    Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an IFN-γ-induced aminopeptidase in the endoplasmic reticulum that trims longer precursors to the antigenic peptides presented on MHC class I molecules. We recently reported that purified ERAP1 trimmed N-extended precursors but spared peptides of 8-9 residues, the length required for binding to MHC class I molecules. Here, we show another remarkable property of ERAP1: that it strongly prefers substrates 9-16 residues long, the lengths of peptid...

  5. Rationally designed inhibitor targeting antigen-trimming aminopeptidases enhances antigen presentation and cytotoxic T-cell responses.

    Science.gov (United States)

    Zervoudi, Efthalia; Saridakis, Emmanuel; Birtley, James R; Seregin, Sergey S; Reeves, Emma; Kokkala, Paraskevi; Aldhamen, Yasser A; Amalfitano, Andrea; Mavridis, Irene M; James, Edward; Georgiadis, Dimitris; Stratikos, Efstratios

    2013-12-01

    Intracellular aminopeptidases endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2), and as well as insulin-regulated aminopeptidase (IRAP) process antigenic epitope precursors for loading onto MHC class I molecules and regulate the adaptive immune response. Their activity greatly affects the antigenic peptide repertoire presented to cytotoxic T lymphocytes and as a result can regulate cytotoxic cellular responses contributing to autoimmunity or immune evasion by viruses and cancer cells. Therefore, pharmacological regulation of their activity is a promising avenue for modulating the adaptive immune response with possible applications in controlling autoimmunity, in boosting immune responses to pathogens, and in cancer immunotherapy. In this study we exploited recent structural and biochemical analysis of ERAP1 and ERAP2 to design and develop phosphinic pseudopeptide transition state analogs that can inhibit this family of enzymes with nM affinity. X-ray crystallographic analysis of one such inhibitor in complex with ERAP2 validated our design, revealing a canonical mode of binding in the active site of the enzyme, and highlighted the importance of the S2' pocket for achieving inhibitor potency. Antigen processing and presentation assays in HeLa and murine colon carcinoma (CT26) cells showed that these inhibitors induce increased cell-surface antigen presentation of transfected and endogenous antigens and enhance cytotoxic T-cell responses, indicating that these enzymes primarily destroy epitopes in those systems. This class of inhibitors constitutes a promising tool for controlling the cellular adaptive immune response in humans by modulating the antigen processing and presentation pathway. PMID:24248368

  6. Aminopeptidase B, a glucagon-processing enzyme: site directed mutagenesis of the Zn2+-binding motif and molecular modelling

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    Etchebest Catherine

    2007-10-01

    Full Text Available Abstract Background Aminopeptidase B (Ap-B; EC 3.4.11.6 catalyzes the cleavage of basic residues at the N-terminus of peptides and processes glucagon into miniglucagon. The enzyme exhibits, in vitro, a residual ability to hydrolyze leukotriene A4 into the pro-inflammatory lipid mediator leukotriene B4. The potential bi-functional nature of Ap-B is supported by close structural relationships with LTA4 hydrolase (LTA4H ; EC 3.3.2.6. A structure-function analysis is necessary for the detailed understanding of the enzymatic mechanisms of Ap-B and to design inhibitors, which could be used to determine the complete in vivo functions of the enzyme. Results The rat Ap-B cDNA was expressed in E. coli and the purified recombinant enzyme was characterized. 18 mutants of the H325EXXHX18E348 Zn2+-binding motif were constructed and expressed. All mutations were found to abolish the aminopeptidase activity. A multiple alignment of 500 sequences of the M1 family of aminopeptidases was performed to identify 3 sub-families of exopeptidases and to build a structural model of Ap-B using the x-ray structure of LTA4H as a template. Although the 3D structures of the two enzymes resemble each other, they differ in certain details. The role that a loop, delimiting the active center of Ap-B, plays in discriminating basic substrates, as well as the function of consensus motifs, such as RNP1 and Armadillo domain are discussed. Examination of electrostatic potentials and hydrophobic patches revealed important differences between Ap-B and LTA4H and suggests that Ap-B is involved in protein-protein interactions. Conclusion Alignment of the primary structures of the M1 family members clearly demonstrates the existence of different sub-families and highlights crucial residues in the enzymatic activity of the whole family. E. coli recombinant enzyme and Ap-B structural model constitute powerful tools for investigating the importance and possible roles of these conserved residues

  7. Aminopeptidase B, a glucagon-processing enzyme: site directed mutagenesis of the Zn2+-binding motif and molecular modelling

    Science.gov (United States)

    Pham, Viet-Laï; Cadel, Marie-Sandrine; Gouzy-Darmon, Cécile; Hanquez, Chantal; Beinfeld, Margery C; Nicolas, Pierre; Etchebest, Catherine; Foulon, Thierry

    2007-01-01

    Background Aminopeptidase B (Ap-B; EC 3.4.11.6) catalyzes the cleavage of basic residues at the N-terminus of peptides and processes glucagon into miniglucagon. The enzyme exhibits, in vitro, a residual ability to hydrolyze leukotriene A4 into the pro-inflammatory lipid mediator leukotriene B4. The potential bi-functional nature of Ap-B is supported by close structural relationships with LTA4 hydrolase (LTA4H ; EC 3.3.2.6). A structure-function analysis is necessary for the detailed understanding of the enzymatic mechanisms of Ap-B and to design inhibitors, which could be used to determine the complete in vivo functions of the enzyme. Results The rat Ap-B cDNA was expressed in E. coli and the purified recombinant enzyme was characterized. 18 mutants of the H325EXXHX18E348 Zn2+-binding motif were constructed and expressed. All mutations were found to abolish the aminopeptidase activity. A multiple alignment of 500 sequences of the M1 family of aminopeptidases was performed to identify 3 sub-families of exopeptidases and to build a structural model of Ap-B using the x-ray structure of LTA4H as a template. Although the 3D structures of the two enzymes resemble each other, they differ in certain details. The role that a loop, delimiting the active center of Ap-B, plays in discriminating basic substrates, as well as the function of consensus motifs, such as RNP1 and Armadillo domain are discussed. Examination of electrostatic potentials and hydrophobic patches revealed important differences between Ap-B and LTA4H and suggests that Ap-B is involved in protein-protein interactions. Conclusion Alignment of the primary structures of the M1 family members clearly demonstrates the existence of different sub-families and highlights crucial residues in the enzymatic activity of the whole family. E. coli recombinant enzyme and Ap-B structural model constitute powerful tools for investigating the importance and possible roles of these conserved residues in Ap-B, LTA4H and M1

  8. Changes in vasopressin-converting aminopeptidase activity in the rat pineal gland during summer : Relationship to vasopressin contents

    OpenAIRE

    Liu, B; Burbach, J. P. H.

    1988-01-01

    Vasopressin (VP)-converting aminopeptidase (VP-AP) activity and VP contents were measured in single rat pineal glands during the summer of two successive years. The peptidase activity decreased significantly in August. The lowest activity (±SEM) of 0.18±0.02 pmol·hour−1 was recorded on August 14, compared to the basal activity of 0.25±0.01 pmol·hour−1 in July and September of 1986. The change with similar percentage occurred in the same period of 1987. The specific activity of the enzyme in t...

  9. [Activity of alanine aminopeptidase in blood and in urine of smoking and non-smoking smelters].

    Science.gov (United States)

    Bizoń, Anna; Stasiak, Karolina; Milnerowicz, Halina

    2010-01-01

    The human body is constantly exposed to xenobiotics. This will include exogenous substances from environmental pollution such as heavy metals and lifestyle such as smoking, which may lead to impaired functioning of many organs. The liver and kidney are the critical organs in the case of a long-term occupational or environmental exposure to heavy metals and tobacco smoke. In diagnostics of liver and kidney damage useful are the methods which determine the activity of enzymes such as alanine aminopeptidase (AAP). AAP is a marker for early detection of acute kidney damage, and presence of AAP derive mainly from proximal tubular brush-border. Activity of AAP in urine allows to assess the damage resulting from the nephrotoxic exposure to heavy metals. In the serum AAP is mainly from hepatic. Activity of AAP may be useful to identify liver cancer. The investigation was shown, that AAP activity in the blood is used to detect hepatic cholestasis and congestive jaundice. The aim of present study was to assess the influence of occupational exposure of copper-foundry workers to heavy metals (arsenic, cadmium, lead) on activity of alanine aminopeptidase in blood and urine. The investigations were performed in blood and urine of 166 subjects: 101 male copper smelters and 65 non-exposed male subjects. The study protocol was approved by Local Bioethics Committee of Wroclaw Medical University (KB No: 469/2008). The data on smoking which had been obtained from a direct personal interview were verified by determination of serum cotinine concentrations. Biological material collected from the control group and smelters was divided into subgroups of nonsmokers and smokers. The concentrations of lead and cadmium were determined in whole blood, whilst the level of arsenic and cadmium were determined in urine using FAAS method (Flame Atomic Absorption Spectrometry) in the acetylate flame on the SOLAAR M6. The activity of AA was determined in blood and in urine. The results showed a 9-fold

  10. Structural bases of coronavirus attachment to host aminopeptidase N and its inhibition by neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Juan Reguera

    Full Text Available The coronaviruses (CoVs are enveloped viruses of animals and humans associated mostly with enteric and respiratory diseases, such as the severe acute respiratory syndrome and 10-20% of all common colds. A subset of CoVs uses the cell surface aminopeptidase N (APN, a membrane-bound metalloprotease, as a cell entry receptor. In these viruses, the envelope spike glycoprotein (S mediates the attachment of the virus particles to APN and subsequent cell entry, which can be blocked by neutralizing antibodies. Here we describe the crystal structures of the receptor-binding domains (RBDs of two closely related CoV strains, transmissible gastroenteritis virus (TGEV and porcine respiratory CoV (PRCV, in complex with their receptor, porcine APN (pAPN, or with a neutralizing antibody. The data provide detailed information on the architecture of the dimeric pAPN ectodomain and its interaction with the CoV S. We show that a protruding receptor-binding edge in the S determines virus-binding specificity for recessed glycan-containing surfaces in the membrane-distal region of the pAPN ectodomain. Comparison of the RBDs of TGEV and PRCV to those of other related CoVs, suggests that the conformation of the S receptor-binding region determines cell entry receptor specificity. Moreover, the receptor-binding edge is a major antigenic determinant in the TGEV envelope S that is targeted by neutralizing antibodies. Our results provide a compelling view on CoV cell entry and immune neutralization, and may aid the design of antivirals or CoV vaccines. APN is also considered a target for cancer therapy and its structure, reported here, could facilitate the development of anti-cancer drugs.

  11. Effects of polymorphic variation on the mechanism of Endoplasmic Reticulum Aminopeptidase 1.

    Science.gov (United States)

    Stamogiannos, Athanasios; Koumantou, Despoina; Papakyriakou, Athanasios; Stratikos, Efstratios

    2015-10-01

    Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) generates antigenic peptides for loading onto Major Histocompatibility Class I molecules (MHCI) and can regulate adaptive immune responses. During the last few years, many genetic studies have revealed strong associations between coding Single Nucleotide Polymorphisms (SNPs) in ERAP1 and common human diseases ranging from viral infections to cancer and autoimmunity. Functional studies have established that these SNPs affect enzyme activity resulting to changes in antigenic peptide processing, presentation by MHCI and cellular cytotoxic responses. These disease-associated polymorphisms are, however, located away from the enzyme's active site and are interspersed to different structural domains. As a result, the mechanism by which these SNPs can affect function remains largely elusive. ERAP1 utilizes a complex catalytic mechanism that involves a large conformational change between inactive and active forms and has the unique property to trim larger peptides more efficiently than smaller ones. We analyzed two of the most consistently discovered disease-associated polymorphisms, namely K528R and Q730E, for their effect on the ability of the enzyme to select substrates based on length and to undergo conformational changes. By utilizing enzymatic and computational analysis we propose that disease-associated SNPs can affect ERAP1 function by influencing: (i) substrate length selection and (ii) the conformational distribution of the protein ensemble. Our results provide novel insight on the mechanisms by which polymorphic variation distal from the active site of ERAP1 can translate to changes in function and contribute to immune system variability in humans. PMID:26224046

  12. Endoplasmic reticulum aminopeptidase-1 functions regulate key aspects of the innate immune response.

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    Yasser A Aldhamen

    Full Text Available Endoplasmic reticulum aminopeptidase-1 (ERAP1 is a multifunctional, ubiquitously expressed enzyme whose peptide-trimming role during antigen processing for presentation by MHC I molecules is well established, however, a role for ERAP1 in modulating global innate immune responses has not been described to date. Here we demonstrate that, relative to wild type mice, mice lacking ERAP1 exhibit exaggerated innate immune responses early during pathogen recognition, as characterized by increased activation of splenic and hepatic NK and NKT cells and enhanced production of pro-inflammatory cytokines such as IL12 and MCP1. Our data also revealed that ERAP1 is playing a critical role in NK cell development and function. We observed higher frequencies of terminally matured NK cells, as well as higher frequencies of licensed NK cells (expressing the Ly49C and Ly49I receptors in ERAP1-KO mice, results that positively correlated with an enhanced NK activation and IFNγ production by ERAP1-KO mice challenged with pro-inflammatory stimuli. Furthermore, during pathogen recognition, ERAP1 regulates IL12 production by CD11c(+ DCs specifically, with increases in IL12 production positively correlated with an increased phagocytic activity of splenic DCs and macrophages. Collectively, our results demonstrate a previously unrecognized, more central role for the ERAP1 protein in modulating several aspects of both the development of the innate immune system, and its responses during the initial stages of pathogen recognition. Such a role may explain why ERAP1 has been implicated by GWAS in the pathogenesis of autoimmune diseases that may be precipitated by aberrant responses to pathogen encounters.

  13. Endoplasmic reticulum aminopeptidase-1 functions regulate key aspects of the innate immune response.

    Science.gov (United States)

    Aldhamen, Yasser A; Seregin, Sergey S; Rastall, David P W; Aylsworth, Charles F; Pepelyayeva, Yuliya; Busuito, Christopher J; Godbehere-Roosa, Sarah; Kim, Sungjin; Amalfitano, Andrea

    2013-01-01

    Endoplasmic reticulum aminopeptidase-1 (ERAP1) is a multifunctional, ubiquitously expressed enzyme whose peptide-trimming role during antigen processing for presentation by MHC I molecules is well established, however, a role for ERAP1 in modulating global innate immune responses has not been described to date. Here we demonstrate that, relative to wild type mice, mice lacking ERAP1 exhibit exaggerated innate immune responses early during pathogen recognition, as characterized by increased activation of splenic and hepatic NK and NKT cells and enhanced production of pro-inflammatory cytokines such as IL12 and MCP1. Our data also revealed that ERAP1 is playing a critical role in NK cell development and function. We observed higher frequencies of terminally matured NK cells, as well as higher frequencies of licensed NK cells (expressing the Ly49C and Ly49I receptors) in ERAP1-KO mice, results that positively correlated with an enhanced NK activation and IFNγ production by ERAP1-KO mice challenged with pro-inflammatory stimuli. Furthermore, during pathogen recognition, ERAP1 regulates IL12 production by CD11c(+) DCs specifically, with increases in IL12 production positively correlated with an increased phagocytic activity of splenic DCs and macrophages. Collectively, our results demonstrate a previously unrecognized, more central role for the ERAP1 protein in modulating several aspects of both the development of the innate immune system, and its responses during the initial stages of pathogen recognition. Such a role may explain why ERAP1 has been implicated by GWAS in the pathogenesis of autoimmune diseases that may be precipitated by aberrant responses to pathogen encounters. PMID:23894499

  14. A Novel Glutamyl (Aspartyl-Specific Aminopeptidase A from Lactobacillus delbrueckii with Promising Properties for Application.

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    Timo Stressler

    Full Text Available Lactic acid bacteria (LAB are auxotrophic for a number of amino acids. Thus, LAB have one of the strongest proteolytic systems to acquit their amino acid requirements. One of the intracellular exopeptidases present in LAB is the glutamyl (aspartyl specific aminopeptidase (PepA; EC 3.4.11.7. Most of the PepA enzymes characterized yet, belonged to Lactococcus lactis sp., but no PepA from a Lactobacillus sp. has been characterized so far. In this study, we cloned a putative pepA gene from Lb. delbrueckii ssp. lactis DSM 20072 and characterized it after purification. For comparison, we also cloned, purified and characterized PepA from Lc. lactis ssp. lactis DSM 20481. Due to the low homology between both enzymes (30%, differences between the biochemical characteristics were very likely. This was confirmed, for example, by the more acidic optimum pH value of 6.0 for Lb-PepA compared to pH 8.0 for Lc-PepA. In addition, although the optimum temperature is quite similar for both enzymes (Lb-PepA: 60°C; Lc-PepA: 65°C, the temperature stability after three days, 20°C below the optimum temperature, was higher for Lb-PepA (60% residual activity than for Lc-PepA (2% residual activity. EDTA inhibited both enzymes and the strongest activation was found for CoCl2, indicating that both enzymes are metallopeptidases. In contrast to Lc-PepA, disulfide bond-reducing agents such as dithiothreitol did not inhibit Lb-PepA. Finally, Lb-PepA was not product-inhibited by L-Glu, whereas Lc-PepA showed an inhibition.

  15. NC2213: a novel methionine aminopeptidase 2 inhibitor in human colon cancer HT29 cells

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    Pati Hari N

    2009-08-01

    Full Text Available Abstract Methionine aminopeptidase 2 (MetAP2 is a bifunctional protein that plays a critical role in the regulation of post-translational processing and protein synthesis. MetAP2 is overexpressed in human colon cancer. In this report we screened various MetAP2 inhibitors and treated HT29 cells with various concentrations of compounds. We evaluated the expression of MetAP2 and pp60c-src expressions in HT29 cells. In addition we also carried out the cell proliferation and cell cycle analysis in the MetAP2 inhibitor-treated HT29 cells. The cell cycle analysis of HT29 treated with 1.0 μM of NC2213 showed an arrest in the G2 phase followed by an induction in the percentage of cells undergoing apoptosis in the sub-G1 phase. Western blot analysis revealed that the MetAP2 expression was dose-dependently decreased when the HT29 cells were treated with the 3,5-bis(benzylidene-4-piperidone derivative (NC2213. In addition, phosphorylation of Src, a myristoylated oncoprotein was significantly decreased by 1.0 μM of NC2213 as revealed by Western blot analysis. Furthermore, NC2213 also inhibits the expression of pp60c-src in HT29 cells. Interestingly, this compound also inhibits the phosphorylation at Tyr416 of pp60c-src while increasing the phosphorylation at Tyr527 of pp60c-src. NC2213 inhibits the growth of HT29 cells by inducing apoptosis and might be useful for the treatment of human colon cancer.

  16. A Novel Glutamyl (Aspartyl)-Specific Aminopeptidase A from Lactobacillus delbrueckii with Promising Properties for Application

    Science.gov (United States)

    Stressler, Timo; Ewert, Jacob; Merz, Michael; Funk, Joshua; Claaßen, Wolfgang; Lutz-Wahl, Sabine; Schmidt, Herbert; Kuhn, Andreas; Fischer, Lutz

    2016-01-01

    Lactic acid bacteria (LAB) are auxotrophic for a number of amino acids. Thus, LAB have one of the strongest proteolytic systems to acquit their amino acid requirements. One of the intracellular exopeptidases present in LAB is the glutamyl (aspartyl) specific aminopeptidase (PepA; EC 3.4.11.7). Most of the PepA enzymes characterized yet, belonged to Lactococcus lactis sp., but no PepA from a Lactobacillus sp. has been characterized so far. In this study, we cloned a putative pepA gene from Lb. delbrueckii ssp. lactis DSM 20072 and characterized it after purification. For comparison, we also cloned, purified and characterized PepA from Lc. lactis ssp. lactis DSM 20481. Due to the low homology between both enzymes (30%), differences between the biochemical characteristics were very likely. This was confirmed, for example, by the more acidic optimum pH value of 6.0 for Lb-PepA compared to pH 8.0 for Lc-PepA. In addition, although the optimum temperature is quite similar for both enzymes (Lb-PepA: 60°C; Lc-PepA: 65°C), the temperature stability after three days, 20°C below the optimum temperature, was higher for Lb-PepA (60% residual activity) than for Lc-PepA (2% residual activity). EDTA inhibited both enzymes and the strongest activation was found for CoCl2, indicating that both enzymes are metallopeptidases. In contrast to Lc-PepA, disulfide bond-reducing agents such as dithiothreitol did not inhibit Lb-PepA. Finally, Lb-PepA was not product-inhibited by L-Glu, whereas Lc-PepA showed an inhibition. PMID:27003449

  17. Lactase-phlorizin hydrolase and aminopeptidase N are differentially regulated in the small intestine of the pig

    DEFF Research Database (Denmark)

    Torp, Niels; Rossi, M; Troelsen, J T;

    1993-01-01

    of lactase-phlorizin hydrolase in the pig during development: (1) a primary regulation at the level of mRNA (predominantly in the ileum); (2) an increased rate of turnover of the enzyme, mainly in the duodenum and proximal jejunum, and most likely due to an increased secretion into the gut lumen......The longitudinal expression of two brush-border enzymes, lactase-phlorizin hydrolase (EC 3.2.1.23/62) and aminopeptidase N (EC 3.4.11.2), was studied in the small intestine of the post-weaned pig. Whereas the level of mRNA, encoding aminopeptidase N (relative to that of beta-actin), only varied...... moderately from the duodenum to the terminal ileum, the amount of lactase-phlorizin hydrolase mRNA exhibited a sharp maximum in the proximal jejunum. For both enzymes, the level of protein synthesis, studied in cultured mucosal explants, correlated well with the level of mRNA, and no major variation in post...

  18. Biochemical and structural analysis of a site directed mutant of manganese dependent aminopeptidase P from Streptomyces lavendulae

    Directory of Open Access Journals (Sweden)

    ARYA NANDAN

    2015-08-01

    Full Text Available Aminopeptidase P (APP removes N-terminal amino acids from peptides and proteins when the penultimate residue is proline. To understand the structure-function relationships of aminopeptidase P of Streptomyces lavendulae, a conserved arginine residue was replaced with lysine (R453K by site-directed mutagenesis. The overexpressed wild and mutant enzymes were of nearly 60 kDa and purified by nickel affinity chromatography. Kinetic analysis of R453K variant using Gly-Pro-pNA as the substrate revealed an increase in Km with a decrease in Vmax, leading to overall decrease in the catalytic efficiency, indicating that the guanidinium group of arginine plays an important role in substrate binding in APP. We constructed three dimensional models for the catalytic domains of wild and mutant enzyme and it revealed an interaction in R453 of native enzyme through hydrogen bonding with the adjacent residues making a substrate binding cavity whereas K453 did not participate in any hydrogen bonding. Hence, R453 in APP of S. lavenduale must be playing a critical role in the hydrolysis of the substrate.

  19. Structure of human aspartyl aminopeptidase complexed with substrate analogue: insight into catalytic mechanism, substrate specificity and M18 peptidase family

    Directory of Open Access Journals (Sweden)

    Chaikuad Apirat

    2012-06-01

    Full Text Available Abstract Backround Aspartyl aminopeptidase (DNPEP, with specificity towards an acidic amino acid at the N-terminus, is the only mammalian member among the poorly understood M18 peptidases. DNPEP has implicated roles in protein and peptide metabolism, as well as the renin-angiotensin system in blood pressure regulation. Despite previous enzyme and substrate characterization, structural details of DNPEP regarding ligand recognition and catalytic mechanism remain to be delineated. Results The crystal structure of human DNPEP complexed with zinc and a substrate analogue aspartate-β-hydroxamate reveals a dodecameric machinery built by domain-swapped dimers, in agreement with electron microscopy data. A structural comparison with bacterial homologues identifies unifying catalytic features among the poorly understood M18 enzymes. The bound ligands in the active site also reveal the coordination mode of the binuclear zinc centre and a substrate specificity pocket for acidic amino acids. Conclusions The DNPEP structure provides a molecular framework to understand its catalysis that is mediated by active site loop swapping, a mechanism likely adopted in other M18 and M42 metallopeptidases that form dodecameric complexes as a self-compartmentalization strategy. Small differences in the substrate binding pocket such as shape and positive charges, the latter conferred by a basic lysine residue, further provide the key to distinguishing substrate preference. Together, the structural knowledge will aid in the development of enzyme-/family-specific aminopeptidase inhibitors.

  20. Reduced activity of CD13/aminopeptidase N (APN) in aggressive meningiomas is associated with increased levels of SPARC.

    Science.gov (United States)

    Mawrin, Christian; Wolke, Carmen; Haase, Daniela; Krüger, Sabine; Firsching, Raimund; Keilhoff, Gerburg; Paulus, Werner; Gutmann, David H; Lal, Anita; Lendeckel, Uwe

    2010-01-01

    Meningiomas are the second most common brain tumors in adults, and meningiomas exhibit a tendency to invade adjacent structures. Compared with high-grade gliomas, little is known about the molecular changes that potentially underlie the invasive behavior of meningiomas. In this study, we examined the expression and function of the membrane alanyl-aminopeptidase [mAAP, aminopeptidase N (APN), CD13, EC3.4.11.2] zinc-dependent ectopeptidase in meningiomas and meningioma cell lines, based on its prior association with tumor invasion in colorectal and renal carcinomas. We found a significant reduction of APNmRNA and protein expression, as well as enzymatic activity, in high-grade meningiomas. While meningioma tumor cell proliferation was not affected by either pharmacologic APN inhibition or siRNA-mediated APN silencing, APN pharmacologic and siRNA knockdown significantly reduced meningioma cell invasion in vitro. Next, we employed pathway-specific cDNA microarray analyses to identify extracellular matrix and adhesion molecules regulated by APN, and found that APN-siRNA knockdown substantially increased the expression of secreted protein, acidic and rich in cysteine (SPARC)/osteonectin. Finally, we demonstrated that SPARC, which has been previously associated with meningioma invasiveness, was increased in aggressive meningiomas. Collectively, these results suggest that APN expression and enzymatic function is reduced in aggressive meningiomas, and that alterations in the balance between APN and SPARC might favor meningioma invasion.

  1. Unlinked genetic loci control the reduced transcription of aminopeptidase N 1 and 3 in the European corn borer and determine tolerance to Bacillus thuringiensis Cry1Ab toxin

    Science.gov (United States)

    Crystalline (Cry) toxins from Bacillus thuringiensis (Bt) control insect feeding damage on crop plants via foliar applications or by expression within transgenic plants, but continued Bt use is threatened by the buildup of insect resistance traits. Aminopeptidase N (apn) gene family members encode m...

  2. Defectively N-glycosylated and non-O-glycosylated aminopeptidase N (CD13) is normally expressed at the cell surface and has full enzymatic activity

    DEFF Research Database (Denmark)

    Norén, K; Hansen, Gert Helge; Clausen, H;

    1997-01-01

    In order to study the effects of the absence of O-glycosylation and modifications of N-glycosylation on a class II membrane protein, pig and human aminopeptidase N (CD13) were stably expressed in the ldl(D) cell line. This cell line carries a UDP-Gal/UDP-GalNAc-epimerase deficiency which blocks...

  3. Molecular characterization and RNA interference of three midgut aminopeptidase N isozymes from bacillus thuringiensis-susceptible and -resistant strains of sugarcane borer diatraea saccharalis

    Science.gov (United States)

    Aminopeptidase N (APN) proteins located at the midgut epithelium of some lepidopterous species have been implicated as receptors for insecticidal proteins from Bacillus thuringiensis. cDNAs of three APN isoforms, DsAPN1, DsAPN2, and DsAPN3, from Cry1Ab-susceptible (Cry1Ab-SS) and -resistant (Cry1Ab-...

  4. Feline and canine coronaviruses are released from the basolateral side of polarized epithelial LLC-PK1 cells expressing the recombinant feline aminopeptidase-N cDNA

    NARCIS (Netherlands)

    Rossen, J W; Kouame, J; Goedheer, A J; Vennema, H; Rottier, P J

    2001-01-01

    In this study feline (FECV and FIPV) and canine (CCoV) coronavirus entry into and release from polarized porcine epithelial LLC-PK1 cells, stably expressing the recombinant feline aminopeptidase-N cDNA, were investigated. Virus entry appeared to occur preferentially through the apical membrane, simi

  5. Dietary fatty acid composition affects aminopeptidase activities in the testes of mice.

    Science.gov (United States)

    Arechaga, Garbiñe; Prieto, Isabel; Segarra, Ana B; Alba, Francisco; Ruiz-Larrea, María B; Ruiz-Sanz, José I; de Gasparo, Marc; Ramirez, Manuel

    2002-04-01

    The autocrine/paracrine control mechanisms of local factors, such as the renin-angiotensin system and the thyrotropin-releasing hormone (TRH), seem to play a relevant role in testicular physiology. It has been proposed that dietary fat composition influences male reproductive function modifying the cholesterol-phospholipid composition of testicular plasma membranes. Modifications in the composition and physical properties of the membranes may lead to alterations in the activities of membrane-bound (M-B) enzymes. We have previously demonstrated that cholesterol and steroid hormones affect aminopeptidase (AP) activities. Dietary fatty acids with different degrees of saturation modified AP activities in the serum of mice and an olive oil supplemented diet influenced the AP activities in the testes of mice. We hypothesized that the modification of dietary fat composition may affect angiotensin- [glutamyl-AP (GluAP), aspartyl-AP (AspAP)] and TRH- [pyroglutamyl-AP (pGluAP)] degrading activities in the testis. In this study, we investigated the effect of diets supplemented with sunflower oil (SFO), fish oil (FO), olive oil (OO), lard (L) or coconut oil (CO) on soluble (Sol) and M-B GluAP, AspAP and pGluAP in mice testis, using arylamides as substrates. Sol GluAP activity did not show differences among groups. However, Sol AspAP and Sol pGluAP progressively decreased with the degree of saturation of the fatty acid used in the diet. In contrast, M-B GluAP progressively increased with the degree of saturation of the fatty acid used in the diet. For M-B AspAP activity, mice fed diets containing FO showed significantly higher levels than those fed diets containing SFO, OO and L but not those containing CO. For M-B pGluAP activity, the highest levels were observed for mice fed diets containing FO and OO. The present data suggest that the type of fat used in the diet may influence the autocrine/paracrine functions of locally synthesized angiotensin peptides and TRH in the testis

  6. A Phase Ib dose-escalation study to evaluate safety and tolerability of the addition of the aminopeptidase inhibitor tosedostat (CHR-2797) to paclitaxel in patients with advanced solid tumours

    NARCIS (Netherlands)

    C.M.L. Herpen, C.M.L. (Carla); F.A.L.M. Eskens (Ferry); M.J.A. de Jonge (Maja); I. Desar; L. Hooftman (Leon); E. Bone (Elisabeth); J.N.H. Timmerbonte (Johanna); J. Verweij (Jaap)

    2010-01-01

    textabstractBackground: This Phase Ib dose-escalating study investigated safety, maximum tolerated dose (MTD), dose-limiting toxicity (DLT), pharmacokinetics (PK) and clinical antitumour activity of tosedostat (CHR-2797), an orally bioavailable aminopeptidase inhibitor, in combination with paclitaxe

  7. A Phase Ib dose-escalation study to evaluate safety and tolerability of the addition of the aminopeptidase inhibitor tosedostat (CHR-2797) to paclitaxel in patients with advanced solid tumours.

    NARCIS (Netherlands)

    Herpen, C.M.L. van; Eskens, F.A.; Jonge, M. de; Desar, I.M.E.; Hooftman, L.; Bone, E.A.; Timmer-Bonte, J.N.H.; Verweij, J.

    2010-01-01

    BACKGROUND: This Phase Ib dose-escalating study investigated safety, maximum tolerated dose (MTD), dose-limiting toxicity (DLT), pharmacokinetics (PK) and clinical antitumour activity of tosedostat (CHR-2797), an orally bioavailable aminopeptidase inhibitor, in combination with paclitaxel. METHODS:

  8. Synthesis, structure-activity relationships and brain uptake of a novel series of benzopyran inhibitors of insulin-regulated aminopeptidase.

    Science.gov (United States)

    Mountford, Simon J; Albiston, Anthony L; Charman, William N; Ng, Leelee; Holien, Jessica K; Parker, Michael W; Nicolazzo, Joseph A; Thompson, Philip E; Chai, Siew Yeen

    2014-02-27

    Peptide inhibitors of insulin-regulated aminopeptidase (IRAP) enhance fear avoidance and spatial memory and accelerate spatial learning in a number of memory paradigms. Using a virtual screening approach, a series of benzopyran compounds was identified that inhibited the catalytic activity of IRAP, ultimately resulting in the identification of potent and specific inhibitors. The present study describes the medicinal chemistry campaign that led to the development of the lead candidate, 3, highlighting the key structural features considered as critical for binding. Furthermore, the in vivo pharmacokinetics and brain uptake of compounds (1 and 3) were assessed in rats and were complemented with in vitro human and rat microsomal stability studies. Following intravenous administration to rodents, 3 exhibits brain exposure, albeit it is rapidly converted to 1, a compound which also exhibits potent inhibition of IRAP.

  9. Cutting Edge: Coding Single Nucleotide Polymorphisms of Endoplasmic Reticulum Aminopeptidase 1 Can Affect Antigenic Peptide Generation In Vitro by Influencing Basic Enzymatic Properties of the Enzyme

    OpenAIRE

    Evnouchidou, Irini; Kamal, Ram P.; Seregin, Sergey S.; Goto, Yoshikuni; Tsujimoto, Masafumi; Hattori, Akira; Voulgari, Paraskevi V; Drosos, Alexandros A.; Amalfitano, Andrea; Ian A York; Stratikos, Efstratios

    2011-01-01

    ER aminopeptidase 1 (ERAP1) customizes antigenic peptide precursors for MHC class I presentation and edits the antigenic peptide repertoire. Coding single nucleotide polymorphisms (SNPs) in ERAP1 were recently linked with predisposition to autoimmune disease, suggesting a link between pathogenesis of autoimmunity and ERAP1-mediated Ag processing. To investigate this possibility, we analyzed the effect that disease-linked SNPs have on Ag processing by ERAP1 in vitro. Michaelis–Menten analysis ...

  10. Comparative expression profiling for human endoplasmic reticulum-resident aminopeptidases 1 and 2 in normal kidney versus distinct renal cell carcinoma subtypes

    OpenAIRE

    Stoehr, Christine; Buettner-Herold, Maike; Kamphausen, Esther; Bertz, Simone; Hartmann, Arndt; Seliger, Barbara

    2013-01-01

    Altered expression of the ER-resident aminopeptidases ERAP1 and ERAP2 might play an important role in shaping the MHC class I-presented peptide repertoire, but their function in tumors has not been determined in detail. Thus, the expression of ERAP1, ERAP2 and HLA class I heavy chain (HC) was analysed in various renal tumor types and corresponding kidney parenchyma by immunohistochemistry. Additionally, comparative expression profilings of untreated versus interferon (IFN)-γ-treated RCC cell ...

  11. Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims MHC class I-presented peptides in vivo and plays an important role in immunodominance

    OpenAIRE

    Ian A York; Brehm, Michael A.; Zendzian, Sophia; Towne, Charles F.; Rock, Kenneth L.

    2006-01-01

    CD8+ T cells respond to short peptides bound to MHC class I molecules. Although most antigenic proteins contain many sequences that could bind to MHC class I, few of these peptides actually stimulate CD8+ T cell responses. Moreover, the T cell responses that are generated often follow a very reproducible hierarchy to different peptides for reasons that are poorly understood. We find that the loss of a single enzyme, endoplasmic reticulum aminopeptidase 1 (ERAP1), in the antigen-processing pat...

  12. Characterization of a salt-tolerant aminopeptidase from marine Bacillus licheniformis SWJS33 that improves hydrolysis and debittering efficiency for soy protein isolate.

    Science.gov (United States)

    Lei, Fenfen; Zhao, Qiangzhong; Sun-Waterhouse, Dongxiao; Zhao, Mouming

    2017-01-01

    An aminopeptidase was isolated from the marine Bacillus licheniformis SWJS33 (BLAP) and purified. According to the tandem mass spectrometry, the enzyme displayed 11% amino acid identity with the aminopeptidase from Bacillus (gi|496687392). BLAP exhibited maximum activity at 60°C and pH 8.0-8.5 and had a molecular mass of 100kDa. The presence of NaCl enabled 50% improvement of enzyme activity with 10-15% NaCl being the best. The observed inactivation by EDTA and bestatin and activation by Co(2+) and Ag(+) indicated that the obtained enzyme was a metalloaminopeptidase. Such an aminopeptidase could further improve the hydrolysis degree of soy protein isolate hydrolysates catalyzed by papain, Alcalase 2.4L or Flavourzyme 500MG from 8.5%, 9.5% or 14.4-18.8%, 18.7% or 20.1%, respectively, while decreasing the bitter intensity score of the SPI hydrolysates catalyzed by Alcalase 2.4L from 3.6 to 0.4. PMID:27507484

  13. Amino-terminal extension present in the methionine aminopeptidase type 1c of Mycobacterium tuberculosis is indispensible for its activity

    Directory of Open Access Journals (Sweden)

    Kumaran Sangaralingam

    2011-07-01

    Full Text Available Abstract Background Methionine aminopeptidase (MetAP is a ubiquitous enzyme in both prokaryotes and eukaryotes, which catalyzes co-translational removal of N-terminal methionine from elongating polypeptide chains during protein synthesis. It specifically removes the terminal methionine in all organisms, if the penultimate residue is non-bulky and uncharged. The MetAP action for exclusion of N-terminal methionine is mandatory in 50-70% of nascent proteins. Such an activity is required for proper sub cellular localization, additional processing and eventually for the degradation of proteins. Results We cloned genes encoding two such metalloproteases (MtMetAP1a and MtMetAP1c present in Mycobacterium tuberculosis and expressed them as histidine-tagged proteins in Escherichia coli. Although they have different substrate preferences, for Met-Ala-Ser, we found, MtMetAP1c had significantly high enzyme turnover rate as opposed to MtMetAP1a. Circular dichroism spectroscopic studies as well as monitoring of enzyme activity indicated high temperature stability (up to 50°C of MtMetAP1a compared to that of the MtMetAP1c. Modelling of MtMetAP1a based on MtMetAP1c crystal structure revealed the distinct spatial arrangements of identical active site amino acid residues and their mutations affected the enzymatic activities of both the proteins. Strikingly, we observed that 40 amino acid long N-terminal extension of MtMetAP1c, compared to its other family members, contributes towards the activity and stability of this enzyme, which has never been reported for any methionine aminopeptidase. Furthermore, mutational analysis revealed that Val-18 and Pro-19 of MtMetAP1c are crucial for its enzymatic activity. Consistent with this observation, molecular dynamic simulation studies of wild-type and these variants strongly suggest their involvement in maintaining active site conformation of MtMetAP1c. Conclusion Our findings unequivocally emphasized that N

  14. Kinetic, spectroscopic, and X-ray crystallographic characterization of the functional E151H aminopeptidase from Aeromonas proteolytica.

    Science.gov (United States)

    Bzymek, Krzysztof P; Moulin, Aaron; Swierczek, Sabina I; Ringe, Dagmar; Petsko, Gregory A; Bennett, Brian; Holz, Richard C

    2005-09-13

    Glutamate151 (E151) has been shown to be catalytically essential for the aminopeptidase from Vibrio proteolyticus (AAP). E151 acts as the general acid/base during the catalytic mechanism of peptide hydrolysis. However, a glutamate residue is not the only residue capable of functioning as a general acid/base during catalysis for dinuclear metallohydrolases. Recent crystallographic characterization of the D-aminopeptidase from Bacillus subtilis (DppA) revealed a histidine residue that resides in an identical position to E151 in AAP. Because the active-site ligands for DppA and AAP are identical, AAP has been used as a model enzyme to understand the mechanistic role of H115 in DppA. Substitution of E151 with histidine resulted in an active AAP enzyme exhibiting a kcat value of 2.0 min(-1), which is over 2000 times slower than r AAP (4380 min(-1)). ITC experiments revealed that ZnII binds 330 and 3 times more weakly to E151H-AAP compared to r-AAP. UV-vis and EPR spectra of CoII-loaded E151H-AAP indicated that the first metal ion resides in a hexacoordinate/pentacoordinate equilibrium environment, whereas the second metal ion is six-coordinate. pH dependence of the kinetic parameters kcat and K(m) for the hydrolysis of L-leucine p-nitroanilide (L-pNA) revealed a change in an ionization constant in the enzyme-substrate complex from 5.3 in r-AAP to 6.4 in E151H-AAP, consistent with E151 in AAP being the active-site general acid/base. Proton inventory studies at pH 8.50 indicate the transfer of one proton in the rate-limiting step of the reaction. Moreover, the X-ray crystal structure of [ZnZn(E151H-AAP)] has been solved to 1.9 A resolution, and alteration of E151 to histidine does not introduce any major conformational changes to the overall protein structure or the dinuclear ZnII active site. Therefore, a histidine residue can function as the general acid/base in hydrolysis reactions of peptides and, through analogy of the role of E151 in AAP, H115 in DppA likely

  15. A role for naturally occurring alleles of endoplasmic reticulum aminopeptidases in tumor immunity and cancer pre-disposition.

    Science.gov (United States)

    Stratikos, Efstratios; Stamogiannos, Athanasios; Zervoudi, Efthalia; Fruci, Doriana

    2014-01-01

    Endoplasmic reticulum aminopeptidase 1 and 2 (ERAP1 and ERAP2) are key components on the pathway that generates antigenic epitopes for presentation to cytotoxic T-lymphocytes (CTLs). Coding single nucleotide polymorphisms (SNPs) in these enzymes have been associated with pre-disposition to several major human diseases including inflammatory diseases with autoimmune etiology, viral infections, and virally induced cancer. The function of these enzymes has been demonstrated to affect CTL and natural killer cell responses toward healthy and malignant cells as well as the production of inflammatory cytokines. Recent studies have demonstrated that SNPs in ERAP1 and ERAP2 can affect their ability to generate or destroy antigenic epitopes and define the immunopeptidome. In this review, we examine the potential role of these enzymes and their polymorphic states on the generation of cytotoxic responses toward malignantly transformed cells. Given the current state-of-the-art, it is possible that polymorphic variation in these enzymes may contribute to the individual's pre-disposition to cancer through altered generation or destruction of tumor antigens that can facilitate tumor immune evasion. PMID:25566501

  16. Concerted in vitro trimming of viral HLA-B27-restricted ligands by human ERAP1 and ERAP2 aminopeptidases.

    Directory of Open Access Journals (Sweden)

    Elena Lorente

    Full Text Available In the classical human leukocyte antigen (HLA class I antigen processing and presentation pathway, the antigenic peptides are generated from viral proteins by multiple proteolytic cleavages of the proteasome (and in some cases other cytosolic proteases and transported to the endoplasmic reticulum (ER lumen where they are exposed to aminopeptidase activity. In human cells, two different ER-resident enzymes, ERAP1 and ERAP2, can trim the N-terminally extended residues of peptide precursors. In this study, the possible cooperative effect of generating five naturally processed HLA-B27 ligands by both proteases was analyzed. We identified differences in the products obtained with increased detection of natural HLA-B27 ligands by comparing double versus single enzyme digestions by mass spectrometry analysis. These in vitro data suggest that each enzyme can use the degradation products of the other as a substrate for new N-terminal trimming, indicating concerted aminoproteolytic activity of ERAP 1 and ERAP2.

  17. Autoimmune disease-associated variants of extracellular endoplasmic reticulum aminopeptidase 1 induce altered innate immune responses by human immune cells.

    Science.gov (United States)

    Aldhamen, Yasser A; Pepelyayeva, Yuliya; Rastall, David P W; Seregin, Sergey S; Zervoudi, Efthalia; Koumantou, Despoina; Aylsworth, Charles F; Quiroga, Dionisia; Godbehere, Sarah; Georgiadis, Dimitris; Stratikos, Efstratios; Amalfitano, Andrea

    2015-01-01

    Endoplasmic reticulum aminopeptidase 1 (ERAP1) gene polymorphisms have been linked to several autoimmune diseases; however, the molecular mechanisms underlying these associations are not well understood. Recently, we demonstrated that ERAP1 regulates key aspects of the innate immune response. Previous studies show ERAP1 to be endoplasmic reticulum-localized and secreted during inflammation. Herein, we investigate the possible roles that ERAP1 polymorphic variants may have in modulating the innate immune responses of human peripheral blood mononuclear cells (hPBMCs) using two experimental methods: extracellular exposure of hPBMCs to ERAP1 variants and adenovirus (Ad)-based ERAP1 expression. We found that exposure of hPBMCs to ERAP1 variant proteins as well as ERAP1 overexpression by Ad5 vectors increased inflammatory cytokine and chemokine production, and enhanced immune cell activation. Investigating the molecular mechanisms behind these responses revealed that ERAP1 is able to activate innate immunity via multiple pathways, including the NLRP3 (NOD-like receptor, pyrin domain-containing 3) inflammasome. Importantly, these responses varied if autoimmune disease-associated variants of ERAP1 were examined in the assay systems. Unexpectedly, blocking ERAP1 cellular internalization augmented IL-1β production. To our knowledge, this is the first report identifying ERAP1 as being involved in modulating innate responses of human immune cells, a finding that may explain why ERAP1 has been genetically associated with several autoimmune diseases. PMID:25591727

  18. Mutation of an aminopeptidase N gene is associated with Helicoverpa armigera resistance to Bacillus thuringiensis Cry1Ac toxin.

    Science.gov (United States)

    Zhang, Shaoping; Cheng, Hongmei; Gao, Yulin; Wang, Guirong; Liang, Gemei; Wu, Kongming

    2009-07-01

    A Cry1Ac-resistant strain (Bt-R) of Helicoverpa armigera, with 2971-fold resistance, was derived by selection with Cry1Ac toxin for 75 generations. We used cDNA-amplified fragment length polymorphism analysis to identify those genes differentially expressed in the Cry1Ac-resistant and -susceptible strains, which revealed 212 differentially expressed transcripts among 2000 screened cDNAs. Among these transcript-derived fragments (TDFs), 37 showed some homology to known sequences, including Aminopeptidase N (APN), which is expressed in the midgut epithelium and has been implicated as a Cry1A subfamily receptor in several moths, including H. armigera. We confirmed the TDF by RT-PCR and identified a deletion mutation of apn1 in the Bt-R strain. We expressed the TDF in bacteria. The partial HaAPN1-96S wild-type protein, bound to Cry1Ac on ligand blots, whereas HaAPN1-BtR did not. This suggested that HaAPN1 is a receptor for Bt Cry1Ac and that its deletion mutation is associated with Cry1Ac resistance in H. armigera. The absence of one binding site is responsible for its resistance to Cry1Ac. We developed an allele-specific PCR to monitor whether the apn1 gene in an H. armigera field population produced a similar mutation. No deleted mutants were found in 2250 individuals collected from the field in 2006-2007. PMID:19376227

  19. Efficient inhibition of tumor angiogenesis and growth by a synthetic peptide blocking S100A4-methionine aminopeptidase 2 interaction.

    Science.gov (United States)

    Ochiya, Takahiro; Takenaga, Keizo; Asagiri, Masataka; Nakano, Kazumi; Satoh, Hitoshi; Watanabe, Toshiki; Imajoh-Ohmi, Shinobu; Endo, Hideya

    2015-01-01

    The prometastatic calcium-binding protein, S100A4, is expressed in endothelial cells, and its downregulation markedly suppresses tumor angiogenesis in a xenograft cancer model. Given that endothelial S100A4 can be a molecular target for inhibiting tumor angiogenesis, we addressed here whether synthetic peptide capable of blocking S100A4-effector protein interaction could be a novel antiangiogenic agent. To examine this hypothesis, we focused on the S100A4-binding domain of methionine aminopeptidase 2, an effector protein, which plays a role in endothelial cell growth. Overexpression of the domain in mouse endothelial MSS31 cells reduced DNA synthesis, and the corresponding synthetic peptide (named NBD) indeed interacted with S100A4 and inhibited capillary formation in vitro and new blood vessel formation in vivo. Intriguingly, a single intra-tumor administration of the NBD peptide in human prostate cancer xenografts significantly reduced vascularity, resulting in tumor regression. Mechanistically, the NBD peptide enhanced assembly of nonmuscle myosin IIA filaments along with Ser1943 phosphorylation, stimulated formation of focal adhesions without phosphorylation of focal adhesion kinase, and provoked G1/S arrest of the cell cycle. Altogether, the NBD peptide is a potent inhibitor for tumor angiogenesis, and is the first example of an anticancer peptide drug developed on the basis of an endothelial S100A4-targeted strategy. PMID:26029719

  20. Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

    Directory of Open Access Journals (Sweden)

    Jenkins Jeremy L

    2001-10-01

    Full Text Available Abstract Background To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays. Results The Bombyx mori (silkworm aminopeptidase N (APN and cadherin-like receptors for Bacillus thuringiensis insecticidal Cry1Aa toxin were purified and their real-time binding affinities for Cry toxins were examined by surface plasmon resonance. Cry1Ab and Cry1Ac toxins did not bind to the immobilized native receptors, correlating with their low toxicities. Cry1Aa displayed moderate affinity for B. mori APN (75 nM, and unusually tight binding to the cadherin-like receptor (2.6 nM, which results from slow dissociation rates. The binding of a hybrid toxin (Aa/Aa/Ac was identical to Cry1Aa. Conclusions These results indicate domain II of Cry1Aa is essential for binding to native B. mori receptors and for toxicity. Moreover, the high-affinity binding of Cry1Aa to native cadherin-like receptor emphasizes the importance of this receptor class for Bt toxin research.

  1. Human cytomegalovirus microRNA miR-US4-1 inhibits CD8+ T cell response by targeting the aminopeptidase ERAP1

    OpenAIRE

    Kim, Sungchul; Lee, Sanghyun; Shin, Jinwook; Kim, Youngkyun; Evnouchidou, Irini; Kim, Donghyun; Kim, Young-Kook; Kim, Young-Eui; Ahn, Jin-Hyun; Riddell, Stanley R; Stratikos, Efstratios; Kim, V. Narry; Ahn, Kwangseog

    2011-01-01

    The major histocompatibility complex (MHC) class I molecules present peptides on the cell surface by CD8+ T cells, which is critical for killing of virally infected or transformed cells. Precursors of MHC class I-presented peptides are trimmed to mature epitopes by endoplasmic reticulum aminopeptidase 1 (ERAP1). The US2-US11 genomic region of human cytomegalovirus (HCMV) is dispensable for viral replication and harbors 3 microRNAs (miRNAs). We show here the HCMV miR-US4-1 specifically down-re...

  2. Endoplasmic reticulum aminopeptidase-1 alleles associated with increased risk of Ankylosing Spondylitis reduce HLA-B27 mediated presentation of multiple antigens

    OpenAIRE

    Seregin, Sergey S.; Rastall, David P. W.; Evnouchidou, Irini; Charles F Aylsworth; Quiroga, Dionisia; Kamal, Ram P.; Godbehere-Roosa, Sarah; Blum, Christopher F.; Ian A York; Stratikos, Efstratios; Amalfitano, Andrea

    2013-01-01

    Ankylosing spondylitis (AS) is a chronic systemic arthritic disease that leads to significant disability and loss of quality of life in the ~0.5% of the worldwide human population it affects. There is currently no cure for AS and mechanisms underlying its pathogenesis remain unclear. AS is highly genetic, with over 70% of the genetic risk being associated with the presence of HLA-B27 and endoplasmic reticulum aminopeptidase-1 (ERAP1) alleles. Furthermore, gene-gene interactions between HLA-B2...

  3. Small-angle neutron scattering reveals the assembly mode and oligomeric architecture of TET, a large, dodecameric aminopeptidase

    Energy Technology Data Exchange (ETDEWEB)

    Appolaire, Alexandre; Girard, Eric; Colombo, Matteo; Durá, M. Asunción [Université Grenoble Alpes, IBS, 38044 Grenoble (France); CNRS, IBS, 38044 Grenoble (France); CEA, IBS, 38044 Grenoble (France); Moulin, Martine; Härtlein, Michael [Institut Laue–Langevin, 38042 Grenoble CEDEX 9 (France); Franzetti, Bruno [Université Grenoble Alpes, IBS, 38044 Grenoble (France); CNRS, IBS, 38044 Grenoble (France); CEA, IBS, 38044 Grenoble (France); Gabel, Frank, E-mail: frank.gabel@ibs.fr [Université Grenoble Alpes, IBS, 38044 Grenoble (France); CNRS, IBS, 38044 Grenoble (France); CEA, IBS, 38044 Grenoble (France); Institut Laue–Langevin, 38042 Grenoble CEDEX 9 (France)

    2014-11-01

    The present work illustrates that small-angle neutron scattering, deuteration and contrast variation, combined with in vitro particle reconstruction, constitutes a very efficient approach to determine subunit architectures in large, symmetric protein complexes. In the case of the 468 kDa heterododecameric TET peptidase machine, it was demonstrated that the assembly of the 12 subunits is a highly controlled process and represents a way to optimize the catalytic efficiency of the enzyme. The specific self-association of proteins into oligomeric complexes is a common phenomenon in biological systems to optimize and regulate their function. However, de novo structure determination of these important complexes is often very challenging for atomic-resolution techniques. Furthermore, in the case of homo-oligomeric complexes, or complexes with very similar building blocks, the respective positions of subunits and their assembly pathways are difficult to determine using many structural biology techniques. Here, an elegant and powerful approach based on small-angle neutron scattering is applied, in combination with deuterium labelling and contrast variation, to elucidate the oligomeric organization of the quaternary structure and the assembly pathways of 468 kDa, hetero-oligomeric and symmetric Pyrococcus horikoshii TET2–TET3 aminopeptidase complexes. The results reveal that the topology of the PhTET2 and PhTET3 dimeric building blocks within the complexes is not casual but rather suggests that their quaternary arrangement optimizes the catalytic efficiency towards peptide substrates. This approach bears important potential for the determination of quaternary structures and assembly pathways of large oligomeric and symmetric complexes in biological systems.

  4. Screening the Medicines for Malaria Venture "Malaria Box" against the Plasmodium falciparum aminopeptidases, M1, M17 and M18.

    Directory of Open Access Journals (Sweden)

    Alessandro Paiardini

    Full Text Available Malaria is a parasitic disease that remains a global health burden. The ability of the parasite to rapidly develop resistance to therapeutics drives an urgent need for the delivery of new drugs. The Medicines for Malaria Venture have compounds known for their antimalarial activity, but not necessarily the molecular targets. In this study, we assess the ability of the "MMV 400" compounds to inhibit the activity of three metalloaminopeptidases from Plasmodium falciparum, PfA-M1, PfA-M17 and PfM18 AAP. We have developed a multiplex assay system to allow rapid primary screening of compounds against all three metalloaminopeptidases, followed by detailed analysis of promising compounds. Our results show that there were no PfM18AAP inhibitors, whereas two moderate inhibitors of the neutral aminopeptidases PfA-M1 and PfA-M17 were identified. Further investigation through structure-activity relationship studies and molecular docking suggest that these compounds are competitive inhibitors with novel binding mechanisms, acting through either non-classical zinc coordination or independently of zinc binding altogether. Although it is unlikely that inhibition of PfA-M1 and/or PfA-M17 is the primary mechanism responsible for the antiplasmodial activity reported for these compounds, their detailed characterization, as presented in this work, pave the way for their further optimization as a novel class of dual PfA-M1/PfA-M17 inhibitors utilising non-classical zinc binding groups.

  5. Structural insights into chaperone-activity enhancement by a K354E mutation in tomato acidic leucine aminopeptidase.

    Science.gov (United States)

    DuPrez, Kevin T; Scranton, Melissa A; Walling, Linda L; Fan, Li

    2016-05-01

    Tomato plants express acidic leucine aminopeptidase (LAP-A) in response to various environmental stressors. LAP-A not only functions as a peptidase for diverse peptide substrates, but also displays chaperone activity. A K354E mutation has been shown to abolish the peptidase activity but to enhance the chaperone activity of LAP-A. To better understand this moonlighting function of LAP-A, the crystal structure of the K354E mutant was determined at 2.15 Å resolution. The structure reveals that the K354E mutation destabilizes an active-site loop and causes significant rearrangement of active-site residues, leading to loss of the catalytic metal-ion coordination required for the peptidase activity. Although the mutant was crystallized in the same hexameric form as wild-type LAP-A, gel-filtration chromatography revealed an apparent shift from the hexamer to lower-order oligomers for the K354E mutant, showing a mixture of monomers to trimers in solution. In addition, surface-probing assays indicated that the K354E mutant has more accessible hydrophobic areas than wild-type LAP-A. Consistently, computational thermodynamic estimations of the interfaces between LAP-A monomers suggest that increased exposure of hydrophobic surfaces occurs upon hexamer breakdown. These results suggest that the K354E mutation disrupts the active-site loop, which also contributes to the hexameric assembly, and destabilizes the hexamers, resulting in much greater hydrophobic areas accessible for efficient chaperone activity than in the wild-type LAP-A. PMID:27139632

  6. Microarray analysis of tomato's early and late wound response reveals new regulatory targets for Leucine aminopeptidase A.

    Directory of Open Access Journals (Sweden)

    Melissa A Scranton

    Full Text Available Wounding due to mechanical injury or insect feeding causes a wide array of damage to plant cells including cell disruption, desiccation, metabolite oxidation, and disruption of primary metabolism. In response, plants regulate a variety of genes and metabolic pathways to cope with injury. Tomato (Solanum lycopersicum is a model for wound signaling but few studies have examined the comprehensive gene expression profiles in response to injury. A cross-species microarray approach using the TIGR potato 10-K cDNA array was analyzed for large-scale temporal (early and late and spatial (locally and systemically responses to mechanical wounding in tomato leaves. These analyses demonstrated that tomato regulates many primary and secondary metabolic pathways and this regulation is dependent on both timing and location. To determine if LAP-A, a known modulator of wound signaling, influences gene expression beyond the core of late wound-response genes, changes in RNAs from healthy and wounded Leucine aminopeptidase A-silenced (LapA-SI and wild-type (WT leaves were examined. While most of the changes in gene expression after wounding in LapA-SI leaves were similar to WT, overall responses were delayed in the LapA-SI leaves. Moreover, two pathogenesis-related 1 (PR-1c and PR-1a2 and two dehydrin (TAS14 and Dhn3 genes were negatively regulated by LAP-A. Collectively, this study has shown that tomato wound responses are complex and that LAP-A's role in modulation of wound responses extends beyond the well described late-wound gene core.

  7. Bacillus thuringiensis Cry1Ca-resistant Spodoptera exigua lacks expression of one of four Aminopeptidase N genes

    Directory of Open Access Journals (Sweden)

    Moar William J

    2005-06-01

    Full Text Available Abstract Background Insecticidal toxins from Bacillus thuringiensis bind to receptors on midgut epithelial cells of susceptible insect larvae. Aminopeptidases N (APNs from several insect species have been shown to be putative receptors for these toxins. Here we report the cloning and expression analysis of four APN cDNAs from Spodoptera exigua. Results Suppression Subtractive Hybridization (SSH was used to construct cDNA libraries of genes that are up-and down-regulated in the midgut of last instar larvae of beet armyworm, S. exigua exposed to B. thuringiensis Cry1Ca toxin. Among the clones from the SSH libraries, cDNA fragments coding for two different APNs were obtained (APN2 and APN4. A similar procedure was employed to compare mRNA differences between susceptible and Cry1Ca resistant S. exigua. Among the clones from this last comparison, cDNA fragments belonging to a third APN (APN1 were detected. Using sequences obtained from the three APN cDNA fragments and degenerate primers for a fourth APN (APN3, the full length sequences of four S. exigua APN cDNAs were obtained. Northern blot analysis of expression of the four APNs showed complete absence of APN1 expression in the resistant insects, while the other three APNs showed similar expression levels in the resistant and susceptible insects. Conclusion We have cloned and characterized four different midgut APN cDNAs from S. exigua. Expression analysis revealed the lack of expression of one of these APNs in the larvae of a Cry1Ca-resistant colony. Combined with previous evidence that shows the importance of APN in the mode of action of B. thuringiensis toxins, these results suggest that the lack of APN1 expression plays a role in the resistance to Cry1Ca in this S. exigua colony.

  8. Involvement of insulin-regulated aminopeptidase in the effects of the renin-angiotensin fragment angiotensin IV: a review.

    Science.gov (United States)

    Stragier, Bart; De Bundel, Dimitri; Sarre, Sophie; Smolders, Ilse; Vauquelin, Georges; Dupont, Alain; Michotte, Yvette; Vanderheyden, Patrick

    2008-09-01

    For decades, angiotensin (Ang) II was considered as the end product and the only bioactive peptide of the renin-angiotensin system (RAS). However, later studies revealed biological activity for other Ang fragments. Amongst those, Ang IV has drawn a lot of attention since it exerts a wide range of central and peripheral effects including the ability to enhance learning and memory recall, anticonvulsant and anti-epileptogenic properties, protection against cerebral ischemia, activity at the vascular level and an involvement in atherogenesis. Some of these effects are AT(1) receptor dependent but others most likely result from the binding of Ang IV to insulin-regulated aminopeptidase (IRAP) although the exact mechanism(s) of action that mediate the Ang IV-induced effects following this binding are until now not fully known. Nevertheless, three hypotheses have been put forward: since Ang IV is an inhibitor of the catalytic activity of IRAP, its in vivo effects might result from a build-up of IRAP's neuropeptide substrates. Second, IRAP is co-localized with the glucose transporter GLUT4 in several tissue types and therefore, Ang IV might interact with the uptake of glucose. A final and more intriguing hypothesis ascribes a receptor function to IRAP and hence an agonist role to Ang IV. Taken together, it is clear that further work is required to clarify the mechanism of action of Ang IV. On the other hand, a wide range of studies have made it clear that IRAP might become an important target for drug development against different pathologies such as Alzheimer's disease, epilepsy and ischemia.

  9. Location of the Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin.

    Science.gov (United States)

    Atsumi, Shogo; Mizuno, Eri; Hara, Hirotaka; Nakanishi, Kazuko; Kitami, Madoka; Miura, Nami; Tabunoki, Hiroko; Watanabe, Ayako; Sato, Ryoichi

    2005-07-01

    We analyzed the binding site on Cry1Aa toxin for the Cry1Aa receptor in Bombyx mori, 115-kDa aminopeptidase N type 1 (BmAPN1) (K. Nakanishi, K. Yaoi, Y. Nagino, H. Hara, M. Kitami, S. Atsumi, N. Miura, and R. Sato, FEBS Lett. 519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking molecule N-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of 508STLRVN513 and 582VFTLSAHV589. These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding. PMID:16000811

  10. Aminopeptidase activity by spoilage bacteria and its relationship to microbial load and sensory attributes of poultry legs during aerobic cold storage.

    Science.gov (United States)

    Guevara-Franco, José Alfredo; Alonso-Calleja, Carlos; Capita, Rosa

    2010-02-01

    The shelf life of poultry legs stored aerobically and the possible role of the aminopeptidase activity of gram-negative bacteria (p-nitroaniline test) as a predictor of poultry spoilage were evaluated on the basis of microbiological and sensory parameters. Chicken legs (n = 30) obtained immediately after evisceration in a local poultry processing plant were kept under aerobic refrigeration (4 +/- 1 degrees C) for 7 days. Microbiological (counts of aerobic bacteria and psychrotrophs) and sensory (odor, color, and general acceptability on a hedonic scale of 1 to 9) parameters and aminopeptidase activity (absorbance at 390 nm [A(390)]) determinations were performed after 0, 1, 3, 5, and 7 days of storage. Aerobic plate counts of 7 log CFU/g and a score of 6 for general acceptability were used as indicators of the end point of shelf life. Strong correlations (r > or = 0.76; P counts, hedonic scores, and A(390) values. Samples were judged as unacceptable (shelf-life end point) after 2 and 4 days on the basis of sensory and microbiological analyses, respectively. A(390) values of 0.52 and 0.89 (corresponding to p-nitroaniline concentrations of 6.25 and 10.7 microg/ml, respectively) are proposed as the upper limits for acceptability on the basis of sensory and microbiological determinations, respectively. However, these recommendations are based on a small set of samples, and their general application is yet to be verified.

  11. A structural insight into the P1S1 binding mode of diaminoethylphosphonic and phosphinic acids, selective inhibitors of alanine aminopeptidases.

    Science.gov (United States)

    Węglarz-Tomczak, Ewelina; Berlicki, Łukasz; Pawełczak, Małgorzata; Nocek, Bogusław; Joachimiak, Andrzej; Mucha, Artur

    2016-07-19

    N'-substituted 1,2-diaminoethylphosphonic acids and 1,2-diaminoethylphosphinic dipeptides were explored to unveil the structural context of the unexpected selectivity of these inhibitors of M1 alanine aminopeptidases (APNs) versus M17 leucine aminopeptidase (LAP). The diaminophosphonic acids were obtained via aziridines in an improved synthetic procedure that was further expanded for the phosphinic pseudodipeptide system. The inhibitory activity, measured for three M1 and one M17 metalloaminopeptidases of different sources (bacterial, human and porcine), revealed several potent compounds (e.g., Ki = 65 nM of 1u for HsAPN). Two structures of an M1 representative (APN from Neisseria meningitidis) in complex with N-benzyl-1,2-diaminoethylphosphonic acid and N-cyclohexyl-1,2-diaminoethylphosphonic acid were determined by the X-ray crystallography. The analysis of these structures and the models of the phosphonic acid complexes of the human ortholog provided an insight into the role of the additional amino group and the hydrophobic substituents of the ligands within the S1 active site region. PMID:27100031

  12. 3,4-diaminobenzoic acid derivatives as inhibitors of the oxytocinase subfamily of M1 aminopeptidases with immune-regulating properties.

    Science.gov (United States)

    Papakyriakou, Athanasios; Zervoudi, Efthalia; Tsoukalidou, Sofia; Mauvais, Francois-Xavier; Sfyroera, Georgia; Mastellos, Dimitrios C; van Endert, Peter; Theodorakis, Emmanuel A; Vourloumis, Dionisios; Stratikos, Efstratios

    2015-02-12

    Members of the oxytocinase subfamily of M1 aminopeptidases (ERAP1, ERAP2, and IRAP) play important roles in both the adaptive and innate human immune responses. Their enzymatic activity can contribute to the pathogenesis of several major human diseases ranging from viral and parasitic infections to autoimmunity and cancer. We have previously demonstrated that diaminobenzoic acid derivatives show promise as selective inhibitors for this group of aminopeptidases. In this study, we have thoroughly explored a series of 3,4-diaminobenzoic acid derivatives as inhibitors of this class of enzymes, achieving submicromolar inhibitors for ERAP2 (IC50 = 237 nM) and IRAP (IC50 = 105 nM). Cell-based analysis indicated that the lead compounds can be effective in downregulating macrophage activation induced by lipopolysaccharide and interferon-γ as well as cross-presentation by bone marrow-derived dendritic cells. Our results indicate that this class of inhibitors may be useful for the targeted downregulation of immune responses. PMID:25635706

  13. Experimental study of the enhancement effect of aminopeptidase N inhibitor ubenimex on the differentiation induction activity of all-trans-retinoic acid in acute promyeiocytic leukemia cells and its mechanism

    Institute of Scientific and Technical Information of China (English)

    钱习军

    2006-01-01

    Objective To investigate the effect of aminopeptidase N inhibitor ubenimex on differentiation induction of alltrans -retinoic acid (ATRA) in acute promyelocytic leukemia (APL) cells and its mechanism. Methods The expression of CD11b was analyzed by flow cytometry and nitroblue-tetrazolium (NBT) reduction assay was per-

  14. Functional interpretation of a non-gut hemocoelic tissue aminopeptidase N (APN) in a lepidopteran insect pest Achaea janata.

    Science.gov (United States)

    Ningshen, Thuirei Jacob; Aparoy, Polamarasetty; Ventaku, Venkat Rao; Dutta-Gupta, Aparna

    2013-01-01

    Insect midgut membrane-anchored aminopeptidases N (APNs) are Zn(++) dependent metalloproteases. Their primary role in dietary protein digestion and also as receptors in Cry toxin-induced pathogenesis is well documented. APN expression in few non-gut hemocoelic tissues of lepidopteran insects has also been reported but their functions are widely unknown. In the present study, we observed specific in vitro interaction of Cry1Aa toxin with a 113 kDa AjAPN1 membrane protein of larval fat body, Malpighian tubule and salivary gland of Achaea janata. Analyses of 3D molecular structure of AjAPN1, the predominantly expressed APN isoform in these non-gut hemocoelic tissues of A. janata showed high structural similarity to the Cry1Aa toxin binding midgut APN of Bombyx mori, especially in the toxin binding region. Structural similarity was further substantiated by in vitro binding of Cry1Aa toxin. RNA interference (RNAi) resulted in significant down-regulation of AjAPN1 transcript and protein expression in fat body and Malpighian tubule but not in salivary gland. Consequently, reduced AjAPN1 expression resulted in larval mortality, larval growth arrest, development of lethal larval-pupal intermediates, development of smaller pupae and emergence of viable defective adults. In vitro Cry1Aa toxin binding analysis of non-gut hemocoelic tissues of AjAPN1 knockdown larvae showed reduced interaction of Cry1Aa toxin with the 113 kDa AjAPN1 protein, correlating well with the significant silencing of AjAPN1 expression. Thus, our observations suggest AjAPN1 expression in non-gut hemocoelic tissues to play important physiological role(s) during post-embryonic development of A. janata. Though specific interaction of Cry1Aa toxin with AjAPN1 of non-gut hemocoelic tissues of A. janata was demonstrated, evidences to prove its functional role as a Cry1Aa toxin receptor will require more in-depth investigation. PMID:24244508

  15. Studying progress on aminopeptidase N of insects%昆虫氨肽酶N的研究进展

    Institute of Scientific and Technical Information of China (English)

    常晓丽; 吴青君; 王少丽; 徐宝云; 张友军

    2011-01-01

    氨肽酶N(aminopeptidase N,APN)是一种偏好水解蛋白或寡肽N端中性氨基酸的酶,广泛分布于动植物中.在昆虫中,APN主要分布于中肠刷状缘膜上,涉及食物蛋白的消化以及Bt毒素对靶标害虫的作用.从APN的结构特征和分类地位、APN的分离纯化及其酶活性位点与结合位点的相关性、APN同工酶的区分特征及系统发育关系、APN同工酶的基因功能、不同APN同工酶在昆虫体内的分布及表达量、APN基因功能最新研究方法和结果以及APN与昆虫抗药性的关系等方面概述了昆虫APN的最新研究进展.鉴于昆虫体内受体的多样性、复杂性及当前研究手段的局限性,对APN功能的全面了解还有赖于研究方法的改进和提高而逐步深入.%Aminopeptidase N (APN) is an enzyme that preferentially cleaves neutral amino acids from the amino-terminus of proteins or oligopeptides. This enzyme is widely distributed in the plants and animals. In insects, APN is most abundant in the brush border membrane of the midgut and is an enzyme involved in the digestion of protein in food and the toxication of Bt for target insects. The recent research results of APNs of insects in the world were summarized. The characteristics,configuration, classification, isolation and purification, relationship between the catalytic and toxinbinding sites, phylogenetic relationship, gene function, the distribution and gene expression level, the recent studying methods and results of APNs and whether they are related between the Bt resistance of insects and APNs were elucidated. It is necessary to further study the function of APNs in view of the effect of other receptors, action complication and limited research techniques.

  16. Functional interpretation of a non-gut hemocoelic tissue aminopeptidase N (APN in a lepidopteran insect pest Achaea janata.

    Directory of Open Access Journals (Sweden)

    Thuirei Jacob Ningshen

    Full Text Available Insect midgut membrane-anchored aminopeptidases N (APNs are Zn(++ dependent metalloproteases. Their primary role in dietary protein digestion and also as receptors in Cry toxin-induced pathogenesis is well documented. APN expression in few non-gut hemocoelic tissues of lepidopteran insects has also been reported but their functions are widely unknown. In the present study, we observed specific in vitro interaction of Cry1Aa toxin with a 113 kDa AjAPN1 membrane protein of larval fat body, Malpighian tubule and salivary gland of Achaea janata. Analyses of 3D molecular structure of AjAPN1, the predominantly expressed APN isoform in these non-gut hemocoelic tissues of A. janata showed high structural similarity to the Cry1Aa toxin binding midgut APN of Bombyx mori, especially in the toxin binding region. Structural similarity was further substantiated by in vitro binding of Cry1Aa toxin. RNA interference (RNAi resulted in significant down-regulation of AjAPN1 transcript and protein expression in fat body and Malpighian tubule but not in salivary gland. Consequently, reduced AjAPN1 expression resulted in larval mortality, larval growth arrest, development of lethal larval-pupal intermediates, development of smaller pupae and emergence of viable defective adults. In vitro Cry1Aa toxin binding analysis of non-gut hemocoelic tissues of AjAPN1 knockdown larvae showed reduced interaction of Cry1Aa toxin with the 113 kDa AjAPN1 protein, correlating well with the significant silencing of AjAPN1 expression. Thus, our observations suggest AjAPN1 expression in non-gut hemocoelic tissues to play important physiological role(s during post-embryonic development of A. janata. Though specific interaction of Cry1Aa toxin with AjAPN1 of non-gut hemocoelic tissues of A. janata was demonstrated, evidences to prove its functional role as a Cry1Aa toxin receptor will require more in-depth investigation.

  17. Identification of a novel aminopeptidase P-like gene (OnAPP) possibly involved in Bt toxicity and resistance in a major corn pest (Ostrinia nubilalis).

    Science.gov (United States)

    Khajuria, Chitvan; Buschman, Lawrent L; Chen, Ming-Shun; Siegfried, Blair D; Zhu, Kun Yan

    2011-01-01

    Studies to understand the Bacillus thuringiensis (Bt) resistance mechanism in European corn borer (ECB, Ostrinia nubilalis) suggest that resistance may be due to changes in the midgut-specific Bt toxin receptor. In this study, we identified 10 aminopeptidase-like genes, which have previously been identified as putative Bt toxin receptors in other insects and examined their expression in relation to Cry1Ab toxicity and resistance. Expression analysis for the 10 aminopeptidase-like genes revealed that most of these genes were expressed predominantly in the larval midgut, but there was no difference in the expression of these genes in Cry1Ab resistant and susceptible strains. This suggested that altered expression of these genes was unlikely to be responsible for resistance in these ECB strains. However, we found that there were changes in two amino acid residues of the aminopeptidase-P like gene (OnAPP) involving Glu(305) to Lys(305) and Arg(307) to Leu(307) in the two Cry1Ab-resistant strains as compared with three Cry1Ab-susceptible strains. The mature OnAPP contains 682 amino acid residues and has a putative signal peptide at the N-terminus, a predicted glycosylphosphatidyl-inositol (GPI)-anchor signal at the C-terminal, three predicted N-glycosylation sites at residues N178, N278 and N417, and an O-glycosylation site at residue T653. We used a feeding based-RNA interference assay to examine the role of the OnAPP gene in Cry1Ab toxicity and resistance. Bioassays of Cry1Ab in larvae fed diet containing OnAPP dsRNA resulted in a 38% reduction in the transcript level of OnAPP and a 25% reduction in the susceptibility to Cry1Ab as compared with larvae fed GFP dsRNA or water. These results strongly suggest that the OnAPP gene could be involved in binding the Cry1Ab toxin in the ECB larval midgut and that mutations in this gene may be associated with Bt resistance in these two ECB strains. PMID:21887358

  18. Endoplasmic reticulum aminopeptidase 1 function and its pathogenic role in regulating innate and adaptive immunity in cancer and major histocompatibility complex class I-associated autoimmune diseases.

    Science.gov (United States)

    Fruci, D; Romania, P; D'Alicandro, V; Locatelli, F

    2014-08-01

    Major histocompatibility complex (MHC) class I molecules present antigenic peptides on the cell surface to alert natural killer (NK) cells and CD8(+) T cells for the presence of abnormal intracellular events, such as virus infection or malignant transformation. The generation of antigenic peptides is a multistep process that ends with the trimming of N-terminal extensions in the endoplasmic reticulum (ER) by aminopeptidases ERAP1 and ERAP2. Recent studies have highlighted the potential role of ERAP1 in reprogramming the immunogenicity of tumor cells in order to elicit innate and adaptive antitumor immune responses, and in conferring susceptibility to autoimmune diseases in predisposed individuals. In this review, we will provide an overview of the current knowledge about the role of ERAP1 in MHC class I antigen processing and how its manipulation may constitute a promising tool for cancer immunotherapy and treatment of MHC class I-associated autoimmune diseases. PMID:25066018

  19. A novel recombinant Leishmania donovani p45, a partial coding region of methionine aminopeptidase, generates protective immunity by inducing a Th1 stimulatory response against experimental visceral leishmaniasis.

    Science.gov (United States)

    Gupta, Reema; Kushawaha, Pramod K; Tripathi, Chandra Dev Pati; Sundar, Shyam; Dube, Anuradha

    2012-05-01

    The development of a vaccine against visceral leishmaniasis (VL) conferring long-lasting immunity remains a challenge. Identification and proteomic characterization of parasite proteins led to the detection of p45, a member of the methionine aminopeptidase family. To our knowledge the present study is the first known report that describes the molecular and immunological characterization of p45. Recombinant Leishmania donovani p45 (rLdp45) induced cellular responses in cured hamsters and generated Th1-type cytokines from peripheral blood mononuclear cells of cured/endemic VL patients. Immunization with rLdp45 exerted considerable prophylactic efficacy (∼85%) supported by an increase in mRNA expression of iNOS, IFN-γ, TNF-α and IL-12 and decrease in TGF-β and IL-4, indicating its potential as a vaccine candidate against VL.

  20. QSAR study of anthranilic acid sulfonamides as inhibitors of methionine aminopeptidase-2 using LS-SVM and GRNN based on principal components.

    Science.gov (United States)

    Shahlaei, Mohsen; Sabet, Razieh; Ziari, Maryam Bahman; Moeinifard, Behzad; Fassihi, Afshin; Karbakhsh, Reza

    2010-10-01

    Quantitative relationships between molecular structure and methionine aminopeptidase-2 inhibitory activity of a series of cytotoxic anthranilic acid sulfonamide derivatives were discovered. We have demonstrated the detailed application of two efficient nonlinear methods for evaluation of quantitative structure-activity relationships of the studied compounds. Components produced by principal component analysis as input of developed nonlinear models were used. The performance of the developed models namely PC-GRNN and PC-LS-SVM were tested by several validation methods. The resulted PC-LS-SVM model had a high statistical quality (R(2)=0.91 and R(CV)(2)=0.81) for predicting the cytotoxic activity of the compounds. Comparison between predictability of PC-GRNN and PC-LS-SVM indicates that later method has higher ability to predict the activity of the studied molecules.

  1. Functional co-localization of monocytic aminopeptidase N/CD13 with the Fc gamma receptors CD32 and CD64

    DEFF Research Database (Denmark)

    Riemann, Dagmar; Tcherkes, Anatolij; Hansen, Gert H;

    2005-01-01

    , but not with the myeloid marker CD33 representing a member of the sialoadhesin family. Our results imply a novel functional role of CD13 and Fc gamma receptors as members of a multimeric receptor complex. Further studies have to be done to elucidate common signaling pathways of these molecules.......Information about the function of aminopeptidase N/CD13 on monocytes is limited. In order to gain more insight into its interaction with other proteins, we have identified molecules that co-localize with the membrane ectoenzyme at the cell surface of monocytes. Using laser scanning and electron...... microscopy as well as fluorescence resonance energy transfer (FRET) measured by flow cytometry we show that monocytic CD13 co-localized with the Fc gamma receptor II/CD32 after Fc receptor ligation by a CD32-specific antibody. FRET was also observed between CD13 and the Fc gamma receptor I/CD64...

  2. 2-Arylbenzothiazole, benzoxazole and benzimidazole derivatives as fluorogenic substrates for the detection of nitroreductase and aminopeptidase activity in clinically important bacteria.

    Science.gov (United States)

    Cellier, Marie; Fabrega, Olivier J; Fazackerley, Elizabeth; James, Arthur L; Orenga, Sylvain; Perry, John D; Salwatura, Vindhya L; Stanforth, Stephen P

    2011-05-01

    A series of 2-(2-nitrophenyl)benzothiazole 7, 2-(2-nitrophenyl)benzoxazole 10 and 2-(2-nitrophenyl)benzimidazole 13 derivatives have been synthesised and assessed as indicators of nitroreductase activity across a range of clinically important Gram negative and Gram positive bacteria. The majority of Gram negative bacteria produced strongly fluorescent colonies with substrates 7 and 10 whereas fluorescence production in Gram positive bacteria was less widespread. The l-alanine 16 and 19 and β-alanine 21 and 23 derivatives have been prepared from 2-(2-aminophenyl)benzothiazole 14 and 2-(2-aminophenyl)benzoxazole 17. These four compounds have been evaluated as indicators of aminopeptidase activity. The growth of Gram positive bacteria was generally inhibited by these substrates but fluorescent colonies were produced with the majority of Gram negative bacteria tested.

  3. Binding of phylogenetically distant Bacillus thuringiensis cry toxins to a Bombyx mori aminopeptidase N suggests importance of Cry toxin's conserved structure in receptor binding.

    Science.gov (United States)

    Shinkawa, A; Yaoi, K; Kadotani, T; Imamura, M; Koizumi, N; Iwahana, H; Sato, R

    1999-07-01

    We investigated the binding proteins for three Cry toxins, Cry1Aa, Cry1Ac, and the phylogenetically distant Cry9Da, in the midgut cell membrane of the silkworm. In a ligand blot experiment, Cry1Ac and Cry9Da bound to the same 120-kDa aminopeptidase N (APN) as Cry1Aa. A competition experiment with the ligand blot indicated that the three toxins share the same binding site on several proteins. The values of the dissociation constants of the three Cry toxins and 120-kDa APN are as low as the case of other Cry toxins and receptors. These results suggest that distantly related Cry toxins bind to the same site on the same proteins, especially with APN. We propose that the conserved structure in these three toxins includes the receptor-binding site. PMID:10387111

  4. Bacillus thuringiensis insecticidal Cry1Aa toxin binds to a highly conserved region of aminopeptidase N in the host insect leading to its evolutionary success.

    Science.gov (United States)

    Nakanishi, K; Yaoi, K; Shimada, N; Kadotani, T; Sato, R

    1999-06-15

    Bacillus thuringiensis insecticidal protein, Cry1Aa toxin, binds to a specific receptor in insect midguts and has insecticidal activity. Therefore, the structure of the receptor molecule is probably a key factor in determining the binding affinity of the toxin and insect susceptibility. The cDNA fragment (PX frg1) encoding the Cry1Aa toxin-binding region of an aminopeptidase N (APN) or an APN family protein from diamondback moth, Plutella xylostella midgut was cloned and sequenced. A comparison between the deduced amino acid sequence of PX frg1 and other insect APN sequences shows that Cry1Aa toxin binds to a highly conserved region of APN family protein. In this paper, we propose a model to explain the mechanism that causes B. thuringiensis evolutionary success and differing insect susceptibility to Cry1Aa toxin. PMID:10366728

  5. cDNA cloning and expression of Bacillus thuringiensis Cry1Aa toxin binding 120 kDa aminopeptidase N from Bombyx mori.

    Science.gov (United States)

    Yaoi, K; Nakanishi, K; Kadotani, T; Imamura, M; Koizumi, N; Iwahana, H; Sato, R

    1999-01-18

    Bacillus thuringiensis Cry1Aa toxin binds to a 120 kDa putative receptor protein in the Bombyx mori midgut. Recently, this protein was purified and identified as glycosyl-phosphatidylinositol (GPI) anchored aminopeptidase N (APN). In this study, a full-length cDNA thought to encode this 120 kDa APN was isolated and sequenced. It has a 2958 bp ORF encoding 986 amino acids. In the deduced amino acid sequence, we identified GPI-anchor and zinc-metallopeptidase signals, which are the same as those of APNs of other insects that are reported to be putative Cry1 toxin receptors. The B. mori APN amino acid sequence also has a high similarity with those of the other APNs. Subsequently, the recombinant APN was expressed by Escherichia coli and its Cry1Aa toxin binding ability was analyzed. Ligand blotting showed that Cry1Aa toxin bound to the recombinant APN. PMID:9931470

  6. The type II secretion system of Legionella pneumophila elaborates two aminopeptidases, as well as a metalloprotease that contributes to differential infection among protozoan hosts.

    Science.gov (United States)

    Rossier, Ombeline; Dao, Jenny; Cianciotto, Nicholas P

    2008-02-01

    Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular parasite of aquatic amoebae and human macrophages. A key factor for L. pneumophila in intracellular infection is its type II protein secretion system (Lsp). In order to more completely define Lsp output, we recently performed a proteomic analysis of culture supernatants. Based upon the predictions of that analysis, we found that L. pneumophila secretes two distinct aminopeptidase activities encoded by the genes lapA and lapB. Whereas lapA conferred activity against leucine, phenylalanine, and tyrosine aminopeptides, lapB was linked to the cleavage of lysine- and arginine-containing substrates. To assess the role of secreted aminopeptidases in intracellular infection, we examined the relative abilities of lapA and lapB mutants to infect human U937 cell macrophages as well as Hartmannella vermiformis and Acanthamoeba castellanii amoebae. Although these experiments identified a dispensable role for LapA and LapB, they uncovered a previously unrecognized role for the type II-dependent ProA (MspA) metalloprotease. Whereas proA mutants were not defective for macrophage or A. castellanii infection, they (but not their complemented derivatives) were impaired for growth upon coculture with H. vermiformis. Thus, ProA represents the first type II effector implicated in an intracellular infection event. Furthermore, proA represents an L. pneumophila gene that shows differential importance among protozoan infection models, suggesting that the legionellae might have evolved some of its factors to especially target certain of their protozoan hosts. PMID:18083880

  7. Two specific membrane-bound aminopeptidase N isoforms from Aedes aegypti larvae serve as functional receptors for the Bacillus thuringiensis Cry4Ba toxin implicating counterpart specificity.

    Science.gov (United States)

    Aroonkesorn, Aratee; Pootanakit, Kusol; Katzenmeier, Gerd; Angsuthanasombat, Chanan

    2015-05-29

    The interaction between Bacillus thuringiensis Cry toxins and their receptors on midgut cells of susceptible insect larvae is the critical determinant in toxin specificity. Besides GPI-linked alkaline phosphatase in Aedes aegypti mosquito-larval midguts, membrane-bound aminopeptidase N (AaeAPN) is widely thought to serve as a Cry4Ba receptor. Here, two full-length AaeAPN isoforms, AaeAPN2778 and AaeAPN2783, predicted to be GPI-linked were cloned and successfully expressed in Spodoptera frugiperda (Sf9) cells as 112- and 107-kDa membrane-bound proteins, respectively. In the cytotoxicity assay, Sf9 cells expressing each of the two AaeAPN isoforms showed increased sensitivity to the Cry4Ba mosquito-active toxin. Double immunolocalization revealed specific binding of Cry4Ba to each individual AaeAPN expressed on the cell membrane surface. Sequence analysis and homology-based modeling placed these two AaeAPNs to the M1 aminopeptidase family as they showed similar four-domain structures, with the most conserved domain II being the catalytic component. Additionally, the most variable domain IV containing negatively charged surface patches observed only in dipteran APNs could be involved in insect specificity. Overall results demonstrated that these two membrane-bound APN isoforms were responsible for mediating Cry4Ba toxicity against AaeAPN-expressed Sf9 cells, suggesting their important role as functional receptors for the toxin counterpart in A. aegypti mosquito larvae. PMID:25871797

  8. The unique functional role of the C-HS hydrogen bond in the substrate specificity and enzyme catalysis of type 1 methionine aminopeptidase.

    Science.gov (United States)

    Reddi, Ravikumar; Singarapu, Kiran Kumar; Pal, Debnath; Addlagatta, Anthony

    2016-07-19

    It is intriguing how nature attains recognition specificity between molecular interfaces where there is no apparent scope for classical hydrogen bonding or polar interactions. Methionine aminopeptidase (MetAP) is one such enzyme where this fascinating conundrum is at play. In this study, we demonstrate that a unique C-HS hydrogen bond exists between the enzyme methionine aminopeptidase (MetAP) and its N-terminal-methionine polypeptide substrate, which allows specific interaction between apparent apolar interfaces, imposing a strict substrate recognition specificity and efficient catalysis, a feature replicated in Type I MetAPs across all kingdoms of life. We evidence this evolutionarily conserved C-HS hydrogen bond through enzyme assays on wild-type and mutant MetAP proteins from Mycobacterium tuberculosis that show a drastic difference in catalytic efficiency. The X-ray crystallographic structure of the methionine bound protein revealed a conserved water bridge and short contacts involving the Met side-chain, a feature also observed in MetAPs from other organisms. Thermal shift assays showed a remarkable 3.3 °C increase in melting temperature for methionine bound protein compared to its norleucine homolog, where C-HS interaction is absent. The presence of C-HS hydrogen bonding was also corroborated by nuclear magnetic resonance spectroscopy through a change in chemical shift. Computational chemistry studies revealed the unique role of the electrostatic environment in facilitating the C-HS interaction. The significance of this atypical hydrogen bond is underscored by the fact that the function of MetAP is essential for any living cell. PMID:27225936

  9. Aminopeptidases from Aspergillus niger

    NARCIS (Netherlands)

    Wijk, van D.

    2004-01-01

    Aspergillusis a filamentous fungus that can grow in many environments, on several substrates at different conditions. In the soil,Aspergillirecycle nutrients by the degradation of plant material. In particular,Aspergilliare known for their capabili

  10. Intestinal Disaccharidases and Aminopeptidase-N in two species of Cinclodes (Passerine: Furnaridae Disacaridasas y aminopeptidasa-N en dos especies de Cinclodes (Paserine: Furnaridae

    Directory of Open Access Journals (Sweden)

    PABLO SABAT

    2000-06-01

    Full Text Available It has been postulated that both digestive capacity and intestinal biochemical features are correlated to dietary habits in birds. Therefore, it would be expected to find biochemical constraint to hydrolyze sugars in those species, which predate exclusively on marine invertebrates. In vitro intestinal activities of these enzymes were studied in Cinclodes nigrofumosus (d' Orbigny and Cinclodes patagonicus (Gmelling. Due to differences in dietary habits between species I predicted the lack of sucrase activity in C. nigrofumosus but not in C. patagonicus. Also, low activities of maltase would be expected in both species. On the other hand due to the considerable amount of proteins and trehalose present in preys, high activities of both trehalase and aminopeptidase-N were also expected. Contrary to previous reports in birds, significant activity of trehalase was found. Also lack of sucrase and small amounts of maltase were observed as well as a significant aminopeptidase-N activity in both species. Although the digestive enzyme activities of C. nigrofumosus and C. patagonicus appear to be correlated with their natural diet, the similarities between species in all enzymes activities suggest an strong effect of phylogenetic inertiaSe ha postulado que en las aves tanto la capacidad digestiva como las características bioquímicas del intestino están correlacionadas con los hábitos dietarios. Por esto, es probable que aquellas especies que se alimentan exclusivamente de invertebrados, posean una restricción bioquímica para hidrolizar azúcares. La actividad intestinal in vitro de esas enzimas se estudió en Cinclodes nigrofumosus (d' Orbigny y Cinclodes patagonicus (Gmelling.Debido a diferencias en los habitos entre las especies, se predice que sacarasa estará ausente en C. nigrofumosus, pero no en C. patagonicus. Además, se debiera encontrar una actividad maltásica baja en ambas especies. Por otro lado, debido a las considerables cantidades de

  11. RNA interference-mediated knockdown of three putative aminopeptidases N affects susceptibility of Spodoptera exigua larvae to Bacillus thuringiensis Cry1Ca.

    Science.gov (United States)

    Ren, Xiang-Liang; Ma, Yan; Cui, Jin-Jie; Li, Guo-Qing

    2014-08-01

    Aminopeptidase N (APN) isoforms in insects have been documented to be involved in the mode of action of insecticidal crystal proteins (Cry) from Bacillus thuringiensis. Here we cloned two novel Seapns from the larval midgut of Spodoptera exigua, a major pest of many crops of economic importance in China. According to a phylogenetic analysis, these two novel SeAPNs, along with the four SeAPN isoforms already described, belong to six different clades. All the six SeAPNs share similar structural features. From N- to C-terminus a signal peptide, a gluzincin aminopeptidase motif, a zinc binding/gluzincin motif, and a glycosylphosphatidylinositol-anchor sequence are located. The six Seapn genes were highly expressed at the larval stage, especially in the larval gut. Ingestion during four consecutive days of double-stranded RNAs (dsRNAs) targeting Seapn1, Seapn2, Seapn3, Seapn4, Seapn5 and Seapn6 significantly reduced corresponding mRNA levels by 55.6%, 45.5%, 43.2%, 56.8%, 45.4%, and 46.0% respectively, compared with those recorded in control larvae fed on non-specific dsRNA (dsegfp). When the larvae that previously ingested phosphate buffered saline (PBS)-, dsegfp-, or six dsSeapns-overlaid diets were then exposed to a diet containing Cry1Ca, the larval mortalities were 71.2%, 69.3%, 52.0%, 77.2%, 43.3%, 62.0%, 65.4% and 53.8% respectively recorded after 6days. ANOVA analysis revealed that the larvae previously fed on dsSeapn1-, dsSeapn3-, and dsSeapn6-overlaid diets had significantly lower mortalities than those previously ingested PBS-, dsegfp-, dsSeapn2-, dsSeapn4- and dsSeapn5-overlaid diets. Thus, these results suggest that SeAPN1, SeAPN3 and SeAPN6 may be candidate receptors for Cry1Ca in S. exigua. PMID:24932922

  12. An alternative methionine aminopeptidase, MAP-A, is required for nitrogen starvation and high-light acclimation in the cyanobacterium Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Drath, Miriam; Baier, Kerstin; Forchhammer, Karl

    2009-05-01

    Methionine aminopeptidases (MetAPs or MAPs, encoded by map genes) are ubiquitous and pivotal enzymes for protein maturation in all living organisms. Whereas most bacteria harbour only one map gene, many cyanobacterial genomes contain two map paralogues, the genome of Synechocystis sp. PCC 6803 even three. The physiological function of multiple map paralogues remains elusive so far. This communication reports for the first time differential MetAP function in a cyanobacterium. In Synechocystis sp. PCC 6803, the universally conserved mapC gene (sll0555) is predominantly expressed in exponentially growing cells and appears to be a housekeeping gene. By contrast, expression of mapA (slr0918) and mapB (slr0786) genes increases during stress conditions. The mapB paralogue is only transiently expressed, whereas the widely distributed mapA gene appears to be the major MetAP during stress conditions. A mapA-deficient Synechocystis mutant shows a subtle impairment of photosystem II properties even under non-stressed conditions. In particular, the binding site for the quinone Q(B) is affected, indicating specific N-terminal methionine processing requirements of photosystem II components. MAP-A-specific processing becomes essential under certain stress conditions, since the mapA-deficient mutant is severely impaired in surviving conditions of prolonged nitrogen starvation and high light exposure. PMID:19359320

  13. Molecular and pathogenic effects of endoplasmic reticulum aminopeptidases ERAP1 and ERAP2 in MHC-I-associated inflammatory disorders: Towards a unifying view.

    Science.gov (United States)

    López de Castro, José A; Alvarez-Navarro, Carlos; Brito, Ariadna; Guasp, Pablo; Martín-Esteban, Adrian; Sanz-Bravo, Alejandro

    2016-09-01

    The inflammatory diseases that are most strongly associated with major histocompatibility Complex class I (MHC-I) alleles are also influenced by endoplasmic reticulum aminopeptidase (ERAP) 1 and/or 2, often in epistasis with the susceptibility MHC-I allele. This review will focus on the four major MHC-I-associated inflammatory disorders: ankylosing spondylitis, birdshot chorioretinopathy, Behçet's disease and psoriasis. The genetics of ERAP1/ERAP2 association and the alterations induced by polymorphism of these enzymes on the risk MHC-I allotypes will be examined. A pattern emerges of analogous effects on peptide length, sequence and affinity of disparate peptidomes, suggesting that similar peptide-mediated mechanisms underlie the pathogenesis and the joint contribution of ERAP1/ERAP2 and MHC-I to distinct inflammatory diseases. Processing of specific antigens, peptide-dependent changes in global properties of the MHC-I molecules, such as folding and stability, or both may be pathogenic. PMID:27522479

  14. Purification of Aminopeptidase N Protein and Differences in cDNAs Encoding APN1 Between Susceptible and Resistant Helicoverpa armigera Strains to Bacillus thuringiensis Toxins

    Institute of Scientific and Technical Information of China (English)

    LIANG Ge-mei; WANG Gui-rong; XU Guang; WU Kong-ming; GUO Yu-yuan

    2004-01-01

    The brush border membrane vesicles (BBMVs) in midgut of Helicoverpa armigera were successfully separated, and most of the Aminopeptidase N (APN) activities in BBMV were preserved. The 3-[(3-chlor-amidopropyl) dimethylammonio]-l-propane-sulphonate (CHAPS)can enhance the dissolution of BBMV, and phosphatidylinositol-specific phosopholipase C (PI-PLC) can cleave the APN from midgut membrane. The APN was primarily purified using a Mono-Q column. The results of immunoblotting showed that the 120 and 170 kDa proteins in the BBMV could bind CrylAc, and 120kDa APN was a glycosylphosphalidylinositol(GPI)anchored protein. Two Bt-resistant strains (Bt-P, Bt-M) were obtained after being selected for more than five years in laboratory using Bt insecticides and Bt transgenic cotton incorporated into diet separately. The resistance of Bt-P and Bt-M were 1 083.3and 48.7 times that of susceptible strain. The genes encoding APN1 in midgut of susceptible and resistant H.armigera were cloned by PCR and RACE techniques. The inferred amino acid sequences of APN1 possessed the common character of APN family in insects. In comparison with APN1 in susceptible strain, three nucleotide mutations were observed in the APN1 of Bt-M strain and resulted in two amino acid replace in the putative protein sequences, and eight nucleotide mutations were observed in Bt-P strain and resulted in five amino acid replace.

  15. Expression of Aminopeptidase N1(APN1),the Main Receptor Protein for Bacillus thuringiensis Cry1A Toxin from Helicoverpa armigera Larval Midgut in Trichoplusia ni cells

    Institute of Scientific and Technical Information of China (English)

    CHANG Hong-lei; LIANG Ge-mei; WANG Gui-rong; YU Hong-kun; GUO Yu-yuan; WU Kong-ming

    2008-01-01

    The aim of this article is to successfully express the Bt(Bacillus thuringiensis)toxin receptor protein located on the internal membrane of larval midgut of cotton bollworm(Helicoverpa armigera Hiibner)within eukaryotic expression system,which is one of the key links for clarifying the relationship between receptor and Bt resistance.The fragments of aminopeptidase N1(APN1)gene without signal peptide in the susceptible and the resistant H. armigera were cloned separately using PCR method,and were separately cloned into pUC 19 vector.After sequencing the gene,the fragments encoding for APN1 without signal peptide were cloned into the Bac-to-Bac baculovirus expression system with transfer vector pFastBacHTB under the polyhedron gene promoter.The recombinant transposing plasmid pFastBacHTB/APN1 was screened and then transformed into Escherichia coli DH10Bac.It was cultured in LB medium,which contained Te, Kan,Ge,X-gal,and IPTG.The resulting recombinant bacmid was transfected into cells of the insect Trichoplusia ni and recombinant baculoviruse was obtained.The lysate of cells infected with recombinant baculoviruse was analyzed by SDS-PAGE and blot analysis.The results showed that the recombinant baculoviruse was fully capable of expressing APN1.The APN1 gene successfully expressed in T. ni cell established the base for continuing the research on its function and relationship of resistance with Bt.

  16. Comparative expression profiling for human endoplasmic reticulum-resident aminopeptidases 1 and 2 in normal kidney versus distinct renal cell carcinoma subtypes.

    Science.gov (United States)

    Stoehr, Christine G; Buettner-Herold, Maike; Kamphausen, Esther; Bertz, Simone; Hartmann, Arndt; Seliger, Barbara

    2013-01-01

    Altered expression of the ER-resident aminopeptidases ERAP1 and ERAP2 might play an important role in shaping the MHC class I-presented peptide repertoire, but their function in tumors has not been determined in detail. Thus, the expression of ERAP1, ERAP2 and HLA class I heavy chain (HC) was analysed in various renal tumor types and corresponding kidney parenchyma by immunohistochemistry. Additionally, comparative expression profilings of untreated versus interferon (IFN)-γ-treated RCC cell lines were performed applying qRT-PCR, Western blot and/or flow cytometry. Normal kidney tissues showed strong ERAP1 staining in the proximal tubules of 57.4 % of cases, in the distal tubules of 94.3 % of cases and in the medulla of 88.6 % of cases, whereas high ERAP2 levels were observed in the medulla of 77.1 % of cases and in both, proximal and distal tubules of about 88 % of cases. Imbalanced, downregulated and RCC subtype-specific ERAP1 or ERAP2 expression was detected in 12.7 % or 43.8 % of samples analyzed, respectively. A coordinated downregulation of ERAPs was found in 4.8 %, an upregulation of ERAP1 or ERAP2 in 22.8 % or 2.0 % of RCC lesions. No association exists between ERAP and HLA class I HC expression for any tissue type. A heterogeneous constitutive ERAP expression pattern was also detected in RCC cell lines with lower ERAP2 than ERAP1 expression levels, which was in 11/17 RCC cell lines inducible by IFN-γ. Conclusively, ERAP1 and ERAP2 might be involved in the development of immune escape mechanisms of RCC. PMID:23696916

  17. Endoplasmic reticulum aminopeptidase-1 alleles associated with increased risk of ankylosing spondylitis reduce HLA-B27 mediated presentation of multiple antigens.

    Science.gov (United States)

    Seregin, Sergey S; Rastall, David P W; Evnouchidou, Irini; Aylsworth, Charles F; Quiroga, Dionisia; Kamal, Ram P; Godbehere-Roosa, Sarah; Blum, Christopher F; York, Ian A; Stratikos, Efstratios; Amalfitano, Andrea

    2013-12-01

    Ankylosing spondylitis (AS) is a chronic systemic arthritic disease that leads to significant disability and loss of quality of life in the ∼0.5% of the worldwide human population it affects. There is currently no cure for AS and mechanisms underlying its pathogenesis remain unclear. AS is highly genetic, with over 70% of the genetic risk being associated with the presence of HLA-B27 and endoplasmic reticulum aminopeptidase-1 (ERAP1) alleles. Furthermore, gene-gene interactions between HLA-B27 and ERAP1 AS risk alleles have recently been confirmed. Here, we demonstrate that various ERAP1 alleles can differentially mediate surface expression of antigens presented by HLA-B27 on human cells. Specifically, for all peptides tested, we found that an ERAP1 variant containing high AS risk SNPs reduced the amount of the peptide presented by HLA-B27, relative to low AS risk ERAP1 variants. These results were further validated using peptide catalysis assays in vitro, suggesting that high AS risk alleles have an enhanced catalytic activity that more rapidly destroys many HLA-B27-destined peptides, a result that correlated with decreased HLA-B27 presentation of the same peptides. These findings suggest that one mechanism underlying AS pathogenesis may involve an altered ability for AS patients harboring both HLA-B27 and high AS risk ERAP1 alleles to correctly display a variety of peptides to the adaptive arm of the immune system, potentially exposing such individuals to higher AS risk due to abnormal display of pathogen or self-derived peptides by the adaptive immune system. PMID:24028501

  18. The proteasome immunosubunits, PA28 and ER-aminopeptidase 1 protect melanoma cells from efficient MART-126-35 -specific T-cell recognition.

    Science.gov (United States)

    Keller, Martin; Ebstein, Frédéric; Bürger, Elke; Textoris-Taube, Kathrin; Gorny, Xenia; Urban, Sabrina; Zhao, Fang; Dannenberg, Tanja; Sucker, Antje; Keller, Christin; Saveanu, Loredana; Krüger, Elke; Rothkötter, Hermann-Josef; Dahlmann, Burkhardt; Henklein, Petra; Voigt, Antje; Kuckelkorn, Ulrike; Paschen, Annette; Kloetzel, Peter-Michael; Seifert, Ulrike

    2015-12-01

    The immunodominant MART-1(26(27)-35) epitope, liberated from the differentiation antigen melanoma antigen recognized by T cells/melanoma antigen A (MART-1/Melan-A), has been frequently targeted in melanoma immunotherapy, but with limited clinical success. Previous studies suggested that this is in part due to an insufficient peptide supply and epitope presentation, since proteasomes containing the immunosubunits β5i/LMP7 (LMP, low molecular weight protein) or β1i/LMP2 and β5i/LMP7 interfere with MART-1(26-35) epitope generation in tumor cells. Here, we demonstrate that in addition the IFN-γ-inducible proteasome subunit β2i/MECL-1 (multicatalytic endopeptidase complex-like 1), proteasome activator 28 (PA28), and ER-resident aminopeptidase 1 (ERAP1) impair MART-1(26-35) epitope generation. β2i/MECL-1 and PA28 negatively affect C- and N-terminal cleavage and therefore epitope liberation from the proteasome, whereas ERAP1 destroys the MART-1(26-35) epitope by overtrimming activity. Constitutive expression of PA28 and ERAP1 in melanoma cells indicate that both interfere with MART-1(26-35) epitope generation even in the absence of IFN-γ. In summary, our results provide first evidence that activities of different antigen-processing components contribute to an inefficient MART-1(26-35) epitope presentation, suggesting the tumor cell's proteolytic machinery might have an important impact on the outcome of epitope-specific immunotherapies. PMID:26399368

  19. Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) Polymorphism Relevant to Inflammatory Disease Shapes the Peptidome of the Birdshot Chorioretinopathy-Associated HLA-A*29:02 Antigen.

    Science.gov (United States)

    Alvarez-Navarro, Carlos; Martín-Esteban, Adrian; Barnea, Eilon; Admon, Arie; López de Castro, José A

    2015-07-01

    Birdshot chorioretinopathy is a rare ocular inflammation whose genetic association with HLA-A*29:02 is the highest between a disease and a major histocompatibility complex (MHC) molecule. It belongs to a group of MHC-I-associated inflammatory disorders, also including ankylosing spondylitis, psoriasis, and Behçet's disease, for which endoplasmic reticulum aminopeptidases (ERAP) 1 and/or 2 have been identified as genetic risk factors. Since both enzymes are involved in the processing of MHC-I ligands, it seems reasonable that common peptide-mediated mechanisms may underlie the pathogenesis of these diseases. In this study, comparative immunopeptidomics was used to characterize >5000 A*29:02 ligands and quantify the effects of ERAP1 polymorphism and expression on the A*29:02 peptidome in human cells. The peptides predominant in an active ERAP1 context showed a higher frequency of nonamers and bulkier amino acid side chains at multiple positions, compared with the peptides predominant in a less active ERAP1 background. Thus, ERAP1 polymorphism has a large influence, shaping the A*29:02 peptidome through length-dependent and length-independent effects. These changes resulted in increased affinity and hydrophobicity of A*29:02 ligands in an active ERAP1 context. The results reveal the nature of the functional interaction between A*29:02 and ERAP1 and suggest that this enzyme may affect the susceptibility to birdshot chorioretinopathy by altering the A*29:02 peptidome. The complexity of these alterations is such that not only peptide presentation but also other potentially pathogenic features could be affected. PMID:25892735

  20. Purification and Characterization of an Aminopeptidase with Highest Preference for Lysine from Paralichthys Olivaceus Skeletal Muscle%牙鲆肌肉赖氨酸氨肽酶的分离纯化与性质研究

    Institute of Scientific and Technical Information of China (English)

    陈曦; 蔡秋凤; 刘光明; 苏文金; 曹敏杰

    2012-01-01

    An aminopeptidase was purified from Paralichthys olivaceus skeletal muscle to homogeneity by ammonium sulfate fractionation and three column chromatographies, including DEAE -Sephacel, Phenyl -Sepharose, and DEAE -Sepharose Fast Flow. The molecular mass of the enzyme was approximately 100 ku. Optimum temperature and pH of the enzyme were 45 ℃ and 7.5, respectively. Metal-chelating agents such as EDTA, EGTA and 1,10-phenanthroline effectively inhibited the enzyme activity. Zn2+, Mn2+ and (or) Co2+ were quite possibly its metal cofactor(s), strongly suggesting the enzyme belong to metalloproteinase family. The enzyme purified in the present study was regarded as a lysine aminopeptidase (LysAP) according to its substrate specificity. Western blot analysis revealed that leucine aminopeptidase from red sea bream and LysAP from Paralichthys olivaceus have high homogeneity in amino acid sequences. Furthermore, LysAP distributes in tissues including skeletal muscle, heart, liver, spleen, stomach, kidney, gut and gill.%通过DEAE-Sephacel阴离子葡聚糖纤维素交换层析,Phenyl Sepharose 6-Fast Flow苯基葡聚糖凝胶疏水层析和DEAE Sepharose-Fast Flow阴离子葡聚糖凝胶交换层析,从牙鲆(Paralichthys olivaceus)肌肉中分离纯化得到1种赖氨酸氨肽酶,并对其生化性质进行研究.牙鲆赖氨酸氨肽酶的分子质量约为100 ku.该酶的最适温度45℃,最适pH 7.5,二价金属离子对酶活性有较大影响,EDTA和EGTA等金属螯合剂对其活性有抑制作用.该酶对荧光合成底物Lys-MCA分解作用最强,而对内肽酶对应的荧光底物没有分解作用.免疫印迹反应结果显示,该酶与鲤鱼肌肉中亮氨酸氨肽酶同源性较高,并在牙鲆多种器官中均有分布.

  1. Effect of Lathyrus sativus and vitamin C on the status of aromatic L-amino acid decarboxylase and dipeptidyl-aminopeptidase-IV in the central and peripheral tissues and serum of guinea pigs

    International Nuclear Information System (INIS)

    Studies on the effect of Lathyrus Sativus seeds (LLS) on aromatic L-amino acid decarboxylase (AADC) and on dipeptidyl-aminopeptidase-IV (DAP-IV) were carried out in the central and peripheral tissues and serum of LSS-treated and LSS plus vitamin C-treated guinea pigs. The feeding of LSS for 35 days decreased the AADC activity significantly in the brain and peripheral tissues, but the activity was recovered to normal level in the most tissues when vitamin C was added with the LSS. DAP-IV activity decreased in the peripheral tissues when treated with LSS, but the vitamin C administration with LSS did not recover the enzyme activity. The DAP-IV activity did not decrease significantly in any of the brain tissues of the LSS-treated group. (author). 18 refs, 2 tabs

  2. Aminopeptidase N isoforms from the midgut of Bombyx mori and Plutella xylostella -- their classification and the factors that determine their binding specificity to Bacillus thuringiensis Cry1A toxin.

    Science.gov (United States)

    Nakanishi, Kazuko; Yaoi, Katsuro; Nagino, Yasushi; Hara, Hirotaka; Kitami, Madoka; Atsumi, Shogo; Miura, Nami; Sato, Ryoichi

    2002-05-22

    Novel aminopeptidase N (APN) isoform cDNAs, BmAPN3 and PxAPN3, from the midguts of Bombyx mori and Plutella xylostella, respectively, were cloned, and a total of eight APN isoforms cloned from B. mori and P. xylostella were classified into four classes. Bacillus thuringiensis Cry1Aa and Cry1Ab toxins were found to bind to specific APN isoforms from the midguts of B. mori and P. xylostella, and binding occurred with fragments that corresponded to the BmAPN1 Cry1Aa toxin-binding region of each APN isoform. The results suggest that APN isoforms have a common toxin-binding region, and that the apparent specificity of Cry1Aa toxin binding to each intact APN isoform seen in SDS-PAGE is determined by factors such as expression level in conjunction with differences in binding affinity. PMID:12023048

  3. 稻纵卷叶螟氨肽酶N基因的克隆及时空表达分析%Cloning and spatio-temporal expression of aminopeptidase N encoding genes in Cnaphalocrocis medinalis (Lepidoptera:Pyralidae)

    Institute of Scientific and Technical Information of China (English)

    杜立啸; 周艳聪; 王兴云; 韩兰芝; 侯茂林; 彭于发

    2014-01-01

    【目的】昆虫中肠氨肽酶 N(Aminopeptidase N,APN)是 Bt 杀虫蛋白的重要受体之一,与Bt 蛋白的杀虫机制及昆虫对 Bt 蛋白的抗性密切相关。为阐明稻纵卷叶螟 Cnaphalocrocis medinalis (Guenée)APN基因的功能及明确Bt蛋白对稻纵卷叶螟的毒力机制,本研究系统开展了稻纵卷叶螟中肠APN基因的克隆及时空表达分析。【方法】通过简并引物 PCR 结合 RACE 技术克隆并获得4条稻纵卷叶螟 APN 基因的 cDNA 序列全长,采用实时定量PCR技术研究了APN基因在稻纵卷叶螟不同虫态及幼虫不同组织中的时空表达情况。【结果】经NCBI同源比对分析,认为这4个基因分属于APN基因家族的不同类别,分别将其命名为CmAPN1(GenBank登录号:HQ853294)、CmAPN2(GenBank登录号:HQ853295)、CmAPN3(GenBank登录号:KJ143755)、CmAPN4(GenBank登录号:HQ853296)。序列分析表明,CmAPN1-4 cDNA序列全长分别为:3698、3478、3150和3149 bp,开放阅读框分别为:3045、2877、3045和2862 bp,分别编码965、958、1014和952个氨基酸。其推导的氨基酸序列具有鳞翅目昆虫氨肽酶 N 的典型结构特征,即含有糖基化位点、N-末端信号肽序列、谷氨酸锌化氨肽酶保守结构GAMEN、锌结合位点 HEX2HX18E、C-末端糖基磷脂酰肌醇(GPI)锚信号肽。实时定量研究表明,CmAPNs在幼虫中的表达量显著高于卵、蛹和成虫;在幼虫中,CmAPN1的表达水平明显低于CmAPN2-4,且其在不同龄期中的表达差异显著;CmAPN2-4的表达量随幼虫龄期的增加而增加;CmAPNs 在幼虫肠道组织中的表达量显著高于其它组织器官,且CmAPN1和CmAPN2分别在中肠和后肠中呈现高水平表达,CmAPN3在前、中肠内均高水平表达;但CmAPN4在各个组织器官中的表达均保持较低水平。【结论】 CmAPNs 基因在稻纵卷叶螟的不同虫态和不同组织中呈现了差异显

  4. Differences between disease-associated endoplasmic reticulum aminopeptidase 1 (ERAP1) isoforms in cellular expression, interactions with tumour necrosis factor receptor 1 (TNF-R1) and regulation by cytokines.

    Science.gov (United States)

    Yousaf, N; Low, W Y; Onipinla, A; Mein, C; Caulfield, M; Munroe, P B; Chernajovsky, Y

    2015-05-01

    Endoplasmic reticulum aminopeptidase 1 (ERAP1) processes peptides for major histocompatibility complex (MHC) class I presentation and promotes cytokine receptor ectodomain shedding. These known functions of ERAP1 may explain its genetic association with several autoimmune inflammatory diseases. In this study, we identified four novel alternatively spliced variants of ERAP1 mRNA, designated as ΔExon-11, ΔExon-13, ΔExon-14 and ΔExon-15. We also observed a rapid and differential modulation of ERAP1 mRNA levels and spliced variants in different cell types pretreated with lipopolysaccharide (LPS). We have studied three full-length allelic forms of ERAP1 (R127-K528, P127-K528, P127-R528) and one spliced variant (ΔExon-11) and assessed their interactions with tumour necrosis factor receptor 1 (TNF-R1) in transfected cells. We observed variation in cellular expression of different ERAP1 isoforms, with R127-K528 being expressed at a much lower level. Furthermore, the cellular expression of full-length P127-K528 and ΔExon-11 spliced variant was enhanced significantly when co-transfected with TNF-R1. Isoforms P127-K528, P127-R528 and ΔExon-11 spliced variant associated with TNF-R1, and this interaction occurred in a region within the first 10 exons of ERAP1. Supernatant-derived vesicles from transfected cells contained the full-length and ectodomain form of soluble TNF-R1, as well as carrying the full-length ERAP1 isoforms. We observed marginal differences between TNF-R1 ectodomain levels when co-expressed with individual ERAP1 isoforms, and treatment of transfected cells with tumour necrosis factor (TNF), interleukin (IL)-1β and IL-10 exerted variable effects on TNF-R1 ectodomain cleavage. Our data suggest that ERAP1 isoforms may exhibit differential biological properties and inflammatory mediators could play critical roles in modulating ERAP1 expression, leading to altered functional activities of this enzyme. PMID:25545008

  5. 内质网氨基肽酶1在银屑病发病中的作用%Roles of endoplasmic reticulum aminopeptidase-1 in the pathogenesis of psoriasis

    Institute of Scientific and Technical Information of China (English)

    潘倩; 姚唯一; 张安平

    2013-01-01

    Psoriasis is a chronic inflammatory skin disease.The pathogenesis of psoriasis remains unclear,and is considered to be associated with genetics,environment and autoimmunity.In recent years,with the progress in genome-wide association studies,numerous susceptibility loci have been successively identified for psoriasis,including the endoplasmic reticulum aminopeptidase-1 (ERAP1) gene.A lot of single nucleotide polymorphisms (SNPs) inside the ERAP1 gene have been repeatedly confirmed to be associated powerfully with psoriasis in nearly all ethnic/racial populations,and there might be some interactions between ERAP1 and HLA-C.ERAP1 is also involved in the trimming of HLA class I molecules to an optimal length and in the cleavage of several cytokine cell surface receptors.Moreover,ERAP1 takes part in immune regulation,in particular the activation of CD8+ T cells,and plays a certain role in the induction and maintenance of psoriatic lesions via cooperating with other inflammatory cells and cytokines.%银屑病是一种慢性炎症性皮肤病,其发病机制尚不清楚,可能涉及遗传、环境及自身免疫等多方面因素.近年来,随着全基因组关联研究的进展,众多银屑病易感位点陆续被发现,内质网氨基肽酶1基因便是其中之一.几乎所有种族人群的研究均表明,内质网氨基肽酶1区域的许多单核苷酸多肽性与银屑病相关,且与HLA-C之间可能存在交互作用.内质网氨基肽酶1参与人白细胞抗原Ⅰ类分子翻译后的加工,并与多种细胞膜表面受体的分裂有关.内质网氨基肽酶1还参与免疫调节,尤其与CD8+T细胞的活化有关,与其他炎性细胞和细胞因子共同作用,诱发或维持银屑病的皮损.

  6. 大螟中肠氨肽酶 N 基因的克隆及表达谱分析%Cloning and expression profiling of aminopeptidase N encoding gene in the larval midgut of Sesamia inferens (Lepidoptera: Noctuidae)

    Institute of Scientific and Technical Information of China (English)

    王兴云; 马文静; 韩兰芝; 侯茂林

    2012-01-01

    昆虫中肠氨肽酶N是Bt毒素的重要受体之一,与Bt毒素的杀虫机制及昆虫Bt抗性的产生密切相关.本研究通过简并引物PCR结合RACE技术克隆并获得大螟Sesamia inferens Bt受体蛋白——氨肽酶N(aminopeptidase N,APN)基因的cDNA序列全长,经NCBI同源比对分析,认为该基因为APN3基因,并将其命名为SiAPN3(GenBank登录号为HQ636624).序列分析表明,该基因的cDNA序列全长为3 411 bp,开放阅读框为3 018 bp,编码1 006个氨基酸;预测蛋白质分子量为114 kD,等电点为4.95;其推导的氨基酸序列中具有鳞翅目昆虫氨肽酶N典型的结构特征,即含有1个N-和12个O-连接的糖基化位点,N-末端具有18个氨基酸的剪切信号肽,谷氨酸锌化氨肽酶保守结构GAMEN,锌结合位点HEXXHX18E,C-末端具有22个氨基酸的糖基磷脂酰肌醇(GPI)锚信号肽.采用实时定量PCR研究了SiAPN3在大螟4龄幼虫肠道不同部位和幼虫不同龄期的转录表达谱.结果表明,该基因在幼虫中肠中的表达量最高,其次为后肠,前肠中表达量最低,且中肠和后肠中的表达量显著高于前肠(P<0.05);SiAPN3在3龄幼虫中的表达量最高,1龄幼虫中最低,虽然3、4日龄之间的表达量没有显著差异,但二者均显著高于其他日龄,1,2和5日龄之间不存在显著差异(P>0.05).该研究为阐明APN基因的功能及大螟对Bt抗性产生的分子机制奠定了基础.%Aminopeptidase N (APN) is located in the brush border membrane vesicles (BBMV) of insect midgut. It is the major receptor of insecticidal crystal proteins from Bacillus thuringiensis ( Bt) and is closely related to the resistance of insects to Bt toxin. To clarify the relationship of APN and the resistance of Sesamia inferens to Bt loxin, the full-length APN-encoding cDNA sequence was cloned from BBMV of S. inferens midgut by degenerate PCR and RACE techniques and named as SiAPN3 (GenBank accession no; HQ636624). The relative expression levels of

  7. Binding of Bt Cry toxins to lepidopteran midgut aminopeptidase N and the relationship between their interactions with Bt resistance%鳞翅目昆虫氨肽酶N与Bt毒素的结合及其与Bt抗性的关系

    Institute of Scientific and Technical Information of China (English)

    马文静; 韩兰芝; 尹新明; 曹广春; 苏丽娟

    2011-01-01

    随着Bt Cry作物在我国的广泛应用和推广,靶标害虫对其抗性风险已成为Bt Cry作物生态安全研究的重要内容.氨肽酶N(Aminopeptidase N,APN)是位于昆虫中肠刷状缘膜囊泡(Brush Border Membrane Vesicles,BBMV)上Bt Cry毒素重要的受体蛋白之一,它与Bt Cry毒素的结合能力决定了Bt Cry毒素的杀虫活性及昆虫对Bt抗性的产生.本文从APN的结构特征与分类、APN与Bt Cry毒素的结合特异性、结合位点、结合过程中的分子互作机制及APN变异导致昆虫抗性产生几方面系统综述了鳞翅目昆虫中肠Bt Cry受体蛋白-氨肽酶N与Bt Cry 毒素的结合及其与Bt抗性关系的研究进展.%With the wide application and popularization of Bt Cry crop, the resistance of target pests a-gainst Bt Cry crop has become the focus of studies on its ecological safety. Aminopeptidase N (APN) is one of Bt-toxin receptor proteins located in brush border membrane vesicles ( BBMV) of insect midgut. Its binding capability with Bt Cry toxin determines the toxin's insecticidal spectrum and insect Bt resistance. This paper systemically reviews the progress of researches on APN structural characteristics and classfication, the binding feature, binding sites and interactions mechanism of Bt Cry toxin and APN as well as Bt resistance resulting from APN mutations.

  8. 家蚕氨肽酶N家族基因在5龄幼虫中肠组织的表达分析%Expression Analysis of Aminopeptidase N Family Genes in Midgut Tissue of the Fifth Instar Bombyx mori Larvae

    Institute of Scientific and Technical Information of China (English)

    王猛; 程晨; 郝碧芳; 徐安英; 沈兴家; 黄金山

    2013-01-01

    Aminopeptidase N (APN) is an enzyme that readily hydrolyzes protein or neutral amino acids at the N-terminal of an oligopeptide.It is mainly distributed in the brush border membrane vesicle of midgut epithelial cells of lepidopterous insects and is the important receptor of Bacillus thuringiensis crystal toxins (Cry).To investigate expression patterns of APN family genes,Real-time PCR was employed to analyze the differential expression of APN family genes in larva migut tissue of different silkworm (Bombyx mori) varieties and the expression level of various family members in larva midgut tissue of the same silkworm variety.The results showed that APN family genes were expressed in larva midgut of all tested silkworm varieties.The expression level of APN genes were significantly different between silkworm varieties having different voltinisms (P <0.05).However,there was little difference between silkworm varieties having the same voltinism (P >0.05).The expression levels of APN2,APN4 and APN5 genes were relatively higher in larva midgut tissue of the same silkworm variety,whereas those of APN1 and APN3 were relatively lower.The obtained results would facilitate further study on functioning mechanism of silkworm APN family genes,and provide theoretical basis for breeding silkworm strains resistant to Bacillus thuringiensis.%氨肽酶N(aminopeptidase N,APN)是一种偏好水解蛋白质或寡肽N端中性氨基酸的酶,在鳞翅目昆虫中主要分布于中肠上皮细胞的刷状缘囊膜上,是苏云金芽孢杆菌(Bacillus thuringiensis,Bt)伴孢晶体(Cry)毒素的重要受体蛋白.为了研究家蚕APN家族基因的表达特征,运用Real-time PCR技术检测分析该家族基因在不同家蚕品种幼虫中肠组织的表达差异以及同一家蚕品种幼虫中肠组织中该家族基因各个成员的表达丰度.在所有供试家蚕品种的幼虫中肠组织均可检测到APN家族基因的表达,但不同化性家蚕品种间APN基因的表

  9. Synthesis and spectroscopic studies on the new Schiff base derived from the 1:2 condensation of 2,6-diformyl-4-methylphenol with 5-aminouracil (BDF5AU) and its transition metal complexes. Influence on biologically active peptides-regulating aminopeptidases.

    Science.gov (United States)

    Hueso-Ureña, Francisco; Illán-Cabeza, Nuria A; Moreno-Carretero, Miguel N; Martínez-Martos, José M; Ramírez-Expósito, María J

    2003-04-01

    The synthesis, spectroscopic (IR, 1H and 13C NMR, UV-Vis-NIR, EPR), magnetic measurements and biological studies of a number of complexes of Co(II), Ni(II), Cu(II), Zn(II), Cd(II), Au(III) and Hg(II) of the Schiff base derived from the 1:2 condensation of 2,6-diformyl-4-methylphenol and 5-aminouracil, ((5-[[(3-[[(2,4-dioxopyrimidin-5(1H,3H)-yl)imino]methyl]-2-hydroxy-5-methylphenyl)methylene]amino]pyrimidine-2,4(1H,3H)-dione, hereafter denoted as BDF5AU) are reported. In all cases, the complexes appear to be monomeric. The deprotonated ligand in the phenolic oxygen atom shows a tridentate coordination mode through the two azomethine nitrogen atoms and the phenolic oxygen atom. The coordination of the neutral ligand takes place through the phenolic oxygen atom and one azomethine nitrogen atom and the carbonylic oxygen atom in fourth position of one uracil ring. The biological properties of some perchlorate complexes on the activity of some neutral, acid, basic and omega aminopeptidases (AP) are assayed, demonstrating a general inhibitory effect. Neutral and basic AP are mainly inhibited by Cu(II), Ni(II) and Cd(II) complexes, although tyrosyl-AP is activated by Zn(II) complex. Glutamyl-AP but not aspartyl-AP is inhibited by all the complexes assayed excepting Zn(II) complex. Finally, omega AP is inhibited by Ni(II) and Cd(II) complexes. PMID:12667703

  10. 银屑病患者梅克尔细胞角蛋白表达及与氨基肽酶N表达的关系%Relationship between cytokeratin expression in Merkel cells and aminopeptidase N expression in patients with psoriasis

    Institute of Scientific and Technical Information of China (English)

    王骏; 刘太华; 刘德芳; 汪晓军

    2011-01-01

    Objective To investigate the expression of three cytokeratins( CK20, CK19, CK18 ) in Merkel cells and their relations with aminopeptidase N( CD13 )expression in psoriatic skin lesion tissues, and to explore the pathogenesis of psoriasis. Methods Immunohistochemical method was employed to detect the expression of CK20, CK19, CK18 and CD13 in skin lesion tissues of patients with psoriasis vulgaris. The relationships among them were statistically analyzed and compared with healthy people. Results Compared with healthy controls, the expression of CK20, CK19, CK18 and CD13 increased obviously in psoriatic skin lesion tissues ( P<0. 01 ). The expression of CK20,CK19 and CK18 was positively correlated with CD13 expression, and the coefficient correlation was 0. 856,0. 838 and 0. 820 respectively( P < 0. 01 ). Conclusion The functional changes of Merkel cells in psoriatic skin lesion tissues maybe play some roles in the pathogenesis of psoriasis.%目的 探索银屑病皮损组织中梅克尔细胞表达角蛋白(CK20、CK19、CK18)的情况及与氨基肽酶N(CD13)表达的关系,探讨银屑病的发病机制.方法 采用免疫组化方法,检测寻常型银屑病患者皮损组织中梅克尔细胞表达CK20、CK19、CK18的情况和表皮层CD13表达的情况,分析CK20、CK19和CK18与CD13 表达的关系,并与正常人对比.结果 与正常人比较,皮损组织中CK20、CK19、CK18 和CD13的表达均升高(P<0.01).CK20、CK19和CK18与CD13的表达呈正相关,相关系数分别为0.856、0.838、0.820(P<0.01).结论 银屑病皮损组织中Merkel细胞变化在银屑病发病中可能发挥一定的作用.

  11. Methionine AminoPeptidase Type-2 Inhibitors Targeting Angiogenesis.

    Science.gov (United States)

    Ehlers, Tedman; Furness, Scott; Robinson, Thomas Philip; Zhong, Haizhen A; Goldsmith, David; Aribser, Jack; Bowen, J Phillip

    2016-01-01

    Angiogenesis has been identified as a crucial process in the development and spread of cancers. There are many regulators of angiogenesis which are not yet fully understood. Methionine aminiopeptidase is a metalloenzyme with two structurally distinct forms in humans, Type-1 (MetAP-1) and Type-2 (MetAP-2). It has been shown that small molecule inhibitors of MetAP-2 suppress endothelial cell proliferation. The initial discovery by Donald Ingber of MetAP-2 inhibition as a potential target in angiogenesis began with a fortuitous observation similar to the discovery of penicillin activity by Sir Alexander Fleming. From a drug design perspective, MetAP-2 is an attractive target. Fumagillin and ovalicin, known natural products, bind with IC50 values in low nanomolar concentrations. Crystal structures of the bound complexes provide 3-dimensional coordinates for advanced computational studies. More recent discoveries have shown other biological activities for MetAP-2 inhibition, which has generated new interests in the design of novel inhibitors. Semisynthetic fumagillin derivatives such as AGM-1470 (TNP-470) have been shown to have better drug properties, but have not been very successful in clinical trials. The rationale and development of novel multicyclic analogs of fumagillin are reviewed. PMID:26369821

  12. 尿胞外体亮氨酸氨基肽酶及二肽基肽酶在糖尿病肾病中的变化%Changes of urinary exosomal leucine aminopeptidase and dipeptidyl peptidase 4 in diabetic nephropathy

    Institute of Scientific and Technical Information of China (English)

    孙爱丽; 胡晓燕; 关广聚; 邓浩萍; 刘元涛; 陈诗鸿; 刘军莉; 邓景惕

    2011-01-01

    AIM: To investigate the changes of urinary exosomal enzymes and the correlation with diabetic nephropathy.METHODS: Thirty - four healthy volunteers and 127 patients of type 2 diabetes mellitus ( T2DM ) were included in the study.The healthy volunteers served as control.The patients with T2DM were divided into 3 groups based on their 24 h urinary albumin/creatinine ratio ( UACR ): 50 patients with microalbuminuria in early DN group ( DN1 ), 34 patients with macroalbuminuria in overt DN group ( DN2 ) and 43 patients without albuminuria in DM group.The levels of urine exosomal leucine aminopeptidase( exosome - LAP ) and exosomal dipeptidyl peptidase 4( exosome - DPP4 ) were determined by enzyme - linked immunosorbent assay ( ELISA ).The following methods were used to determine the biochemical parameters: liquid chromatography for glycated hemoglobin ( HbA1c ), chemical modification method for cholesterol ( CH ), Jaffe - kinetic assay for creatinine ( CR ) and urease - GLDH method for blood urea nitrogen ( BUN ).Multiple stepwise linear regressions were used to analyze the relationship of exosome - LAP or exosome - DPP4 with HbA1c, CH, UACR, CR and BUN.RESULTS: The levels of exosome - LAP and exosome - DPP4 in DM, DN1 and DN2 groups were significantly higher than those in control group ( P <0.01 ).The exosome - LAP in DN2 group was significantly higher than that in DM group.Correlation analysis showed that the levels of urinary exosome - LAP and exosome - DPP4 were positively correlated with HbA1c, CH, UACR, CR and BUN.Multiple stepwise linear regression analysis showed that CH and UACR were independent determinants for exosome - LAP ( P <0.01 ), and UACR and HbAlc were independent determinants for exosome - DPP4 ( P <0.01 ).CONCLUSION: Urine exosome - LAP and exosome - DPP4 are correlated with the severity of diabetic nephropathy.These parameters may serve as clinical markers for the diagnosis and prognosis evaluation of diabetic nephropathy.%目的:探讨 2

  13. STRUCTURE OF A MICROSPORIDIAN METHIONINE AMINOPEPTIDASE TYPE 2 COMPLEXED WITH FUMAGILLIN AND TNP470

    OpenAIRE

    Alvarado, John Jeff; NEMKAL, ANJANA; Sauder, J. Michael; RUSSELL, MARIJANE; Akiyoshi, Donna E.; Shi, Wuxian; Almo, Steven C.; Weiss, Louis M.

    2009-01-01

    Microsporidia are protists that have been reported to cause infections in both vertebrates and invertebrates. They have emerged as human pathogens particularly in patients that are immunnosuppressed and cases of gastrointestinal infection, encephalitis, keratitis, sinusitis, myositis and disseminated infection are well described in the literature. While benzimidazoles are active against many species of Microsporidia, these drugs do not have significant activity against Enterocytozoon bieneusi...

  14. Association between endoplasmic reticulum aminopeptidase-1 (ERAP-1 and susceptibility to ankylosing spondylitis in Iran.

    Directory of Open Access Journals (Sweden)

    Mahdi Mahmoudi

    2012-12-01

    Full Text Available Ankylosing Spondylitis (AS is an inflammatory arthritis, which affects mainly spine and sacroiliac joints. According to recent studies, ERAP1 is the second most common candidate gene for  AS susceptibility after HLA-B27. The  aim of this study was to  determine the association of ERAP1 gene polymorphisms with AS in Iranian population.The study group comprised 387 Iranian AS patients and 316 healthy controls from Iran.Using Real Time PCR allelic discrimination method,  we genotyped four SNPs (rs30187, rs469876, rs13167972 and rs27434 of ERAP1.We found  that  rs30187 and rs27434 were significantly associated with AS in Iranian population (P=6×10-5, P=7×10-3,  respectively. The rs30187 T/T genotype was associated with AS compared  with C/C  genotype (P=1.5×10-5.  The  rs27434 G/G genotype was inversely associated with  AS (P=5×10-3.  Two  specific haplotypes  including:  rs30187/ rs469876/ rs13167972/ rs27434 TAAA and CAGG  were associated with increased and decreased risk of AS in Iranian population, respectively.These results indicated that ERAP1 SNPs and haplotypes were associated with AS in Iranian population.

  15. 21 CFR 184.1985 - Aminopeptidase enzyme preparation derived from lactococcus lactis.

    Science.gov (United States)

    2010-04-01

    ...) and other peptidases that hydrolyze milk proteins. The preparation is produced by pure culture... HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) DIRECT FOOD SUBSTANCES...) and 1 CFR part 51. Copies are available from the National Academy Press, 2101 Constitution Ave....

  16. Organelles involved in the intracellular transport of newly synthesized aminopeptidase N and their acidity

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Danielsen, E M; Sjöström, H;

    1989-01-01

    in the microvillar membrane, the Golgi complex, apical small smooth vesicles, and various acidic lysosomal/endosomal-like organelles. By culturing mucosal explants in the presence of either cycloheximide or (3-(2,4-dinitroanilino)-3-amino-N-methylpropylamine) (DAMP) it was demonstrated that the apical small smooth...

  17. Correlation Between the Clinical Diagnosis of Bacterial Vaginosis and the Results of a Proline Aminopeptidase Assay

    Directory of Open Access Journals (Sweden)

    George H. Nelson

    1994-01-01

    Full Text Available Objective: The object of this study was to develop a simple and inexpensive test for detection of bacterial vaginosis (BV in pregnant patients and to test its accuracy in a clinic population.

  18. The standalone aminopeptidase PepN catalyzes the maturation of blasticidin S from leucylblasticidin S.

    Science.gov (United States)

    Yu, Guiyang; Li, Li; Liu, Xiangyang; Liu, Guang; Deng, Zixin; Zabriskie, Mark T; Jiang, Ming; He, Xinyi

    2015-01-01

    The peptidyl nucleoside blasticidin S (BS) isolated from Streptomyces griseochromogenes was the first non-mercurial fungicide used on a large scale to prevent rice blast. In the biosynthesis of BS, leucylblasticidin S (LBS) was suggested as the penultimate metabolite with 20-fold less inhibitory activity than the final product BS. Incomplete conversion of LBS to BS at a variable efficiency ranging from 10% to 90% was observed either in the native strain S. griseochromogenes or a heterologous producer Streptomyces lividans WJ2. In this study, we determined that maturation of BS from LBS is not a spontaneous process but is governed by a standalone peptidase PepN, which hydrolyzes LBS in a pH-sensitive way with most appropriate of pH 7~8 but is inactive when the pH is below 5 or above 10. PepN1 and PepN2, two neighboring PepN homologs from Streptomyces lividans were purified in E. coli but displayed ca.100-fold difference in LBS hydrolytic activity. Overexpression of pepN1 in WJ2 enhanced BS yield by 100% and lowered the ratio of LBS to BS from 2:1 to 2:3. This work presents the expansion of the biological role for PepN in antibiotic maturation and the first report of hydrolysis of beta amide linkage by this conserved enzyme. PMID:26621790

  19. Structural Basis For Antigenic Peptide Precursor Processing by the Endoplasmic Reticulum Aminopeptidase ERAP1

    OpenAIRE

    Nguyen, Tina T.; Chang, Shih-Chung; Evnouchidou, Irini; Ian A York; Zikos, Christos; Rock, Kenneth L.; Goldberg, Alfred L.; Stratikos, Efstratios; Stern, Lawrence J.

    2011-01-01

    ERAP1 trims antigen precursors to fit into MHC class I proteins. To perform this function, ERAP1 has unique substrate preferences, trimming long peptides while sparing shorter ones. To identify the structural basis for ERAP1's unusual properties, we determined the X-ray crystal structure of human ERAP1 bound to bestatin. The structure reveals an open conformation with a large interior compartment. An extended groove originating from the enzyme's catalytic center can accommodate long peptides ...

  20. Association between endoplasmic reticulum aminopeptidase-1 (ERAP-1) and susceptibility to ankylosing spondylitis in Iran.

    OpenAIRE

    Mahdi Mahmoudi; Ahmad Reza Jamshidi; Ali Akbar Amirzargar; Elham Farhadi; Keramat Nourijelyani; Sasan Fallahi; Mona Oraei; Sahar Noori; Mohammad Hossein Nicknam

    2012-01-01

    Ankylosing Spondylitis (AS) is an inflammatory arthritis, which affects mainly spine and sacroiliac joints. According to recent studies, ERAP1 is the second most common candidate gene for  AS susceptibility after HLA-B27. The  aim of this study was to  determine the association of ERAP1 gene polymorphisms with AS in Iranian population.The study group comprised 387 Iranian AS patients and 316 healthy controls from Iran.Using Real Time PCR allelic discrimination method,  we genotyped four SNPs ...

  1. p53 increases MHC class I expression by upregulating the endoplasmic reticulum aminopeptidase ERAP1

    OpenAIRE

    Wang, Bei; Niu, Dandan; Lai, Liyun; Ren, Ee Chee

    2013-01-01

    The p53 tumour suppressor has an important role in cancer cells. Here we show that p53 regulates expression of major histocompatibility complex I on the cell surface. We show that the tumour cell line HCT116, which lacks p53 exhibits significantly lower major histocompatibility complex I expression than its wild-type counterpart. Using a combination of chromatin immunoprecipitation sequencing and gene expression analysis, we demonstrate that p53 upregulates expression of endoplasmic reticulum...

  2. Inhibition of the methionine aminopeptidase 2 enzyme for the treatment of obesity

    OpenAIRE

    Joharapurkar AA; Dhanesha NA; Jain

    2014-01-01

    Amit A Joharapurkar, Nirav A Dhanesha, Mukul R Jain Department of Pharmacology and Toxicology, Zydus Research Centre, Cadila Healthcare Limited, Ahmedabad, India Abstract: Worldwide prevalence of obesity has nearly doubled since 1980. Obesity is the result of interactions among the environmental factors, genetic predisposition, and human behavior. Even modest weight reduction in obese patients provides beneficial health outcomes. For effective weight reduction, a drug should either increase ...

  3. Porcine aminopeptidase N mediated polarized infection by porcine epidemic diarrhea virus in target cells

    International Nuclear Information System (INIS)

    Infection of polarized intestinal epithelial cells by porcine epidemic diarrhea virus (PEDV) was characterized. Indirect immunofluorescence assay, real-time PCR, and transmission electron microscopy confirmed PEDV can be successfully propagated in immortalized swine small intestine epithelial cells (IECs). Infection involved porcine aminpeptidase N (pAPN), a reported cellular receptor for PEDV, transient expression of pAPN and siRNA targeted pAPN increased and decreased the infectivity of PEDV in IECs, respectively. Subsequently, polarized entry into and release from both Vero E6 and IECs was analyzed. PEDV entry into polarized cells and pAPN grown on membrane inserts occurs via apical membrane. The progeny virus released into the medium was also quantified which demonstrated that PEDV is preferentially released from the apical membrane. Collectively, our data demonstrate that pAPN, the cellular receptor for PEDV, mediates polarized PEDV infection. These results imply the possibility that PEDV infection may proceed by lateral spread of virus in intestinal epithelial cells. - Highlights: • PEDV infection of polarized intestinal epithelial cells (IECs) was characterized. • Porcine aminpeptidase N (pAPN) facilitated PEDV infection in IECs. • PEDV entry into and release from polarized cell via its apical membrane. • PEDV infection may proceed by lateral spread of virus in IECs

  4. Alkaline phosphatases and aminopeptidases are altered in a Cry11Aa resistant strain of Aedes aegypti.

    Science.gov (United States)

    Lee, Su-Bum; Aimanova, Karlygash G; Gill, Sarjeet S

    2014-11-01

    Bacillus thuringiensis subsp. israelensis (Bti) is widely used for the biological control of mosquito populations. However, the mechanism of Bti toxins is still not fully understood. To further elucidate the mechanism of Bti toxins, we developed an Aedes aegypti resistant strain that shows high-level resistance to Cry11Aa toxin. After 27 selections with Cry11Aa toxin, the larvae showed a 124-fold resistance ratio for Cry11Aa (strain G30). G30 larvae showed cross-resistance to Cry4Aa (66-fold resistance), less to Cry4Ba (13-fold), but not to Cry11Ba (2-fold). Midguts from these resistant larvae did not show detectable difference in the processing of the Cry11Aa toxin compared to that in susceptible larvae (WT). Brush border membrane vesicles (BBMV) from resistant larvae bound slightly less Cry11Aa compared to WT BBMV. To identify potential proteins associated with Cry11A resistance, not only transcript changes in the larval midgut were analyzed using Illumina sequencing and qPCR, but alterations of previously identified receptor proteins were investigated using immunoblots. The transcripts of 375 genes were significantly increased and those of 208 genes were down regulated in the resistant larvae midgut compared to the WT. None of the transcripts for previously identified receptors of Cry11Aa (Aedes cadherin, ALP1, APN1, and APN2) were altered in these analyses. The genes for the identified functional receptors in resistant larvae midgut did not contain any mutation in their sequences nor was there any change in their transcript expression levels compared to WT. However, ALP proteins were expressed at reduced levels (∼ 40%) in the resistant strain BBMV. APN proteins and their activity were also slightly reduced in resistance strain. The transcript levels of ALPs (AAEL013330 and AAEL015070) and APNs (AAEL008158, AAEL008162) were significantly reduced. These results strongly suggest that ALPs and APNs could be associated with Cry11Aa resistance in Ae. aegypti. PMID:25242559

  5. MOLECULAR CLONING AND ANALYSIS OF THE CRYPTOSPORIDIUM PARVUM AMINOPEPTIDASE N GENE. (R829180)

    Science.gov (United States)

    Cryptosporidium parvum proteases have been associated with release of infective sporozoites from oocysts, and their specific inhibition blocks parasite excystation in vitro. Additionally, proteases have been implicated in the processing of parasite adhesion molecules fo...

  6. MOLECULAR CLONING AND ANALYSIS OF THE CRYPTOSPORIDIUM PARVUM AMINOPEPTIDASE N GENE. (R828035)

    Science.gov (United States)

    Cryptosporidium parvum proteases have been associated with release of infective sporozoites from oocysts, and their specific inhibition blocks parasite excystation in vitro. Additionally, proteases have been implicated in the processing of parasite adhesion molecules fo...

  7. Porcine aminopeptidase N mediated polarized infection by porcine epidemic diarrhea virus in target cells

    Energy Technology Data Exchange (ETDEWEB)

    Cong, Yingying; Li, Xiaoxue; Bai, Yunyun [College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030 (China); Lv, Xiaonan [College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030 (China); CAS Key Lab for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience & Technology of China, Beijing 100090 (China); Herrler, Georg [Institute for Virology, University of Veterinary Medicine, Hannover D-30559 (Germany); Enjuanes, Luis [Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid (Spain); Zhou, Xingdong [College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030 (China); Qu, Bo [Faculty of Life Sciences, Northeast Agricultural University, Harbin 150030 (China); Meng, Fandan [Institute for Virology, University of Veterinary Medicine, Hannover D-30559 (Germany); Cong, Chengcheng [College Animal Husbandry and Veterinary Medicine, Shenyang Agricultural University, Shenyang 110161 (China); Ren, Xiaofeng; Li, Guangxing [College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030 (China)

    2015-04-15

    Infection of polarized intestinal epithelial cells by porcine epidemic diarrhea virus (PEDV) was characterized. Indirect immunofluorescence assay, real-time PCR, and transmission electron microscopy confirmed PEDV can be successfully propagated in immortalized swine small intestine epithelial cells (IECs). Infection involved porcine aminpeptidase N (pAPN), a reported cellular receptor for PEDV, transient expression of pAPN and siRNA targeted pAPN increased and decreased the infectivity of PEDV in IECs, respectively. Subsequently, polarized entry into and release from both Vero E6 and IECs was analyzed. PEDV entry into polarized cells and pAPN grown on membrane inserts occurs via apical membrane. The progeny virus released into the medium was also quantified which demonstrated that PEDV is preferentially released from the apical membrane. Collectively, our data demonstrate that pAPN, the cellular receptor for PEDV, mediates polarized PEDV infection. These results imply the possibility that PEDV infection may proceed by lateral spread of virus in intestinal epithelial cells. - Highlights: • PEDV infection of polarized intestinal epithelial cells (IECs) was characterized. • Porcine aminpeptidase N (pAPN) facilitated PEDV infection in IECs. • PEDV entry into and release from polarized cell via its apical membrane. • PEDV infection may proceed by lateral spread of virus in IECs.

  8. Expression and Purification of Active Recombinant Cathepsin C (Dipeptidyl Aminopeptidase I of Kuruma Prawn Marsupenaeus japonicus in Insect Cells

    Directory of Open Access Journals (Sweden)

    Gao-Feng Qiu

    2009-01-01

    Full Text Available Cathepsin C (CTSC is a lysosomal cysteine protease belonging to the papain superfamily. Our previous study showed that CTSC precursor (zymogen is localized exclusively in cortical rods (CRs of mature oocyte in the kuruma prawn Marsupenaeus japonicus, suggesting that CTSC might have roles on regulating release and/or formation of a jelly layer. In this study, enzymically active CTSC of the kuruma prawn was prepared by recombinant expression in the High Five insect cell line. The recombinant enzyme with a polyhistidine tag at its C-terminus was considered to be initially secreted into the culture medium as an inactive form of zymogen, because Western blot with anti-CTSC antibody detected a 51 kDa protein corresponding to CTSC precursor. After purification by affinity chromatography on nickel-iminodiacetic acid resin, the enzyme displayed three forms of 51, 31, and 30 kDa polypeptides. All of the forms can be recognized by antiserum raised against C-terminal polyhistidine tag, indicating that the 31 and 30 kDa forms were generated from 51 kDa polypeptide by removal of a portion of the N-terminus of propeptide. Following activation at pH 5.5 and 37∘C for 40 hours under native conditions, the recombinant CTSC (rCTSC exhibited increased activity against the synthetic substrate Gly-Phe-β-naphthylamide and optimal pH at around 5. The purified rCTSC will be useful for further characterization of its exact physiological role on CRs release and/or formation of a jelly layer in kuruma prawn.

  9. Autoimmune disease-associated variants of extracellular endoplasmic reticulum aminopeptidase 1 induce altered innate immune responses by human immune cells

    OpenAIRE

    Aldhamen, Yasser A; Pepelyayeva, Yuliya; Rastall, David P. W.; Seregin, Sergey S.; Zervoudi, Efthalia; Koumantou, Despoina; Charles F Aylsworth; Quiroga, Dionisia; Godbehere, Sarah; Georgiadis, Dimitris; Stratikos, Efstratios; Amalfitano, Andrea

    2015-01-01

    ERAP1 gene polymorphisms have been linked to several autoimmune diseases; however, the molecular mechanisms underlying these associations are not well understood. Recently, we have demonstrated that ERAP1 regulates key aspects of the innate immune response. Moreover, previous studies show ERAP1 to be ER-localized and secreted during inflammation. Herein, we investigate the possible roles that ERAP1 polymorphic variants may have in modulating innate immune responses of human PBMCs using two ex...

  10. Functional Interaction of the Ankylosing Spondylitis-associated Endoplasmic Reticulum Aminopeptidase 1 Polymorphism and HLA-B27 in Vivo*

    OpenAIRE

    García-Medel, Noel; Sanz-Bravo, Alejandro; Nguyen, Dung; Galocha, Begoña; Gómez-Molina, Patricia; Martín-Esteban, Adrián; Alvarez-Navarro, Carlos; de Castro, José A. López

    2012-01-01

    The association of ERAP1 with ankylosing spondylitis (AS)1 among HLA-B27-positive individuals suggests that ERAP1 polymorphism may affect pathogenesis by altering peptide-dependent features of the HLA-B27 molecule. Comparisons of HLA-B*27:04-bound peptidomes from cells expressing different natural variants of ERAP1 revealed significant differences in the size, length, and amount of many ligands, as well as in HLA-B27 stability. Peptide analyses suggested that the mechanism of ERAP1/HLA-B27 in...

  11. Efficient inhibition of tumor angiogenesis and growth by a synthetic peptide blocking S100A4-methionine aminopeptidase 2 interaction

    OpenAIRE

    Ochiya, Takahiro; Takenaga, Keizo; Asagiri, Masataka; Nakano, Kazumi; Satoh, Hitoshi; Watanabe, Toshiki; Imajoh-Ohmi, Shinobu; Endo, Hideya

    2015-01-01

    The prometastatic calcium-binding protein, S100A4, is expressed in endothelial cells, and its downregulation markedly suppresses tumor angiogenesis in a xenograft cancer model. Given that endothelial S100A4 can be a molecular target for inhibiting tumor angiogenesis, we addressed here whether synthetic peptide capable of blocking S100A4-effector protein interaction could be a novel antiangiogenic agent. To examine this hypothesis, we focused on the S100A4-binding domain of methionine aminopep...

  12. Yeast Interacting Proteins Database: YHR113W, YHR113W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YHR113W - Cytoplasmic aspartyl aminopeptidase; cleaves unblocked N-terminal acidic ...YHR113W - Cytoplasmic aspartyl aminopeptidase; cleaves unblocked N-terminal acidi...ame - Bait description Cytoplasmic aspartyl aminopeptidase; cleaves unblocked N-terminal acidic amino acid r...y description Cytoplasmic aspartyl aminopeptidase; cleaves unblocked N-terminal acidic amino acid residues f

  13. Hypothalamus-pituitary-thyroid axis disruption in rats with breast cancer is related to an altered endogenous oxytocin/insulin-regulated aminopeptidase (IRAP) system.

    Science.gov (United States)

    Carrera-González, María Pilar; Ramírez-Expósito, María Jesús; de Saavedra, Jose Manuel Arias; Sánchez-Agesta, Rafael; Mayas, María Dolores; Martínez-Martos, Jose Manuel

    2011-06-01

    Associations of breast cancer with diseases of the thyroid have been repeatedly reported, but the mechanism underlying this association remains to be elucidated. It has been reported that oxytocin (OXT) attenuates the thyroid-stimulating hormone (TSH) release in response to thyrotrophin-releasing hormone (TRH) and decreased plasma levels of TSH as well as the thyroid hormones by an effect mediated by the central nervous system. Oxytocinase (IRAP) is the regulatory proteolytic enzyme reported to hydrolyze OXT. Changes in IRAP activity have been reported in both human breast cancer and N-methyl-nitrosourea (NMU)-induced rat mammary tumours. Here, we measure IRAP activity fluorometrically using cystyl-β-naphthylamide as the substrate, in the hypothalamus-pituitary-thyroid axis together with the circulating levels of OXT, and its relationship with circulating levels of TSH and free thyroxine (fT4), as markers of thyroid function in control rats and rats with breast cancer induced by NMU. We found decreased thyroid function in rats with breast cancer induced by NMU, supported by the existence of lower serum circulating levels of both TSH and fT4 than their corresponding controls. Concomitantly, we found a decrease of hypothalamic IRAP activity and an increase in circulating levels of OXT. We propose that breast cancer increases OXT pituitary release by decreasing its hypothalamic catabolism through IRAP activity, probably due to the alteration of the estrogenic endocrine status. Thus, high circulating levels of OXT decreased TSH release from the pituitary, and therefore, of thyroid hormones from the thyroid, supporting the association between breast cancer and thyroid function disruption.

  14. Atg21 is a phosphoinositide binding protein required for efficient lipidation and localization of Atg8 during uptake of aminopeptidase I by selective autophagy

    NARCIS (Netherlands)

    Strømhaug, Per E; Reggiori, Fulvio; Guan, Ju; Wang, Chao-Wen; Klionsky, Daniel J; Reggiori, Fulvio

    2004-01-01

    Delivery of proteins and organelles to the vacuole by autophagy and the cytoplasm to vacuole targeting (Cvt) pathway involves novel rearrangements of membrane resulting in the formation of vesicles that fuse with the vacuole. The mechanism of vesicle formation and the origin of the membrane are comp

  15. Investigations and design of pyridine-2-carboxylic acid thiazol-2-ylamide analogs as methionine aminopeptidase inhibitors using 3D-QSAR and molecular docking

    DEFF Research Database (Denmark)

    Hauser, Alexander Sebastian

    2014-01-01

    -dimensional quantitative structure–activity relationship (3D-QSAR) studies were carried out on a series of pyridine-2-carboxylic acid thiazol-2-ylamide-based MetAP inhibitors using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) techniques. The models were...... complexes, four new pyridine-2-carboxylic acid thiazol-2-ylamide analogs were designed. These analogs exhibit significantly better predicted activity than the reported molecules. The present work has implications for the development of novel antibiotics as potent MetAP inhibitors....

  16. Domain III of the Bacillus thuringiensis delta-endotoxin Cry1Ac is involved in binding to Manduca sexta brush border membranes and to its purified aminopeptidase N

    NARCIS (Netherlands)

    Maagd, de R.A.; Bakker, P.L.; Masson, L.; Adang, M.J.; Sangadala, S.; Stiekema, W.; Bosch, D.

    1999-01-01

    Three types of binding assays were used to study the binding of Bacillus thuringiensis delta-endotoxin Cry1Ac to brush border membrane vesicle (BBMV) membranes and a purified putative receptor of the target insect Manduca sexta. Using hybrid proteins consisting of Cry1Ac and the related Cry1C protei

  17. Proteolyse in Synechocystis sp. PCC 6803 : Funktion der Methionin-Aminopeptidase 2 und FtsH2-Protease für die Photosystem II-Stressresistenz

    OpenAIRE

    Drath, Miriam

    2008-01-01

    Methionin-Aminopeptidasen sind essentielle und haben physiologisch wichtige Bedeutung in der post- und co-translationellen regulation von Proteinen in Pro- und Eukaryonten. Zu Beginn dieser Dissertation war bekannt, dass die Methionin-Aminopeptidasen (MetAP) 1-3 in Synechocystis sp. PCC 6803 funktionale Aminopeptidasen sind, allerdings waren die physiologischen Funktionen unbekannt. Die Analyse der MetAP2-defizienten Mutante in Synechocystis sp. PCC 6803 stellte ein Ziel dieser Arbeit dar un...

  18. Peptide handling by HLA-B27 subtypes influences their biological behavior, association with ankylosing spondylitis and susceptibility to endoplasmic reticulum aminopeptidase 1 (ERAP1).

    Science.gov (United States)

    García-Medel, Noel; Sanz-Bravo, Alejandro; Alvarez-Navarro, Carlos; Gómez-Molina, Patricia; Barnea, Eilon; Marcilla, Miguel; Admon, Arie; de Castro, José A López

    2014-12-01

    HLA-B27 is strongly associated with ankylosing spondylitis (AS). We analyzed the relationship between structure, peptide specificity, folding, and stability of the seven major HLA-B27 subtypes to determine the role of their constitutive peptidomes in the pathogenicity of this molecule. Identification of large numbers of ligands allowed us to define the differences among subtype-bound peptidomes and to elucidate the peptide features associated with AS and molecular stability. The peptides identified only in AS-associated or high thermostability subtypes with identical A and B pockets were longer and had bulkier and more diverse C-terminal residues than those found only among non-AS-associated/lower-thermostability subtypes. Peptides sequenced from all AS-associated subtypes and not from non-AS-associated ones, thus strictly correlating with disease, were very rare. Residue 116 was critical in determining peptide binding, thermodynamic properties, and folding, thus emerging as a key feature that unified HLA-B27 biology. HLA-B27 ligands were better suited to TAP transport than their N-terminal precursors, and AS-associated subtype ligands were better than those from non-AS-associated subtypes, suggesting a particular capacity of AS-associated subtypes to bind epitopes directly produced in the cytosol. Peptides identified only from AS-associated/high-thermostability subtypes showed a higher frequency of ERAP1-resistant N-terminal residues than ligands found only in non-AS-associated/low-thermostability subtypes, reflecting a more pronounced effect of ERAP1 on the former group. Our results reveal the basis for the relationship between peptide specificity and other features of HLA-B27, provide a unified view of HLA-B27 biology and pathogenicity, and suggest a larger influence of ERAP1 polymorphism on AS-associated than non-AS-associated subtypes. PMID:25187574

  19. Peptide Handling by HLA-B27 Subtypes Influences Their Biological Behavior, Association with Ankylosing Spondylitis and Susceptibility to Endoplasmic Reticulum Aminopeptidase 1 (ERAP1)*

    OpenAIRE

    García-Medel, Noel; Sanz-Bravo, Alejandro; Alvarez-Navarro, Carlos; Gómez-Molina, Patricia; Barnea, Eilon; Marcilla, Miguel; Admon, Arie; de Castro, José A. López

    2014-01-01

    HLA-B27 is strongly associated with ankylosing spondylitis (AS). We analyzed the relationship between structure, peptide specificity, folding, and stability of the seven major HLA-B27 subtypes to determine the role of their constitutive peptidomes in the pathogenicity of this molecule. Identification of large numbers of ligands allowed us to define the differences among subtype-bound peptidomes and to elucidate the peptide features associated with AS and molecular stability. The peptides iden...

  20. Isolierung und Charakterisierung der ER-residenten Aminopeptidase ERMP1 und Untersuchung ihrer Funktion in der Prozessierung MHC I restringierter CTL Epitope

    OpenAIRE

    Akkad, Nadja

    2011-01-01

    Die effiziente Generierung von Peptid-Epitopen aus zelleigenen oder viralen Proteinen für die Präsentation auf „Major Histocompatibility Complex I“ (MHC I) Molekülen ist essentiell für die Aktivierung des adaptiven Immunsystems und die Effektorfunktion der CD8+ zytotoxischen T-Zellen (CTLs). CTLs erkennen diese Peptide in Kontext mit MHC I Molekülen über ihren spezifischen T-Zellrezeptor (TCR). Die Generierung dieser Epitope ist das Resultat eines komplexen proteolytischen Prozesses, der im Z...

  1. Molecular cloning of a preprohormone from sea anemones containing numerous copies of a metamorphosis-inducing neuropeptide: a likely role for dipeptidyl aminopeptidase in neuropeptide precursor processing

    DEFF Research Database (Denmark)

    Leviev, I; Grimmelikhuijzen, C J

    1995-01-01

    Neuropeptides are an important group of hormones mediating or modulating neuronal communication. Neuropeptides are especially abundant in evolutionarily "old" nervous systems, such as those of cnidarians, the lowest animal group having a nervous system. Cnidarians often have a life cycle including...... a polyp, a medusa, and a planula larva stage. Recently, a neuropeptide, ... the precursor protein for this metamorphosis-inducing neuropeptide from sea anemones. The precursor protein is 514-amino acid residues long and contains 10 copies of the immature, authentic neuropeptide (Gln-Gln-Pro-Gly-Leu-Trp-Gly). All neuropeptide copies are preceded by Xaa-Pro or Xaa-Ala sequences...

  2. Syntheses,Characterization and Equilibrium between Mono—and Aqua—bridged Dicobalt(Ⅱ)Complexes.A Structural Model for Methionine Aminopeptidase

    Institute of Scientific and Technical Information of China (English)

    YE,Bao-Hui; CHEN,Xiao-Ming

    2003-01-01

    A monomeric complex[Co(Im)2(O2CMe)2](1) and a novel aquabridged dimeric complex [Co2(μ-H2O)(μ-O2CMe)2(Im)4-(O2CMe)2](2) (Im=imidazole) have been synthesized and characterized.Complexes 1 and 2 coexisted in solution.Pure forms of either complex can be obtained from the same solution by controlling the crystallization conditions.All two complexes possess a carboxylate-Im-cobalt(Ⅱ) triad system analogous to the carboxylate-histidine-metal triad systems that have been found in many zinc enzymes and coblat(Ⅱ)-substituted enzymes.In2,two Co2+ ions are conected by a wate nolecule in a bridging fasion with Co……Co[0.3687(1)nm],Co-OH2[0.2159(3)nm],and Co-OH2-Co[0.3687(1)nm],Co-OH2[0.2159(3)nm],and Co-OH2-Co[117.2(3)°],in which the water molecule is further stabilized by two intramolecular hydrogen bonds with the oxygens of the terminal monodentate acetate groups with the distance of 0……0[0.2609(7)nm].The terminal monodentate acetate groups display quite abnormal geometry due to the strong "pulling effect"on the carboxylates by intermolecular and intramolecular hydrogen bonds.Complex 2 showed weak antiferromagnetic coupling at low temperature with g=2.22 and J=-1.60 cm-1.

  3. A Comparative Study of Recombinant Expression of Three Aminopeptidases in Escherichia coli%3种氨肽酶基因在大肠杆菌中的重组表达与比较研究

    Institute of Scientific and Technical Information of China (English)

    张洁; 张梁; 黄武宁; 石贵阳

    2013-01-01

    PCR扩增Bacillus licheniformis 14580、Bacillus subtilis 168、Geobacillus stearothermophilus IFO12589氨肽酶基因,分别酶切连接表达质粒pET-28a,构建重组表达载体pET28a-BLAP、pET28a-BSAP、pET28a-GSAP,酶活力检测表明3个氨肽酶基因均在大肠杆菌宿主BL21(DE3)中获得重组表达.进一步对3株重组菌的氨肽酶粗酶液反应条件进行比较研究,结果表明:重组氨肽酶BSAP与GSAP的粗酶活较高,达到1500U/L以上;BLAP、BSAP、GSAP粗酶的最适酶反应温度分别为50、75、60℃,BSAP温度稳定性最好,在30~70℃时比较稳定;3种重组氨肽酶的最适pH值都是9.0,pH值在8.5~9.0时比较稳定;0.1mmol/L Co2+对酶活有较强的激活作用,BSAP的相对酶活力最高达到195.6%,其他二价金属离子对酶活均有不同程度的抑制,其中Zn2+抑制作用最大;重组质粒pET28a-BSAP、pET28a-GSAP在大肠杆菌中较pET28a-BLAP稳定.

  4. Human methionine aminopeptidase-2,a new target of anti-cancer drugs%新的抗癌药物作用靶点甲硫氨酰氨肽酶-2

    Institute of Scientific and Technical Information of China (English)

    鲁衍强; 叶其壮

    2002-01-01

    夫马洁林是目前已知的最有潜力的天然血管生成抑制剂之一.该类药物通过共价抑制甲硫氨酰氨肽酶-2的活性,而特异性地抑制血管内皮细胞的生长和增殖.根据甲硫氨酰氨肽酶-2的结构以及催化和抑制机制,可以设计和合成新型的血管生成抑制剂;通过最新发展的活性检测方法,可以建立高通量药物筛选模型,以期找到新一代高选择性、高亲和性、高生物利用度的甲硫氨酰氨肽酶-2的小分子抑制剂,用于抗癌新药的开发.

  5. New rapid identification test for Clostridium difficile.

    OpenAIRE

    Aspinall, S T; Dealler, S. F.

    1992-01-01

    AIMS: A set of five tests were developed and tested for their ability to confirm the identity of C difficile colonies within 30 minutes. METHODS: The relevant substrates were incorporated into four filter paper squares attached to a plastic carrier (Diffstrip), five enzymes/products (prolyl aminopeptidase, galactosidase, leucine aminopeptidase, acid phosphatase and indole). The strips were inoculated, incubated for 20 minutes, and reagents added. RESULTS: 96.4% (212 of 220) strains of C diffi...

  6. Yeast Interacting Proteins Database: YHR113W, YOL082W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available cargo proteins aminopeptidase I (Lap4p) and alpha-mannosidase (Ams1p) to the phagophore assembly site for packaging...vt) pathway; delivers cargo proteins aminopeptidase I (Lap4p) and alpha-mannosidase (Ams1p) to the phagophore assembly site for packa...ging into Cvt vesicles Rows with this prey as prey (6) Rows with this prey as bait

  7. The Specificity of Trimming of MHC Class I-Presented Peptides in the Endoplasmic Reticulum1

    OpenAIRE

    Hearn, Arron; Ian A York; Rock, Kenneth L.

    2009-01-01

    Aminopeptidases in the endoplasmic reticulum (ER) can cleave antigenic peptides and in so doing either create or destroy MHC class I-presented epitopes. However the specificity of this trimming process overall and of the major ER aminopeptidase ERAP1 in particular is not well understood. This issue is important because peptide trimming influences the magnitude and specificity of CD8 T cell responses. By systematically varying the N-terminal flanking sequences of peptides in a cell free bioche...

  8. The Internal Sequence of the Peptide-Substrate Determines Its N-Terminus Trimming by ERAP1

    OpenAIRE

    Evnouchidou, Irini; Momburg, Frank; Papakyriakou, Athanasios; Chroni, Angeliki; Leondiadis, Leondios; Chang, Shih-Chung; Stratikos, Efstratios; Goldberg, Alfred L.

    2008-01-01

    Background: Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims N-terminally extended antigenic peptide precursors down to mature antigenic peptides for presentation by major histocompatibility complex (MHC) class I molecules. ERAP1 has unique properties for an aminopeptidase being able to trim peptides in vitro based on their length and the nature of their C-termini. Methodology/Principal Findings: In an effort to better understand the molecular mechanism that ERAP1 uses to trim peptides, w...

  9. 血清GPDA、AFu活力联合检测对原发性肝癌的诊断价值%Evaluation of combined detection of glycyl proline dipeptidyl aminopeptidase and α-L-fucosidases in the diagnosis of primary hepatic carcinoma

    Institute of Scientific and Technical Information of China (English)

    宋书卫; 陈国军

    2001-01-01

    目的评价血清甘氨酰脯氨酸二肽氨基肽酶(GPDA)、α-L-岩藻糖苷酶(AFu)联合检测在原发性肝癌诊断中的临床意义.方法收集42例健康人血清和78例原发性肝癌(PHC)及150例非PHC患者的血清.分别测定GPDA、AFu、AFP含量,以GPDA≥136U/L、AFu≥172nkat/L(均以健康组x±3S)为PHC阳性预示值.AFP≥400ng/ml为PHC预示值.结果PHC患者血清GPDA(156.2±39.2),AFu(262.0±88.6)均显著高于健康对照组(P<0.001).对PHC的诊断阳性率GPDA达到64.1%,AFu达到75.6%,AFP仅为59%,若以其中二项或三项阳性作为诊断PHC,则阳性率为89.7%,特异性为91.9%.结论血清GPDA、AFu联合检测可明显提高PHC的阳性检出率.

  10. Optimization of aminopeptidase-producing condition for nitrite-resistant Staphylococcus saprophyticus RG-2 isolated from Rugao ham%如皋火腿中耐亚硝酸盐腐生葡萄球菌RG-2产氨肽酶条件的优化

    Institute of Scientific and Technical Information of China (English)

    汪淼; 王敏; 张培培; 吴雪燕; 葛庆丰; 吴满刚; 于海

    2013-01-01

    氨肽酶能从多肽链的N端顺序水解并释放氨基酸,可广泛用于肉制品加工以增加肉品游离氨基酸,提高发酵肉制品品质.实验利用从如皋火腿中分离得到的l株产氨肽酶腐生葡萄球RG-2,通过单因素试验和响应曲面设计研究了pH、温度、NaCl浓度等因素对菌株RG-2产氨肽酶能力的影响.实验结果表明,最佳产氨肽酶条件为pH6.8、温度41℃、NaCl浓度1%,此条件下氨肽酶酶活达到23.85 U/mg.

  11. Exogenous and endogenous protease inhibitors in the gut of the fall armyworm larvae, Spodoptera frugiperda.

    Science.gov (United States)

    Lwalaba, Digali; Weidlich, Sandy; Hoffmann, Klaus H; Woodring, Joseph

    2010-06-01

    A dose-dependent inhibition of endogenous trypsin and aminopeptidase occurs in the lumen of Spodoptera frugiperda after feeding L6 larvae exogenous inhibitors soybean trypsin inhibitor (SBTI), tosyl-L-lysine chloromethyl ketone-HCl (TLCK), or bestatin, respectively, for 3 days. TLCK inhibits trypsin in tissue extracts and in secretions more strongly than SBTI. The aminopeptidase released into the lumen (containing the peritrophic membrane) is strongly inhibited by bestatin, but the membrane-bound enzyme is not. A bound enzyme may be more resistant to an inhibitor than unbound. A cross-class elevation of aminopeptidase activity occurs in response to ingested trypsin inhibitor, but there was no cross-class effect of aminopeptidase inhibitor (bestatin) on trypsin activity. An endogenous trypsin and aminopeptidase inhibitor is present in the lumen and ventricular cells. The strength of the endogenous trypsin inhibition seems to be in the same range as that resulting from ingestion of the exogenous inhibitor SBTI. In some insect species, considerable trypsin secretion occurs in unfed as well as in fed animals, and endogenous protease inhibitors might function to protect the ventricular epithelium by inactivation of trypsin when less food is available. PMID:20513059

  12. The efficiency of human cytomegalovirus pp65(495-503) CD8+ T cell epitope generation is determined by the balanced activities of cytosolic and endoplasmic reticulum-resident peptidases.

    Science.gov (United States)

    Urban, Sabrina; Textoris-Taube, Kathrin; Reimann, Barbara; Janek, Katharina; Dannenberg, Tanja; Ebstein, Frédéric; Seifert, Christin; Zhao, Fang; Kessler, Jan H; Halenius, Anne; Henklein, Petra; Paschke, Julia; Cadel, Sandrine; Bernhard, Helga; Ossendorp, Ferry; Foulon, Thierry; Schadendorf, Dirk; Paschen, Annette; Seifert, Ulrike

    2012-07-15

    Control of human CMV (HCMV) infection depends on the cytotoxic activity of CD8(+) CTLs. The HCMV phosphoprotein (pp)65 is a major CTL target Ag and pp65(495-503) is an immunodominant CTL epitope in infected HLA-A*0201 individuals. As immunodominance is strongly determined by the surface abundance of the specific epitope, we asked for the components of the cellular Ag processing machinery determining the efficacy of pp65(495-503) generation, in particular, for the proteasome, cytosolic peptidases, and endoplasmic reticulum (ER)-resident peptidases. In vitro Ag processing experiments revealed that standard proteasomes and immunoproteasomes generate the minimal 9-mer peptide epitope as well as N-terminal elongated epitope precursors of different lengths. These peptides are largely degraded by the cytosolic peptidases leucine aminopeptidase and tripeptidyl peptidase II, as evidenced by increased pp65(495-503) epitope presentation after leucine aminopeptidase and tripeptidyl peptidase II knockdown. Additionally, with prolyl oligopeptidase and aminopeptidase B we identified two new Ag processing machinery components, which by destroying the pp65(495-503) epitope limit the availability of the specific peptide pool. In contrast to cytosolic peptidases, silencing of ER aminopeptidases 1 and 2 strongly impaired pp65(495-503)-specific T cell activation, indicating the importance of ER aminopeptidases in pp65(495-503) generation. Thus, cytosolic peptidases primarily interfere with the generation of the pp65(495-503) epitope, whereas ER-resident aminopeptidases enhance such generation. As a consequence, our experiments reveal that the combination of cytosolic and ER-resident peptidase activities strongly shape the pool of specific antigenic peptides and thus modulate MHC class I epitope presentation efficiency. PMID:22706083

  13. Regulation of Proteolytic Enzyme Activity in Lactococcus lactis

    OpenAIRE

    Meijer, W.; Marugg, J D; Hugenholtz, J

    1996-01-01

    Two different Lactococcus lactis host strains, L. lactis subsp. lactis MG1363 and L. lactis subsp. cremoris SK1128, both containing plasmid pNZ521, which encodes the extracellular serine proteinase (PrtP) from strain SK110, were used to study the medium and growth-rate-dependent activity of three different enzymes involved in the proteolytic system of lactococci. The activity levels of PrtP and both the intracellular aminopeptidase PepN and the X-prolyl-dipeptidyl aminopeptidase PepXP were st...

  14. Dimeric assembly of enterocyte brush border enzymes

    DEFF Research Database (Denmark)

    Danielsen, E M

    1994-01-01

    The noncovalent, dimeric assembly of small intestinal brush border enzymes was studied by sedimentation analysis in density gradients of extracts of pulse-labeled pig jejunal mucosal explants. Like aminopeptidase N (EC 3.4.11.2), sucrase-isomaltase (EC 3.2.1.48-10), aminopeptidase A (EC 3...... appearance of the liposome-reconstituted enzyme [Norén et al. (1986) J. Biol. Chem. 261, 12306-12309], showing only the inner, membrane-anchored domains of the monomers to be in close contact with one another while the outer domains are far apart. In contrast to the other brush border enzymes studied...

  15. Translational control of an intestinal microvillar enzyme

    DEFF Research Database (Denmark)

    Danielsen, E M; Cowell, G M; Sjöström, H;

    1986-01-01

    The rates of biosynthesis of adult and foetal pig small-intestinal aminopeptidase N (EC 3.4.11.2) were compared to determine at which level the expression of the microvillar enzyme is developmentally controlled. In organ-cultured explants, the rate of biosynthesis of foetal aminopeptidase N is only...... about 3% of the adult rate. The small amount synthesized occurs in a high-mannose-glycosylated, membrane-bound, form that is processed to the mature, complex-glycosylated, form at a markedly slower rate than that of the adult enzyme. Extracts of total RNA from adult and foetal intestine contained...

  16. TR146 cells grown on filters as a model of human buccal epithelium

    DEFF Research Database (Denmark)

    Mørck Nielsen, H; Rømer Rassing, M; Nielsen, Hanne Mørck

    2000-01-01

    The objective of the present study was to characterise the TR146 cell culture model as an in vitro model of human buccal mucosa with respect to the enzyme activity in the tissues. For this purpose, the contents of aminopeptidase, carboxypeptidase and esterase in homogenate supernatants of the TR146...... of the three enzymes in the TR146 homogenate supernatants was in the same range as the activity in homogenate supernatants of human buccal epithelium. In the TR146 cell culture model, the activity of aminopeptidase (13.70+/-2.10 nmol/min per mg protein) was approx. four times the activity of carboxypeptidase...

  17. Prognostic significance of aberrantly silenced ANPEP expression in prostate cancer

    DEFF Research Database (Denmark)

    Sørensen, Karina Dalsgaard; Abildgaard, Mette Opstrup; Haldrup, Christa;

    2013-01-01

    Background:Novel biomarkers for prostate cancer (PC) are urgently needed. This study investigates the expression, epigenetic regulation, and prognostic potential of ANPEP in PC.Methods:Aminopeptidase N (APN; encoded by ANPEP) expression was analysed by immunohistochemistry using tissue microarrays...

  18. Oxytocin biotransformation in the rat limbic brain: Characterization of peptidase activities and significance in the formation of oxytocin fragments

    NARCIS (Netherlands)

    Burbach, J.P.H.; Kloet, E.R. de; Wied, D. de

    1980-01-01

    The enzymatic conversion of oxytocin by brain peptidases has been studied. Oxytocin was incubated with synaptosomal plasma membranes (SPM) isolated from the rat brain. Qualitative studies using a microdansylation technique revealed two types of oxytocin converting peptidases, e.g. aminopeptidase and

  19. Sequence Classification: 550696 [

    Lifescience Database Archive (English)

    Full Text Available ine phosphatase isozyme conversion, aminopeptidase || http://www.ncbi.nlm.nih.gov/protein/30064110 ... ...Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|30064110|ref|NP_838281.1| alkal

  20. Sequence Classification: 295442 [

    Lifescience Database Archive (English)

    Full Text Available ine phosphatase isozyme conversion, aminopeptidase || http://www.ncbi.nlm.nih.gov/protein/15803270 ... ...Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|15803270|ref|NP_289302.1| alkal

  1. Sequence Classification: 554823 [

    Lifescience Database Archive (English)

    Full Text Available ine phosphatase isozyme conversion, aminopeptidase || http://www.ncbi.nlm.nih.gov/protein/24114048 ... ...Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|24114048|ref|NP_708558.1| alkal

  2. Sequence Classification: 559112 [

    Lifescience Database Archive (English)

    Full Text Available ine phosphatase isozyme conversion, aminopeptidase || http://www.ncbi.nlm.nih.gov/protein/74313319 ... ...Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|74313319|ref|YP_311738.1| alkal

  3. Sequence Classification: 290273 [

    Lifescience Database Archive (English)

    Full Text Available ine phosphatase isozyme conversion protein; aminopeptidase || http://www.ncbi.nlm.nih.gov/protein/16130660 ... ...Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|16130660|ref|NP_417233.1| alkal

  4. Vector analysis of ecoenzyme activities reveal constraints on coupled C, N and P dynamics

    Science.gov (United States)

    We developed a quantitative method for estimating resource allocation strategies of microbial communities based on the proportional activities of four, key extracellular enzymes, 1,4-ß-glucosidase (BG), leucine amino-peptidase (LAP), 1,4-ß-N-acetylglucosaminidase (NAG...

  5. Yeast Interacting Proteins Database: YPL091W, YOL082W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available aminopeptidase I (Lap4p) and alpha-mannosidase (Ams1p) to the phagophore assembly site for packaging into Cv...he phagophore assembly site for packaging into Cvt vesicles Rows with this prey as prey Rows with this prey

  6. Digestive enzymes in the gut and salivary gland of the larvae of Chilo auricilius Ddgn.

    Science.gov (United States)

    Somadder, K; Shrivastava, M

    1980-02-15

    Amylase, alpha- and beta-glucosidase, alpha- and beta-galactosidase, beta-fructosidase, trypsin, aminotripeptidase, leucine-aminopeptidase, prolinase, prolidase glycyl-L-leucine dipeptidase and glygylglycine dipeptidase are present in the 3rd instar larvae of Chilo auricilius. PMID:6989621

  7. Yeast Interacting Proteins Database: YKL103C, YOL082W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available he peptidase family M18; often used as a marker protein in studies of autophagy and cytosol to vacuole targe... as a marker protein in studies of autophagy and cytosol to vacuole targeting (CVT) pathway Rows with this b...on Vacuolar aminopeptidase yscI; zinc metalloproteinase that belongs to the peptidase family M18; often used

  8. 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.)

  9. Evidence for biosynthesis of lactase-phlorizin hydrolase as a single-chain high-molecular weight precursor

    DEFF Research Database (Denmark)

    Skovbjerg, H; Danielsen, E M; Noren, Ove;

    1984-01-01

    Precursor forms of lactase-phlorizin hydrolase, sucrase-isomaltase and aminopeptidase N were studied by pulse-labelling of organ-cultured human intestinal biopsies. After labelling the biopsies were fractionated by the Ca2+-precipitation method and the enzymes isolated by immunoprecipitation. The...

  10. Effect of dietary administration of probiotics on growth and intestine functionality of juvenile Senegalese sole (Solea senegalensis, Kaup 1858)

    NARCIS (Netherlands)

    Saenz de Rodriganez, M.A.; Diaz-Rosales, P.; Chabrillon, M.; Smidt, H.; Arijo, S.; Leon-Rubio, J.M.; Alarcon, F.J.; Balebona, M.C.; Morinigo, M.A.; Cara, J.B.; Moyano, F.J.

    2009-01-01

    The effects of the dietary administration of two bacterial probiotic strains (Ppd11 and Pdp13) from the Alteromonadaceae family for 60 days, were assessed by measuring growth and feed efficiency, activities of leucine aminopeptidase and alkaline phosphatase and structural changes in the intestine of

  11. Comparative expression of the mRNA for three intestinal hydrolases during postnatal development in the rat

    DEFF Research Database (Denmark)

    Freund, J N; Torp, N; Duluc, I;

    1990-01-01

    The distribution of the mRNA for intestinal aminopeptidase-N, lactase-phlorizin hydrolase and sucrase-isomaltase was compared during rat postnatal development as well as along the longitudinal axis of the intestinal tract including small-intestine and colon. We found out that each mRNA exhibited ...

  12. Adaptation of intestinal hydrolases to starvation in rats: effect of thyroid function

    DEFF Research Database (Denmark)

    Galluser, M; Belkhou, R; Freund, J N;

    1991-01-01

    The effects of long-term starvation on the activities of sucrase, lactase, and aminopeptidase, and on their respective mRNA were determined in the small intestine of thyroidectomized and sham-operated adult rats. Thyroidectomy reduced the protein loss at the level of the intestinal brush border m...

  13. Preparation and use of recombinant protein G-gold complexes as markers in double labelling immunocytochemistry

    DEFF Research Database (Denmark)

    Balslev, Y; Hansen, Gert Helge

    1989-01-01

    was performed on ultracryosections of pig small intestine using antibodies directed against aminopeptidase N and sucrase-isomaltase. The labelling efficiency of RPGG was compared to that of protein A-gold conjugates (PAG) in different compartments of the enterocyte. Quantification showed that the labelling...

  14. Sequence Classification: 893876 [

    Lifescience Database Archive (English)

    Full Text Available TMB Non-TMH TMB TMB Non-TMB TMB >gi|6325306|ref|NP_015374.1| Peripheral membrane protein required for delive...ry of aminopeptidase I (Lap4p) to the vacuole in the cytoplasm-to-vacuole targeting

  15. Biosynthesis of intestinal microvillar proteins. Role of the Golgi complex and microtubules

    DEFF Research Database (Denmark)

    Danielsen, E M; Cowell, G M; Poulsen, S S

    1983-01-01

    The effect of monensin and colchicine on the biogenesis of aminopeptidase N (EC 3.4.11.2), aminopeptidase A (EC 3.4.11.7), dipeptidyl peptidase IV (EC 3.4.14.5), sucrase (EC 3.2.1.48)-isomaltase (EC 3.2.1.10) and maltase-glucoamylase (EC 3.2.1.20) was studied in organ-cultured pig small-intestina......The effect of monensin and colchicine on the biogenesis of aminopeptidase N (EC 3.4.11.2), aminopeptidase A (EC 3.4.11.7), dipeptidyl peptidase IV (EC 3.4.14.5), sucrase (EC 3.2.1.48)-isomaltase (EC 3.2.1.10) and maltase-glucoamylase (EC 3.2.1.20) was studied in organ-cultured pig small...... final destination. These findings suggest the involvement of the Golgi complex in the post-translational processing and transport of microvillar enzymes. The presence in the growth medium of colchicine (50 micrograms/ml) caused a significant inhibition of the appearance of newly synthesized enzymes in...... the microvillar membrane during a 3 h labelling period. Since synthesis and post-translational modification of the microvillar enzymes were largely unaffected by colchicine, the results obtained suggest that microtubules play a role in the final transport of the enzymes from the Golgi complex to the...

  16. Fluorometric assay using naphthylamide substrates for assessing novel venom peptidase activities.

    Science.gov (United States)

    Gasparello-Clemente, Elaine; Silveira, Paulo Flávio

    2002-11-01

    In the present study we examined the feasibility of using the fluorometry of naphthylamine derivatives for revealing peptidase activities in venoms of the snakes Bothrops jararaca, Bothrops alternatus, Bothrops atrox, Bothrops moojeni, Bothrops insularis, Crotalus durissus terrificus and Bitis arietans, of the scorpions Tityus serrulatus and Tityus bahiensis, and of the spiders Phoneutria nigriventer and Loxosceles intermedia. Neutral aminopeptidase (APN) and prolyl-dipeptidyl aminopeptidase IV (DPP IV) activities were presented in all snake venoms, with the highest levels in B. alternatus. Although all examined peptidase activities showed relatively low levels in arthropod venoms, basic aminopeptidase (APB) activity from P. nigriventer venom was the exception. Compared to the other peptidase activities, relatively high levels of acid aminopeptidase (APA) activity were restricted to B. arietans venom. B. arietans also exhibited a prominent content of APB activity which was lower in other venoms. Relatively low prolyl endopeptidase and proline iminopeptidase activities were, respectively, detectable only in T. bahiensis and B. insularis. Pyroglutamate aminopeptidase activity was undetectable in all venoms. All examined peptidase activities were undetectable in T. serrulatus venom. In this study, the specificities of a diverse array of peptidase activities from representative venoms were demonstrated for the first time, with a description of their distribution which may contribute to guiding further investigations. The expressive difference between snake and arthropod venoms was indicated by APN and DPP IV activities while APA and APB activities distinguished the venom of B. arietans from those of Brazilian snakes. The data reflected the relatively uniform qualitative distribution of the peptidase activities investigated, together with their unequal quantitative distribution, indicating the evolutionary divergence in the processing of peptides in these different

  17. Elective cesarean delivery affects gut maturation and delays microbial colonization but does not increase necrotizing enterocolitis in preterm pigs

    DEFF Research Database (Denmark)

    Siggers, R. H.; Thymann, Thomas; Jensen, Bent B.;

    2008-01-01

    Although preterm birth and formula feeding increase the risk of necrotizing enterocolitis (NEC), the influences of cesarean section (CS) and vaginal delivery (VD) are unknown. Therefore, gut characteristics and NEC incidence and severity were evaluated in preterm pigs (92% gestation) delivered...... by CS or VD. An initial study showed that newborn CS pigs (n 6) had decreased gastric acid secretion, absorption of intact proteins, activity of brush-border enzymes and pancreatic hydrolases, plasma cortisol, rectal temperature, and changes in blood chemistry, indicating impaired respiratory function......, and increased brush-border enzyme activities (lactase, aminopeptidases) compared with VD pigs. In particular, VD-FORM pigs showed reduced mucosal proportions, reduced lactase and aminopeptidases, and increased proinflammatory cytokine IL-6 compared with CS-FORM (P 0.06). Despite the initial improvement...

  18. Diet-induced hypercholesterolemia impaired testicular steroidogenesis in mice through the renin-angiotensin system.

    Science.gov (United States)

    Martínez-Martos, José M; Arrazola, Marce; Mayas, María D; Carrera-González, María P; García, María J; Ramírez-Expósito, María J

    2011-08-01

    Hypercholesterolemia and low testosterone concentrations in men are associated with a high risk factor for atherosclerosis. It is known that cholesterol serves as the major precursor for the synthesis of the sex hormones. The bioactive peptides of the renin-angiotensin-system localized in the gonads play a key role in the relation between cholesterol and testosterone by modulating steroidogenesis and inhibiting testosterone production. In the present work, we evaluated the effects of diet-induced hypercholesterolemia on circulating testosterone levels and its relationship with the testicular RAS-regulating specific aminopeptidase activities in male mouse. A significant decrease in serum circulating levels of testosterone was observed after induced hypercholesterolemia. The changes found in aminopeptidase activities suggest a role of Ang III and Ang IV in the regulation of steroidogenesis.

  19. Identification of multiple risk variants for ankylosing spondylitis through high-density genotyping of immune-related loci.

    Science.gov (United States)

    Cortes, Adrian; Hadler, Johanna; Pointon, Jenny P; Robinson, Philip C; Karaderi, Tugce; Leo, Paul; Cremin, Katie; Pryce, Karena; Harris, Jessica; Lee, Seunghun; Joo, Kyung Bin; Shim, Seung-Cheol; Weisman, Michael; Ward, Michael; Zhou, Xiaodong; Garchon, Henri-Jean; Chiocchia, Gilles; Nossent, Johannes; Lie, Benedicte A; Førre, Øystein; Tuomilehto, Jaakko; Laiho, Kari; Jiang, Lei; Liu, Yu; Wu, Xin; Bradbury, Linda A; Elewaut, Dirk; Burgos-Vargas, Ruben; Stebbings, Simon; Appleton, Louise; Farrah, Claire; Lau, Jonathan; Kenna, Tony J; Haroon, Nigil; Ferreira, Manuel A; Yang, Jian; Mulero, Juan; Fernandez-Sueiro, Jose Luis; Gonzalez-Gay, Miguel A; Lopez-Larrea, Carlos; Deloukas, Panos; Donnelly, Peter; Bowness, Paul; Gafney, Karl; Gaston, Hill; Gladman, Dafna D; Rahman, Proton; Maksymowych, Walter P; Xu, Huji; Crusius, J Bart A; van der Horst-Bruinsma, Irene E; Chou, Chung-Tei; Valle-Oñate, Raphael; Romero-Sánchez, Consuelo; Hansen, Inger Myrnes; Pimentel-Santos, Fernando M; Inman, Robert D; Videm, Vibeke; Martin, Javier; Breban, Maxime; Reveille, John D; Evans, David M; Kim, Tae-Hwan; Wordsworth, Bryan Paul; Brown, Matthew A

    2013-07-01

    Ankylosing spondylitis is a common, highly heritable inflammatory arthritis affecting primarily the spine and pelvis. In addition to HLA-B*27 alleles, 12 loci have previously been identified that are associated with ankylosing spondylitis in populations of European ancestry, and 2 associated loci have been identified in Asians. In this study, we used the Illumina Immunochip microarray to perform a case-control association study involving 10,619 individuals with ankylosing spondylitis (cases) and 15,145 controls. We identified 13 new risk loci and 12 additional ankylosing spondylitis-associated haplotypes at 11 loci. Two ankylosing spondylitis-associated regions have now been identified encoding four aminopeptidases that are involved in peptide processing before major histocompatibility complex (MHC) class I presentation. Protective variants at two of these loci are associated both with reduced aminopeptidase function and with MHC class I cell surface expression.

  20. Caveolae/lipid rafts in fibroblast-like synoviocytes: ectopeptidase-rich membrane microdomains

    DEFF Research Database (Denmark)

    Riemann, D; Hansen, Gert Helge; Niels-Christiansen, L;

    2001-01-01

    -lymphocytes, cholesterol depletion of synoviocytes greatly reduced their capability to induce an early lymphocytic expression of aminopeptidase N/CD13. We propose caveolae/rafts to be peptidase-rich 'hot-spot' regions of the synoviocyte plasma membrane required for functional cell-cell interactions with lymphocytes......Membrane peptidases play important roles in cell activation, proliferation and communication. Human fibroblast-like synoviocytes express considerable amounts of aminopeptidase N/CD13, dipeptidyl peptidase IV/CD26, and neprilysin/CD10, transmembrane proteins previously proposed to be involved...... in the regulation of intra-articular levels of neuropeptides and chemotactic mediators as well as in adhesion and cell-cell interactions. Here, we report these peptidases in synoviocytes to be localized predominantly in glycolipid- and cholesterol-rich membrane microdomains known as 'rafts'. At the ultrastructural...

  1. Involvement of detergent-insoluble complexes in the intracellular transport of intestinal brush border enzymes

    DEFF Research Database (Denmark)

    Danielsen, E M

    1995-01-01

    , and their insolubility increased to that of the steady-state level soon after they achieved their mature, complex glycosylation, i.e., after passage through the Golgi complex. Detergent-insoluble complexes isolated by density gradient centrifugation were highly enriched in brush border enzymes, and the enrichment......-insoluble complexes commonly known as glycolipid "rafts". Thus, aminopeptidase N (EC 3.4.11.2), aminopeptidase A (EC 3.4.11.7), dipeptidyl peptidase IV (EC 3.4.14.5), and sucrase-isomaltase (EC 3.2.1.48-10) were 34-48% detergent-insoluble. Maltase-glucoamylase (EC 3.2.1.20) was markedly less detergent-insoluble (20......%), and lactase-phlorizin hydrolase (EC 3.2.1.23-62) was essentially fully soluble in detergent. In radioactively labeled, mucosal explants, the newly synthesized brush border enzymes began to associate with detergent-insoluble complexes while still in their transient, high mannose-glycosylated form...

  2. The enzymes of the hepatobiliary tract: a biochemical and clinical comparison.

    Science.gov (United States)

    Batsakis, J G

    1974-01-01

    The "biliary tract" enzymes (leucine aminopeptidase, gamma-glutamyltranspeptidase and 5'-nucleotidase) in serum reflect to varying degrees, obstruction, proliferation, inflammation and neoplasia involving the hepatobiliary duct system. Their use is directed towards two purposes: (1) as non-electrophoretic assays to evaluate the source of an elevated non-specific alkaline phosphatase and (2) to offer greater sensitivity and specificity for space-occuping lesions in the liver. In appropriate clinical states, any of the three enzymes offer these advantages and there is little to chose among them. Selection of the assay to use in the clinical laboratory then becomes based on non-clinical factors, i.e., technical ease, apparent substrate specifities, etc. With these additional factors and despite some shortcomings, our selection is leucine aminopeptidase.

  3. Estudios histológico e histoquímico del sistema digestivo de la almeja catarina Argopecten ventricosus (Sowerby, 1842)

    OpenAIRE

    Cáceres, C.; Alarcón, F.J.

    2002-01-01

    Histological and histochemical studies on the digestive system of Argopecten ventricosus (Sowerby, 1842) was carried out. Lips show a ciliated columnar epithelium with glycoproteins and acid and alkaline phosphatases, and α-D glucosidase activity. Esophagus and lips epitheliums are similar. Acid and alkaline phosphatases, α-D glucosidase and leucil aminopeptidase activities were detected. The epithelium of the stomach is ciliated and contains glands which secrete glycoproteins. Acid and alkal...

  4. Berry and Citrus Phenolic Compounds Inhibit Dipeptidyl Peptidase IV: Implications in Diabetes Management

    OpenAIRE

    Junfeng Fan; Johnson, Michelle H.; Mary Ann Lila; Gad Yousef; Elvira Gonzalez de Mejia

    2013-01-01

    Beneficial health effects of fruits and vegetables in the diet have been attributed to their high flavonoid content. Dipeptidyl peptidase IV (DPP-IV) is a serine aminopeptidase that is a novel target for type 2 diabetes therapy due to its incretin hormone regulatory effects. In this study, well-characterized anthocyanins (ANC) isolated from berry wine blends and twenty-seven other phenolic compounds commonly present in citrus, berry, grape, and soybean, were individually investigated for thei...

  5. Comparison among Different Gilthead Sea Bream (Sparus aurata) Farming Systems: Activity of Intestinal and Hepatic Enzymes and 13C-NMR Analysis of Lipids

    OpenAIRE

    Vincenzo Zonno; Francesco Paolo Fanizzi; Carlo Storelli; Giorgia Bressani; Pascali, Sandra A. De; Laura Del Coco; Paride Papadia

    2009-01-01

    In order to evaluate differences in general health and nutritional values of gilthead sea bream (Sparus aurata), the effects of semi-intensive, land-based tanks and sea-cages intensive rearing systems were investigated, and results compared with captured wild fish. The physiological state was determined by measuring the activity of three different intestinal digestive enzymes: alkaline phosphatase (ALP), leucine aminopeptidase (LAP) and maltase; and the activity of the hepatic ALP. Also, the ...

  6. An Assessment of those Metabolites Considered to be of Value in the Diagnosis of Exposure to Radiation

    International Nuclear Information System (INIS)

    Certain metabolites excreted in urine have been examined in cases of human beings exposed to X- and y-irradiation for therapeutic reasons. These patients were receiving doses of up to 5000 r over a period of three to five weeks. Urinary excretion of creatinine, creatine, total α-amino-nitrogen, β-amino-isobutyric acid, pyrrole-2-carboxylic acid, 5-hydroxyindoleacetic acid and indoxyl-sulphate were followed quantitatively during the whole course of treatment. None of the patients were exposed to total-body irradiation but it is apparent that changes in metabolic function as a result of radiation-induced damage can be reflected in urinary excretion. The biochemical rationale and interpretation of these excretion patterns is discussed in relation to that part of the body irradiated, but it is concluded at this stage of the investigation that quantitative assessment of biological damage is not possible. However, such tests can be useful as a guide to the clinician on the form of therapy. Preliminary tests on changes in certain plasma metabolites in rats exposed to single sub-lethal doses of X-irradiation are worthy of extrapolation to human studies. Plasma concentrations of 5-hydroxytryptamine, uric acid, SGOT, SGPT, globulin fractions of protein, creatine phosphokinase, leucine aminopeptidase, lysyl aminopeptidase, kallikrein, kallidin, zinc and manganese have been measured. Qualitative yet significant changes were observed in plasma levels of uric acid, leucine aminopeptidase, lysyl aminopeptidase and manganese after irradiation. The significance of this data is discussed. Emphasis in these studies has been placed upon methods which are simple and quick to perform. (author)

  7. Association of ERAP1 Allelic Variants with Risk of Ankylosing Spondylitis

    OpenAIRE

    Zvyagin, I.; Dorodnykh, V.; Mamedov, I.; Staroverov, D.; Bochkova, A.; Rebrikov, D.; Lebedev, Y.

    2010-01-01

    Ankylosing spondylitis (AS) belongs to a group of autoimmune diseases affecting the axial skeleton. Beside the hla-b*27 allele, several other human genes that control the variety processes of immune homeostasis are considered to be associated with AS manifestation in different human populations. Among strong associated non-MHC genes erap 1 encoding the endoplasmic reticulum aminopeptidase 1 isoform was recently identified by single nucleotide polymorphisms (SNPs) meta analysis. In our study w...

  8. In vivo role of ER-associated peptidase activity in tailoring peptides for presentation by MHC class Ia and class Ib molecules

    OpenAIRE

    Yan, Jingbo; Parekh, Vrajesh V.; Mendez-Fernandez, Yanice; Olivares-Villagómez, Danyvid; Dragovic, Srdjan; Hill, Timothy; Roopenian, Derry C.; Joyce, Sebastian; Van Kaer, Luc

    2006-01-01

    Endoplasmic reticulum (ER)-associated aminopeptidase (ERAP)1 has been implicated in the final proteolytic processing of peptides presented by major histocompatibility complex (MHC) class I molecules. To evaluate the in vivo role of ERAP1, we have generated ERAP1-deficient mice. Cell surface expression of the class Ia molecules H-2Kb and H-2Db and of the class Ib molecule Qa-2 was significantly reduced in these animals. Although cells from mutant animals exhibited reduced capacity to present s...

  9. ERAP1 genetic variations associated with HLA-B27 interaction and disease severity of syndesmophytes formation in Taiwanese ankylosing spondylitis

    OpenAIRE

    Wang, Chin-Man; Ho, Huei-Huang; Chang, Su-Wei; Wu, Yeong-Jian Jan; Lin, Jing-Chi; Chang, Pi-Yueh; Wu, Jianming; Chen, Ji-Yih

    2012-01-01

    Introduction Ankylosing spondylitis (AS) is a familial, heritable disease specified by syndesmophyte formation leading to an ankylosed spine. Endoplasmic reticulum aminopeptidase 1 (ERAP1) genetic variations have been widely proved to be associated with AS in several ethnic populations. The aim of this study was to investigate whether ERAP1 single nucleotide polymorphisms (SNPs) are associated with AS susceptibility and disease severity in Taiwanese. Methods Four ERAP1 SNPs (rs27037, rs27980,...

  10. A functional variant in ERAP1 predisposes to multiple sclerosis

    OpenAIRE

    Guerini, F R; Cagliani, R.; De Forni, D; C. Agliardi; Caputo, D.; A. Cassinotti; Galimberti, D.; Fenoglio, C; M. Biasin; R. Asselta; Scarpini, E.; Comi, G P; Bresolin, N.; M. S. Clerici; Sironi, M

    2012-01-01

    The ERAP1 gene encodes an aminopeptidase involved in antigen processing. A functional polymorphism in the gene (rs30187, Arg528Lys) associates with susceptibility to ankylosying spondylitis (AS), whereas a SNP in the interacting ERAP2 gene increases susceptibility to another inflammatory autoimmune disorder, Crohn's disease (CD). We analysed rs30187 in 572 Italian patients with CD and in 517 subjects suffering from multiple sclerosis (MS); for each cohort, an independent sex- and age-matched ...

  11. Genetic Association with ERAP1 in Psoriasis Is Confined to Disease Onset after Puberty and Not Dependent on HLA-C*06

    OpenAIRE

    Lysell, Josefin; Padyukov, Leonid; Kockum, Ingrid; Nikamo, Pernilla; Ståhle, Mona

    2012-01-01

    HLA-C remains the strongest susceptibility candidate gene in psoriasis. Evidence for interaction between HLA-C and endoplasmic reticulum aminopeptidase 1 (ERAP1) confined to individuals carrying the HLA-C risk allele was recently reported. Psoriasis displays wide variation, and genetic heterogeneity is likely to contribute to clinical diversity. Age at disease onset is a putative discriminator, and separating psoriasis into early- (

  12. Naturally occurring ERAP1 haplotypes encode functionally distinct alleles with fine substrate specificity

    OpenAIRE

    Reeves, Emma; Edwards, Christopher J.; Elliott, Tim; James, Edward

    2013-01-01

    The aminopeptidase, ERAP1, trims peptides for MHC class I presentation, influencing the degree and specificity of CD8+ T cell responses. Single nucleotide polymorphisms (SNP) within the exons encoding ERAP1 are associated with autoimmune diseases and cervical carcinoma, but it is not known whether they act independently or as disease-associated haplotypes. We sequenced ERAP1 from 20 individuals and show that SNP occur as distinct haplotypes in the human population, and that these haplotypes e...

  13. Immune surveillance for ERAAP dysfunction

    OpenAIRE

    Nagarajan, Niranjana A.; Shastri, Nilabh

    2013-01-01

    The ER aminopeptidase associated with antigen processing, ERAAP (or ERAP1), is essential for trimming peptides that are presented by MHC class I molecules. ERAP1 is inhibited by human cytomegalovirus, and ERAP1 polymorphisms are associated with autoimmune diseases. How the immune system detects ERAAP dysfunction, however, is unknown. We have shown previously that ERAAP-deficient cells present an immunogenic pMHC I repertoire, that elicits CD8+ T cell response in WT mice. Additionally, we disc...

  14. Functionally distinct ERAP1 allotype combinations distinguish individuals with Ankylosing Spondylitis

    OpenAIRE

    Reeves, Emma; Colebatch-Bourn, Alexandra; Elliott, Tim; Edwards, Christopher J.; James, Edward

    2014-01-01

    The immune system performs surveillance to identify infected or cancerous cells through recognition of small protein fragments called antigenic peptides on their surface. To do this, the peptides must be cut to a specific length by an enzyme called endoplasmic reticulum aminopeptidase 1 (ERAP1). Variation in this enzyme has recently been linked to the inflammatory rheumatic disease Ankylosing Spondylitis (AS). We have found that ERAP1 is highly polymorphic in humans and that specific combinat...

  15. Cholesterol depletion of enterocytes. Effect on the Golgi complex and apical membrane trafficking

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Niels-Christiansen, L L; Thorsen, Evy;

    2000-01-01

    Intestinal brush border enzymes, including aminopeptidase N and sucrase-isomaltase, are associated with "rafts" (membrane microdomains rich in cholesterol and sphingoglycolipids). To assess the functional role of rafts in the present work, we studied the effect of cholesterol depletion on apical......-associated. Our results implicate the Golgi complex/trans-Golgi network in raft formation and suggest a close relationship between this event and apical membrane trafficking....

  16. Identification and reproducibility of diagnostic DNA markers for tuber starch and yield optimization in a novel association mapping population of potato (Solanum tuberosum L.)

    OpenAIRE

    Schönhals, E. M.; Ortega, F.; Barandalla, L.; Aragones, A.; Ruiz de Galarreta, J.I.; Liao, J.-C.; Sanetomo, R.; Walkemeier, B.; Tacke, E.; Ritter, E.; Gebhardt, C.

    2016-01-01

    Key message SNPs in candidate genes Pain - 1, InvCD141 (invertases), SSIV (starch synthase), StCDF1 (transcription factor), LapN (leucine aminopeptidase), and cytoplasm type are associated with potato tuber yield, starch content and/or starch yield. Abstract Tuber yield (TY), starch content (TSC), and starch yield (TSY) are complex characters of high importance for the potato crop in general and for industrial starch production in particular. DNA markers associated with superior alleles of ge...

  17. New insights into the role of sex steroid hormones in pregnancy: possible therapeutic approach by sex steroid hormones for the treatment of both preeclampsia and preterm labor.

    Science.gov (United States)

    Mizutani, S; Mizutani, E

    2015-03-01

    Fetal peptide hormones are essential for the development of fetus, which increase in accordance with pregnancy term. Concentration of these hormones within the feto-placental unit is normally higher than that of maternal circulation. Since these hormones are biologically active, the leakage of these hormones into the maternal circulation is regulated by degradation activity by placental aminopeptidases, in order to maintain the balance between carriage of pregnancy and onset of labor.Because the concentration of these hormones, being regulated by the amount of endogenous production and by physiological degradation by enzymes in the blood and tissue, the balance between production and degradation is a definitive element for maintaining normal gestation and term delivery.The changes of the balance between fetal angiotensin II (A-II) and vasopressin (AVP) andA-II and AVP degrading enzymes, between aminopeptidase A (APA) and placental leucine aminopeptidase( P-LAP) - in the placenta and maternal blood due to fetal stress such as hypoxia - are the provable causes of preeclampsia or preterm labor.Induction of APA and P-LAP by estradiol benzoate (E2) and progesterone (P) from placenta has been demonstrated. They are involved in the regulation of fetal peptide hormones via placental aminopeptidases in homeostasis of pregnancy.Recently it was shown that both APA and P-LAP could be potentially safe and effective drugs for preeclampsia and preterm labor. The authors' proposed sex steroid treatment with dose increasing manner by gestational week (sex steroid treatment) for severe preeclampsia and preterm labor could be candidates replacing conventional treatments. In light of lacking safe and effective medication, the proposed sex steroid treatment is worthwhile for the prospective controlled studies for the treatment of both preeclampsia and preterm labor.

  18. Relationship between carbon catabolite repression and the biosynthesis regulation of the prolidase PepQ from Lactobacillus delbrueckii ssp. bulgaricus CNRZ 397

    OpenAIRE

    Lamarque, Mauld; Morel, Fabienne; Bissardon, Isabelle; Galinier, Anne; Portalier, Raymond; Atlan, Danièle

    2001-01-01

    International audience Lactobacillus delbrueckii ssp. bulgaricus CNRZ 397 (L. bulgaricus) displays several enzymes specific of proline-containing peptides. We focused on the prolidase PepQ which specifically cleaves X-Pro dipeptides. PepQ biosynthesis was previously shown to be independent of the peptide concentration of the culture medium in contrast to the cell surface proteinase PrtB and several aminopeptidases. Regulation of PepQ biosynthesis can be explained by the genetic organizatio...

  19. Evaluation of the Strep-A-Fluor identification method for group A streptococci.

    OpenAIRE

    Wasilauskas, B L; Hampton, K D

    1984-01-01

    Strep-A-Fluor (Bio Spec, Inc., Dublin, Calif.) is a new test designed for the rapid identification of group A streptococci. A filter paper strip impregnated with a synthetic substrate is used to detect a specific aminopeptidase present in group A streptococci by UV fluorescence. In a blind study, 305 beta-hemolytic streptococcal isolates were correctly categorized as group A or non-group A.

  20. Evaluation of the Strep-A-Fluor identification method for group A streptococci.

    Science.gov (United States)

    Wasilauskas, B L; Hampton, K D

    1984-12-01

    Strep-A-Fluor (Bio Spec, Inc., Dublin, Calif.) is a new test designed for the rapid identification of group A streptococci. A filter paper strip impregnated with a synthetic substrate is used to detect a specific aminopeptidase present in group A streptococci by UV fluorescence. In a blind study, 305 beta-hemolytic streptococcal isolates were correctly categorized as group A or non-group A. PMID:6394625

  1. Allium sativum aqueous extract prevents potassium dichromate-induced nephrotoxicity and lipid oxidation in rats

    OpenAIRE

    Sergio L. Becerra-Torres; César Soria-Fregozo; Fernando Jaramillo-Juárez; José L. Moreno-Hernández-Duque

    2014-01-01

    Context: The potassium dichromate (K2Cr2O7) induces nephrotoxicity by oxidative stress mechanisms. Aims: To study the potential protection of an aqueous extract of Allium sativum against the K2Cr2O7-induced nephrotoxicity and lipid oxidation in rats. Methods: Twenty four hours after treatment, biomarkers such as proteinuria, creatinine clearance, malondialdehyde production, specific enzyme activity of gamma glutamyl transpeptidase and alanine aminopeptidase, and renal clearance of para-...

  2. Some properties of the intestinal proteases of the rabbitfish, Siganus canaliculatus (Park).

    Science.gov (United States)

    Sabapathy, U; Teo, L H

    1995-06-01

    Some properties of the intestinal proteases of the rabbitfish were examined. At 25°C, both trypsin and chymotrypsin showed pH optima of 8.0. Leucine aminopeptidase, however, displayed maximum activity in the pH range, 7.0-9.0. Leucine aminopeptidase had the highest optimum temperature (60°C), and chymotrypsin, the lowest (30°C). The optimum temperature of trypsin was 55°C. The activation energy, Ea, was found to be 8.24 for trypsin and 8.50 kcal mol(-1) for chymotrypsin. The Ea for leucine aminopeptidase was 6.29 kcal mol(-1) above 40°C and 1.73 kcal mol(-1) below 40°C. Substrate concentration-velocity plots showed that all three enzymes followed Michaelis-Menten kinetics; the Km and Vmax were estimated for the three enzymes. The effects of various protease inhibitors on enzyme activity were also examined and confirmed the protease classes to which each enzyme belonged. The three proteases examined have similar properties to proteases in other fishes.

  3. Isolation of the Binding Protein of Periplocoside E from BBMVs in Midgut of the Oriental Amyworm Mythimna separata Walker (Lepidoptera: Noctuidae) through Affinity Chromatography.

    Science.gov (United States)

    Feng, Mingxing; He, Zhenyu; Wang, Yuanyuan; Yan, Xiufang; Zhang, Jiwen; Hu, Zhaonong; Wu, Wenjun

    2016-01-01

    Periplocosides, which are insecticidal compounds isolated from the root bark of Periploca sepium Bunge, can affect the digestive system of insects. However, the mechanism though which periplocosides induces a series of symptoms remains unknown. In this study, affinity chromatography was conducted by coupling periplocoside E-semi-succinic acid ester with epoxy amino hexyl (EAH) sepharose 4B. Sodium dodecyl sulfonate-polyacrylamide gelelectrophoresis (SDS-PAGE) was performed to analyze the fraction eluted by periplocoside E. Eight binding proteins (luciferin 4-monooxygenase, aminopeptidase N, aminopeptidase N3, nicotinamide adenine dinucleotide health (NADH) dehydrogenase subunit 5, phosphatidylinositol 3-phosphate 3-phosphatase myotubularin, actin, uncharacterized family 31 glucosidase KIAA1161, and 2OG-Fe(2) oxygenase superfamily protein) were obtained and identified through liquid chromatography/quadrupole-time of flight-mass spectrometry (LC/Q-TOF-MS) analysis of the midgut epithelium cells of Mythimna separata larvae. Aminopeptidase N and N3 are potential putative targets of periplocosides. This study establishes the foundation for further research on the mechanism of action and target localization of periplocosides in agricultural pests. PMID:27153092

  4. Preparation of Antioxidant Enzymatic Hydrolysates from Honeybee-Collected Pollen Using Plant Enzymes

    Directory of Open Access Journals (Sweden)

    Margarita D. Marinova

    2010-01-01

    Full Text Available Enzymatic hydrolysates of honeybee-collected pollen were prepared using food-grade proteinase and aminopeptidases entirely of plant origin. Bromelain from pineapple stem was applied (8 mAU/g substrate in the first hydrolysis stage. Aminopeptidase (0.05 U/g substrate and proline iminopeptidase (0.03 U/g substrate from cabbage leaves (Brassica oleracea var. capitata, and aminopeptidase (0.2 U/g substrate from chick-pea cotyledons (Cicer arietinum L. were involved in the additional hydrolysis of the peptide mixtures. The degree of hydrolysis (DH, total phenolic contents, and protein contents of these hydrolysates were as follows: DH (about 20–28%, total phenolics (15.3–27.2 μg/mg sample powder, and proteins (162.7–242.8 μg/mg sample powder, respectively. The hydrolysates possessed high antiradical scavenging activity determined with DPPH (42–46% inhibition. The prepared hydrolysates of bee-collected flower pollen may be regarded as effective natural and functional dietary food supplements due to their remarkable content of polyphenol substances and significant radical-scavenging capacity with special regard to their nutritional-physiological implications.

  5. Structure of microvillar enzymes in different phases of their life cycles

    DEFF Research Database (Denmark)

    Sjöström, H; Norén, Ove; Danielsen, E M;

    1983-01-01

    Structural changes have been studied during the life cycles of three glycosidases: sucrase-isomaltase (EC 3.2.48-10), lactase-phlorizin hydrolase (EC 3.2.1.23-62), maltase-glucoamylase (EC 3.2.1.20); and three peptidases: aminopeptidase A (EC 3.4.11.7), aminopeptidase N (EC 3.4.11.2) and dipeptid...... by pancreatic proteases to smaller peptides, and sucrase-isomaltase may lose its sucrase polypeptide, while both enzymes remain bound to the membrane....... peptidase IV (EC 3.4.14.5). The final forms of the enzymes can be divided into at least two groups: the sucrase-isomaltase type, characterized as dimers, which are asymmetric in their hydrophilic parts, have two types of active site and anchor only on one subunit; and the aminopeptidase N type......, characterized as dimers, which are symmetric in their hydrophilic part, have only one type of active site and anchor on both subunits. These enzymes are likely to be synthesized on rough endoplasmic reticulum and simultaneously glycosylated into endoglycosidase H-sensitive forms. They are later reglycosylated...

  6. Interspecies differences in membrane-associated protease activities of thyrocytes and their relevance for thyroid cancer studies

    Directory of Open Access Journals (Sweden)

    Fröhlich Eleonore

    2012-05-01

    Full Text Available Abstract Background To understand the role of proteases involved in human thyroid cancer progression and tissue invasion, thyrocytes from other species could potentially be used provided their characteristics are similar. It is not known whether dipeptidyl peptidase IV and aminopeptidase N activities, which are overexpressed in human thyroid cancer, are, as in human, also absent in normal thyrocytes of other species, making them suitable models for studies on the regulation of these proteases. Methods To assess the role of these proteases, activity was measured in thyroid tissue of human, mouse, rat, porcine, bovine and ovine origin. The lysosomal protease, dipeptidyl peptidase II, was used for comparison. Results Murine, rat, ovine, bovine and human thyrocytes all lacked dipeptidyl peptidase IV and aminopeptidase N activity, but porcine thyrocytes were found to possess both. In contrast, lysosomal dipeptidyl peptidase II was strongly expressed in all species. These activity patterns were maintained in cultured cells. Cultured porcine thyrocytes formed follicles with typical morphology upon stimulation with TSH but differed from human thyrocytes in their response to thiamazole. Conclusions These species differences in the expression of dipeptidyl peptidase IV and aminopeptidase N, indicate that porcine thyrocytes cannot be considered appropriate for the study of proteases in human cancer development.

  7. The internal sequence of the peptide-substrate determines its N-terminus trimming by ERAP1.

    Directory of Open Access Journals (Sweden)

    Irini Evnouchidou

    Full Text Available BACKGROUND: Endoplasmic reticulum aminopeptidase 1 (ERAP1 trims N-terminally extended antigenic peptide precursors down to mature antigenic peptides for presentation by major histocompatibility complex (MHC class I molecules. ERAP1 has unique properties for an aminopeptidase being able to trim peptides in vitro based on their length and the nature of their C-termini. METHODOLOGY/PRINCIPAL FINDINGS: In an effort to better understand the molecular mechanism that ERAP1 uses to trim peptides, we systematically analyzed the enzyme's substrate preferences using collections of peptide substrates. We discovered strong internal sequence preferences of peptide N-terminus trimming by ERAP1. Preferences were only found for positively charged or hydrophobic residues resulting to trimming rate changes by up to 100 fold for single residue substitutions and more than 40,000 fold for multiple residue substitutions for peptides with identical N-termini. Molecular modelling of ERAP1 revealed a large internal cavity that carries a strong negative electrostatic potential and is large enough to accommodate peptides adjacent to the enzyme's active site. This model can readily account for the strong preference for positively charged side chains. CONCLUSIONS/SIGNIFICANCE: To our knowledge no other aminopeptidase has been described to have such strong preferences for internal residues so distal to the N-terminus. Overall, our findings indicate that the internal sequence of the peptide can affect its trimming by ERAP1 as much as the peptide's length and C-terminus. We therefore propose that ERAP1 recognizes the full length of its peptide-substrate and not just the N- and C- termini. It is possible that ERAP1 trimming preferences influence the rate of generation and the composition of antigenic peptides in vivo.

  8. Effects of tetracycline administration on the proteomic profile of pig muscle samples (L. dorsi)

    DEFF Research Database (Denmark)

    Gratacos-Cubarsi, M.; Castellari, M.; Hortos, M.;

    2008-01-01

    Effect of tetracycline (TC) administration on the proteomic profile of pig muscle was evaluated by 2D electrophoresis and MALDI-TOF mass spectrometry. The TC content at slaughter was determined in L. dorsi samples by HPLC-DAD. Mean residual concentration of TC in the muscle of treated animals......, glycerol-3-phosphate dehydrogenase 1 (G3PD1), phosphoglycerate kinase 1, novelprotein (0610037L13Rik), leucine aminopeptidase 3 (LAP), and hypothetical protein isoform 2. Results show that proteomics could be a useful tool to reveal pharmacological treatments with TC, even if the possible uses...

  9. Apical Plasma Membrane Proteins and Endolyn-78 Travel through a Subapical Compartment in Polarized WIF-B Hepatocytes

    OpenAIRE

    Ihrke, Gudrun; Martin, Greg V.; Shanks, Michael R.; Schrader, Michael; Schroer, Trina A.; Hubbard, Ann L.

    1998-01-01

    We studied basolateral-to-apical transcytosis of three classes of apical plasma membrane (PM) proteins in polarized hepatic WIF-B cells and then compared it to the endocytic trafficking of basolaterally recycling membrane proteins. We used antibodies to label the basolateral cohort of proteins at the surface of living cells and then followed their trafficking at 37°C by indirect immunofluorescence. The apical PM proteins aminopeptidase N, 5′nucleotidase, and the polymeric IgA receptor were ef...

  10. Microvillar membrane microdomains exist at physiological temperature. Role of galectin-4 as lipid raft stabilizer revealed by "superrafts"

    DEFF Research Database (Denmark)

    Braccia, Anita; Villani, Maristella; Immerdal, Lissi;

    2003-01-01

    and the transmembrane aminopeptidase N, whereas the peripheral lipid raft protein annexin 2 was essentially absent. In conclusion, in the microvillar membrane, galectin-4, functions as a core raft stabilizer/organizer for other, more loosely raft-associated proteins. The superraft analysis might be applicable to other...... rafts prepared by the two protocols were morphologically different but had essentially similar profiles of protein- and lipid components, showing that raft microdomains do exist at 37 degrees C and are not "low temperature artifacts." We also employed a novel method of sequential detergent extraction...

  11. Endocytic trafficking from the small intestinal brush border probed with FM dye

    DEFF Research Database (Denmark)

    Hansen, Gert H; Rasmussen, Karina; Niels-Christiansen, Lise-Lotte;

    2009-01-01

    The small intestinal brush border functions as the body's main portal for uptake of dietary nutrients and simultaneously acts as the largest permeability barrier against pathogens. To enable this, the digestive enzymes of the brush border are organized in lipid raft microdomains stabilized by cross...... localized deeper into the cytoplasm of the enterocytes. Two major raft-associated brush border enzymes, alkaline phosphatase and aminopeptidase N, were excluded from endocytosis. We propose that the terminal web cytoskeleton, by inhibiting traffic from apical early endosomes further into the cell......, contributes to the overall permeability barrier of the gut. Key words: FM dye, small intestine, brush border, endocytosis....

  12. Proteome Analysis of Pyloric Ceca: A Methodology for Fish Feed Development?

    DEFF Research Database (Denmark)

    Wulff, Tune; Petersen, Jørgen; Nørrelykke, Mette R.;

    2012-01-01

    to investigate feed effects on fish by analyzing protein changes in the fish gut. The workflow was used to study the effect of substituting fish meal in fish feed by alternative sources of protein. Rainbow trout divided into five groups were fed for 72 days with feeds varying in protein composition. By two...... identified, including proteins involved in digestion (trypsinogen, carboxylic ester hydrolase, and aminopeptidase). The many expression changes indicated that the trout, when adapting to differences in feed formulation, alter the protein composition of the gut....

  13. Enzymatic activity of "high-mannose" glycosylated forms of intestinal microvillar hydrolases

    DEFF Research Database (Denmark)

    Sjöström, H; Norén, Ove; Danielsen, E M

    1985-01-01

    glycosylated form, whereas no difference was observed for the two other enzymes. The change in glycosylation from high-mannose to complex form thus seems to be of importance for the enzymatic activity of sucrase-isomaltase either by direct structural involvement or by a general stabilization effect......The "high-mannose" glycosylated forms of aminopeptidase N (EC 3.4.11.2), maltase-glucoamylase (EC 3.2.1.20), and sucrase-isomaltase (EC 3.2.1.48, EC 3.2.1.10) have been purified. The high-mannose glycosylated form of sucrase-isomaltase was found to have a lower specific activity than the complex...

  14. 第八届中国药理学会SERVIER青年药理学工作者奖获奖者论文摘要

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Effect of Amyloid Precursor Protein 17mer Peptide on Microtubule Structure and Tau Protein Hyperphosphorylation in Hippocampal Neurons of Experimental Diabetic Mice,Naunyn Schmiedebergs Arch Pharmacol. 2003; 368 (6) : 457-62. The serotonergic system may be involved in the sleep-inducing action of oleamide in rats.,Intercellular adhesion molecule-1 and vascular endothelial growth factor expression kinetics in macrophage-derived foam cells,Mutations at the S1 Site of Methionine Aminopeptidases from Escherichia coli and Homo sapiens Reveal the Residues Critical for Substrate Specificity……

  15. Report for an accreditation to supervise research

    International Nuclear Information System (INIS)

    After indications of his scientific publications, of his published posters, and of his activities of supervision of students and researchers, the author proposes an overview of his research works since 1987. His first works related biochemical and immuno-cyto-chemical characterisation of the neutral aminopeptidase (a marker in epithelial cells), and then (for his research thesis), to the study of membrane mechanisms during zinc intestinal absorption, and after that, to a study of divalent cation transport mechanisms. More recent works (1994-2000) addressed molecular mechanisms of water and solute transports by aqua-porins. Project on a medium term are presented. Published articles are proposed in appendix

  16. Simple enzymatic procedure for l‐carnosine synthesis: whole‐cell biocatalysis and efficient biocatalyst recycling

    OpenAIRE

    Heyland, Jan; Antweiler, Nicolai; Lutz, Jochen; Heck, Tobias; Geueke, Birgit; Kohler, Hans‐Peter E.; Blank, Lars M.; Schmid, Andreas

    2009-01-01

    Summary β‐Peptides and their derivates are usually stable to proteolysis and have an increased half‐life compared with α‐peptides. Recently, β‐aminopeptidases were described as a new enzyme class that enabled the enzymatic degradation and formation of β‐peptides. As an alternative to the existing chemical synthesis routes, the aim of the present work was to develop a whole‐cell biocatalyst for the synthesis and production of β‐peptides using this enzymatic activity. For the optimization of th...

  17. Acyl-CoA-binding protein, Acb1p, is required for normal vacuole function and ceramide synthesis in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Færgeman, Nils J.; Feddersen, Søren; Christiansen, Janne K;

    2004-01-01

    In the present study, we show that depletion of acyl-CoA-binding protein, Acb1p, in yeast affects ceramide levels, protein trafficking, vacuole fusion and structure. Vacuoles in Acb1p-depleted cells are multi-lobed, contain significantly less of the SNAREs (soluble N -ethylmaleimide......-sensitive fusion protein attachment protein receptors) Nyv1p, Vam3p and Vti1p, and are unable to fuse in vitro. Mass spectrometric analysis revealed a dramatic reduction in the content of ceramides in whole-cell lipids and in vacuoles isolated from Acb1p-depleted cells. Maturation of yeast aminopeptidase I and...

  18. Biosynthesis of intestinal microvillar proteins. Processing of N-linked carbohydrate is not required for surface expression

    DEFF Research Database (Denmark)

    Danielsen, Erik Michael; Cowell, G M

    1986-01-01

    microvillar enzymes, it does not receive post-translational O-linked carbohydrate. Castanospermine suppressed the synthesis of the four enzymes, but did not block their transport to the microvillar membrane, showing that processing of N-linked carbohydrate is not required for microvillar expression......Castanospermine, an inhibitor of glucosidase I, the initial enzyme in the trimming of N-linked carbohydrate, was used to study the importance of carbohydrate processing in the biosynthesis of microvillar enzymes in organ-cultured pig intestinal explants. For aminopeptidase N (EC 3...

  19. PLAP efficiently generates mature antigenic peptides in vitro but in patterns distinct from ERAP11

    OpenAIRE

    Georgiadou, Dimitra; Hearn, Arron; Evnouchidou, Irini; Chroni, Angeliki; Leondiadis, Leondios; Ian A York; Rock, Kenneth L.; Stratikos, Efstratios

    2010-01-01

    All three members of the oxytocinase sub-family of M1 aminopeptidases, ERAP1 (ERAAP), ERAP2 and PLAP (IRAP), have been implicated in the generation of MHC class I-presented peptides. ERAP1 and 2 trim peptides in the endoplasmic reticulum for direct presentation whereas PLAP has been recently implicated in cross presentation. The best characterized member of the family, ERAP1, has unique enzymatic properties that fit well with its role in antigen processing. ERAP1 can trim a large variety of l...

  20. Biosynthesis of intestinal microvillar proteins. Further characterization of the intracellular processing and transport

    DEFF Research Database (Denmark)

    Danielsen, E M; Cowell, G M

    1984-01-01

    The effect of tunicamycin on synthesis and intracellular transport of pig small intestinal aminopeptidase N (EC 3.4.11.2), sucrase-isomaltase (EC 3.2.1.48-10) and maltase-glucoamylase (EC 3.2.1.20) was studied by labelling of mucosal explants with [35S]methionine. The expression of the microvilla...... presence of tunicamycin. The complex forms were also sensitive to endo F but did not coincide with the high mannose forms after treatment, indicating that the size difference cannot alone be ascribed to processing of N-linked carbohydrate....

  1. A 1-year study of the activities of seven hydrolases in a communal wastewater treatment plant: trends and correlations.

    Science.gov (United States)

    Kreutz, Jennifer Anna; Böckenhüser, Ina; Wacht, Marion; Fischer, Klaus

    2016-08-01

    The activities of seven hydrolytic enzymes (L-alanine aminopeptidase, esterase, α-and β-glucosidase, phosphomonoesterase, phosphodiesterase, sulfatase) were monitored during 1 year in parallel and serial treatment units of the biological stage of a communal wastewater treatment plant. The spatial homogeneity of enzyme activities was high (coefficients of variation  0.8) and highly significant (p plant effluent, dry matter content of activated sludge, and sludge volume, were found. The esterase activity was least correlated with other enzymes and often showed deviating dependencies on process parameters, raising questions concerning its appropriateness as a sum parameter for enzymatic and heterotrophic activity.

  2. Peptides in the nervous systems of cnidarians: structure, function, and biosynthesis

    DEFF Research Database (Denmark)

    Grimmelikhuijzen, C J; Leviev, I; Carstensen, Kathrine

    1996-01-01

    molecule. In addition to well-known, "classical" processing enzymes, novel prohormone processing enzymes must be present in cnidarian neurons. These include a processing enzyme hydrolyzing at the C-terminal sides of acidic (Asp and Glu) residues and a dipeptidyl aminopeptidase digesting at the C......-terminal sides of N-terminally located X-Pro and X-Ala sequences. All this shows that the primitive nervous systems of cnidarians are already quite complex, and that neuropeptides play a central role in the physiology of these animals....

  3. Faecal excretion of brush border membrane enzymes in patients with clostridium difficile diarrhoea

    Directory of Open Access Journals (Sweden)

    Katyal R

    2002-01-01

    Full Text Available PURPOSE: To look for the presence of intestinal brush border membrane (BBM enzymes in the faecal samples of patients with Clostridium difficile association. METHODS: One hundred faecal samples were investigated for C.difficile toxin (CDT. Simultaneous assays for faecal excretion of intestinal BBM enzymes viz., disaccharidases, alkaline phosphatase (AP and leucine aminopeptidase (LAP were also done. RESULTS: C.difficile toxin was detected in 25 (25% of the samples with a titre ranging from 10 to 160. No significant difference (p>0.05 was seen between the CDT positive and negative groups with any of the disaccharidases studied. However, significant increase (pC.difficile diarrhoea.

  4. Biosynthesis of intestinal microvillar proteins. Low temperature arrests both processing and intracellular transport

    DEFF Research Database (Denmark)

    Danielsen, E M; Hansen, Gert Helge; Cowell, G M

    1989-01-01

    mannose-glycosylated form characteristic of the newly synthesized enzymes prior to the molecular processing taking place in the Golgi complex. The general morphology of the enterocyte was unaffected by culture at low temperature except for the Golgi complex where the cisternae appeared condensed and...... surrounded by numerous vesicles of 50 to 55 nm. Both molecular processing and microvillar expression could be restored by shifting the temperature to 37 degrees C. Culture at low temperature did not induce any missorting of newly synthesized aminopeptidase N, but both molecular processing and microvillar...

  5. Vimentin binds IRAP and is involved in GLUT4 vesicle trafficking

    International Nuclear Information System (INIS)

    Research highlights: → Vimentin is shown to bind to the N-terminus of insulin-responsive aminopeptidase (IRAP), a major cargo protein of GLUT4 vesicles in 3T3-L1 adipocytes. → GLUT4 translocation to the plasma membrane by insulin is decreased in vimentin-depleted adipocytes. → An interaction between vimentin and IRAP functions to sequester GLUT4 vesicles to the peri-nuclear region of the cell. -- Abstract: Insulin-responsive aminopeptidase (IRAP) and GLUT4 are two major cargo proteins of GLUT4 storage vesicles (GSVs) that are translocated from a postendosomal storage compartment to the plasma membrane (PM) in response to insulin. The cytoplasmic region of IRAP is reportedly involved in retention of GSVs. In this study, vimentin was identified using the cytoplasmic domain of IRAP as bait. The validity of this interaction was confirmed by pull-down assays and immunoprecipitation in 3T3-L1 adipocytes. In addition, it was shown that GLUT4 translocation to the PM by insulin was decreased in vimentin-depleted adipocytes, presumably due to dispersing GSVs away from the cytoskeleton. These findings suggest that the IRAP binding protein, vimentin, plays an important role in retention of GSVs.

  6. Vimentin binds IRAP and is involved in GLUT4 vesicle trafficking

    Energy Technology Data Exchange (ETDEWEB)

    Hirata, Yohko [Department of Nutrition and Metabolism, Institute of Health Biosciences, Tokushima University, Tokushima (Japan); Hosaka, Toshio, E-mail: hosaka@nutr.med.tokushima-u.ac.jp [Department of Public Health and Applied Nutrition, Institute of Health Biosciences, Tokushima University, Tokushima (Japan); Iwata, Takeo [Department of Medical Pharmacology, Institute of Health Biosciences, Tokushima University, Tokushima (Japan); Le, Chung T.K.; Jambaldorj, Bayasgalan; Teshigawara, Kiyoshi; Harada, Nagakatsu; Sakaue, Hiroshi [Department of Nutrition and Metabolism, Institute of Health Biosciences, Tokushima University, Tokushima (Japan); Sakai, Tohru [Department of Public Health and Applied Nutrition, Institute of Health Biosciences, Tokushima University, Tokushima (Japan); Yoshimoto, Katsuhiko [Department of Medical Pharmacology, Institute of Health Biosciences, Tokushima University, Tokushima (Japan); Nakaya, Yutaka [Department of Nutrition and Metabolism, Institute of Health Biosciences, Tokushima University, Tokushima (Japan)

    2011-02-04

    Research highlights: {yields} Vimentin is shown to bind to the N-terminus of insulin-responsive aminopeptidase (IRAP), a major cargo protein of GLUT4 vesicles in 3T3-L1 adipocytes. {yields} GLUT4 translocation to the plasma membrane by insulin is decreased in vimentin-depleted adipocytes. {yields} An interaction between vimentin and IRAP functions to sequester GLUT4 vesicles to the peri-nuclear region of the cell. -- Abstract: Insulin-responsive aminopeptidase (IRAP) and GLUT4 are two major cargo proteins of GLUT4 storage vesicles (GSVs) that are translocated from a postendosomal storage compartment to the plasma membrane (PM) in response to insulin. The cytoplasmic region of IRAP is reportedly involved in retention of GSVs. In this study, vimentin was identified using the cytoplasmic domain of IRAP as bait. The validity of this interaction was confirmed by pull-down assays and immunoprecipitation in 3T3-L1 adipocytes. In addition, it was shown that GLUT4 translocation to the PM by insulin was decreased in vimentin-depleted adipocytes, presumably due to dispersing GSVs away from the cytoskeleton. These findings suggest that the IRAP binding protein, vimentin, plays an important role in retention of GSVs.

  7. Visualization of enzyme activities inside earthworm pores

    Science.gov (United States)

    Hoang, Duyen; Razavi, Bahar S.

    2015-04-01

    In extremely dynamic microhabitats as bio-pores made by earthworm, the in situ enzyme activities are assumed as a footprint of complex biotic interactions. Our study focused on the effect of earthworm on the enzyme activities inside bio-pores and visualizing the differences between bio-pores and earthworm-free soil by zymography technique (Spohn and Kuzyakov, 2013). For the first time, we aimed at quantitative imaging of enzyme activities in bio-pores. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). After two weeks when bio-pore systems were formed by earthworms, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine-aminopeptidase, and phosphatase. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. However, the differences in activity of cellobiohydrolase and leucine aminopeptidase between bio-pore and bulk soil were less pronounced. This demonstrated an applicability of zymography approach to monitor and to distinguish the in situ activity of hydrolytic enzymes in soil biopores.

  8. Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

    Directory of Open Access Journals (Sweden)

    Z. Liu

    2010-05-01

    Full Text Available Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures was enhanced to 60% of a total of 10 cultures (initiated from 8 distinct fetal small intestines, allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39 and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV, may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.

  9. Technological characterization of lactococci isolated from traditional Chinese fermented milks.

    Science.gov (United States)

    Ma, C L; Zhang, L W; Yi, H X; Du, M; Han, X; Zhang, L L; Feng, Z; Zhang, Y C; Li, Q

    2011-04-01

    To screen lactic acid bacteria for starter cultures in cheese production, 21 Lactococcus strains previously isolated from natural fermented milk and koumiss made by herdsman families in the Xinjiang, Gansu, and Qinghai provinces of China were evaluated for optimal growth temperature, acidification activity, proteolytic activity, aminopeptidase activity, and autolytic activity. All isolates presented low acidification rates, and the pH value did not reach 5.3 after 6 h of inoculation in sterile reconstituted skim milk at 30°C. Strains X9C2 and T7C showed the highest proteolytic activity of 24.67 and 23.58 mg of glycine/L of milk, respectively. For aminopeptidase activity, strains X9C2 and T1C2 displayed the highest activities of 30.56 and 27.70 U/mg of protein using L-leucine-p-nitroanilide as substrate, respectively. Autolytic activity in simulated cheese-like buffer ranged from 7.45 to 34.76%, and strains Q14C2 and Q16C showed the highest values of 34.76 and 34.20%, respectively. Collectively, one main finding is that some technological characteristics of Lactococcus isolates from Chinese traditional fermented products varied greatly. Some isolates with potentially important properties could be valuable for application as starter cultures of cheese or could constitute a mixed culture. PMID:21426956

  10. Development of digestive enzyme activity in spotted rose snapper, Lutjanus guttatus (Steindachner, 1869) larvae.

    Science.gov (United States)

    Moguel-Hernández, I; Peña, R; Nolasco-Soria, H; Dumas, S; Zavala-Leal, I

    2014-06-01

    We describe digestive enzyme activity during the larval development of spotted rose snapper, Lutjanus guttatus. Trypsin, chymotrypsin, leucine aminopeptidase, pepsin, amylase, lipase, and acid and alkaline phosphatase activities were evaluated using spectrophotometric techniques from hatching through 30 days. The spotted rose snapper larvae present the same pattern of digestive enzyme activity previously reported for other species in which pancreatic (i.e., trypsin, chymotrypsin, amylase, and lipase) and intestinal (i.e., acid and alkaline phosphatases and leucine aminopeptidase) enzymatic activities are present from hatching allowing the larvae to digest and absorb nutrients in the yolk-sac and live prey by the time of first feeding. The digestive and absorption capacity of the spotted rose snapper increases during the larval development. A significant increase in individual activity of all enzymes occurs at 20 DAH, and around 25 DAH, the juvenile-type of digestion is observed with the appearance of pepsin secreted by the stomach, suggesting that maturation of the digestive function occurs around 20-25 DAH. Our results are in agreement with a previous suggestion that early weaning may be possible from 20 DAH. However, the patterns of enzymatic activities reported in our study should be considered during the formulation of an artificial diet for early weaning of the spotted rose snapper.

  11. Odontogenic keratocysts: a clinical and histological study with special reference to enzyme histochemistry.

    Science.gov (United States)

    Magnusson, B C

    1978-02-01

    Of a total of 1,420 odontogenic cysts, 52 (3.3%) were diagnosed as odontogenic keratocysts. Clinical and histological findings in these 52 cysts are reported. Frozen sections of 26 of the keratocysts were incubated to show the following enzyme activities: NADH2- and NADPH2-diaphorase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, acid phosphatase, leucine aminopeptidase and ATPase. Furthermore, keratinization was studied with the rhodamine B method and lipids with the oil red O, the OTAN and the acid hematein methods. Sections from epidermis, oral mucosa, radicular cysts, residual cysts and follicular cysts served as reference material. The oxidative enzymes showed strong activity in the keratocyst epithelium which contrasted with weak activity in the reference cysts. Acid phosphatase activity was weak in all epithelia except that in keratocysts, which displayed a marked activity. In the fibrous capsule of the keratocyst a high activity of leucine aminopeptidase was recorded. This high activity contrasted with a weak activity in the reference material. The significance of the histochemical results in relation to the aggressive behavior of the keratocyst is discussed. PMID:148497

  12. Prolonged ingestion of prehydrolyzed whey protein induces little or no change in digestive enzymes, but decreases glutaminase activity in exercising rats.

    Science.gov (United States)

    Nery-Diez, Ana Cláudia C; Carvalho, Iara R; Amaya-Farfán, Jaime; Abecia-Soria, Maria Inés; Miyasaka, Célio K; Ferreira, Clécio da S

    2010-08-01

    Because consumption of whey protein hydrolysates is on the increase, the possibility that prolonged ingestion of whey protein hydrolysates affect the digestive system of mammals has prompted us to evaluate the enzymatic activities of pepsin, leucine-aminopeptidase, chymotrypsin, trypsin, and glutaminase in male Wistar rats fed diets containing either a commercial whey isolate or a whey protein hydrolysate with medium degree of hydrolysis and to compare the results with those produced by physical training (sedentary, sedentary-exhausted, trained, and trained-exhausted) in the treadmill for 4 weeks. The enzymatic activities were determined by classical procedures in all groups. No effect due to the form of the whey protein in the diet was seen in the activities of pepsin, trypsin, chymotrypsin, and leucine-aminopeptidase. Training tended to increase the activity of glutaminase, but exhaustion promoted a decrease in the trained animals, and consumption of the hydrolysate decreased it even further. The results are consistent with the conclusion that chronic consumption of a whey protein hydrolysate brings little or no modification of the proteolytic digestive system and that the lowering of glutaminase activity may be associated with an antistress effect, counteracting the effect induced by training in the rat. PMID:20482282

  13. Tyrosine sulfation, a post-translational modification of microvillar enzymes in the small intestinal enterocyte

    DEFF Research Database (Denmark)

    Danielsen, E M

    1987-01-01

    Protein sulfation in small intestinal epithelial cells was studied by labelling of organ cultured mucosal explants with [35S]-sulfate. Six bands in SDS-PAGE became selectively labelled; four, of 250, 200, 166 and 130 kd, were membrane-bound and two, of 75 and 60 kd, were soluble. The sulfated mem...... sulfated. Most if not all the sulfate was bound to tyrosine residues rather than to the carbohydrate of the microvillar enzymes, showing that this type of modification can occur on plasma membrane proteins as well as on secretory proteins.......Protein sulfation in small intestinal epithelial cells was studied by labelling of organ cultured mucosal explants with [35S]-sulfate. Six bands in SDS-PAGE became selectively labelled; four, of 250, 200, 166 and 130 kd, were membrane-bound and two, of 75 and 60 kd, were soluble. The sulfated...... membrane-bound components were all enriched in the microvillar fraction but either absent or barely detectable in intracellular or basolateral membranes. Immunopurification of sucrase-isomaltase, maltase-glucoamylase, aminopeptidase N and aminopeptidase A showed that these microvillar enzymes become...

  14. CHANGES IN LEVELS OF ACTIVITY OF SERINE PROTEASES ACCOMPANY THE EXPOSURE OF COMMON BEAN (PHASEOLUS VULGARIS L. TO WATER DEFICIT

    Directory of Open Access Journals (Sweden)

    M. Budič

    2008-09-01

    Full Text Available A wide variety of proteolytic enzymes exist in plants. On their levels depends protein turnover, a fundamental component in plant development and adaptation to environmental conditions. Cysteine proteases have frequently been reported to be influenced by drought, but only a few serine proteases (SP, among them the trypsin-like enzyme and two aminopeptidases from bean leaves (Bartels and Sunkar, 2005; Hieng et al., 2004. Our starting point was to identify proteolytic activities assigned to SPs that change with drought and then to characterize the corresponding proteases. A quantitative, analytical one-step method was used to separate endopeptidases and aminopeptidases active against a range of substrates in leaf extracts of plants grown in the field (FC. The influence of drought was determined for those of these activities which were confirmed as SPs, based on their inhibition by specific inhibitors. Under water deficit in plants grown under controlled conditions (CC their levels changed in different ways. The levels of SP activities in FC plants, observed during a period of relative drought, were similar to those measured in mildly stressed CC plants. The partial characterisations of some of these SPs will be presented. Our results point to a number of roles for different SPs in the plant response to water stress, which could range from enhanced protein turnover to limited proteolysis at specific sites.

  15. Functionally distinct ERAP1 allotype combinations distinguish individuals with Ankylosing Spondylitis.

    Science.gov (United States)

    Reeves, Emma; Colebatch-Bourn, Alexandra; Elliott, Tim; Edwards, Christopher J; James, Edward

    2014-12-01

    For more than 40 y, expression of HLA-B27 has been strongly associated with the chronic inflammatory disease Ankylosing Spondylitis (AS); however, the mechanisms underlying this association are still unknown. Single nucleotide polymorphisms within the aminopeptidase endoplasmic reticulum aminopeptidase 1 (ERAP1), which is essential for trimming peptides before they are presented to T cells by major histocompatibility complex (MHC) class I molecules, have been linked with disease. We show that ERAP1 is a highly polymorphic molecule comprising allotypes of single nucleotide polymorphisms. The prevalence of specific ERAP1 allotypes is different between AS cases and controls. Both chromosomal copies of ERAP1 are codominantly expressed, and analysis of allotype pairs provided clear stratification of individuals with AS versus controls. Functional analyses demonstrated that ERAP1 allotype pairs seen in AS cases were poor at generating optimal peptide ligands for binding to murine H-2K(b) and -D(b) and the AS-associated HLA-B*2705. We therefore provide strong evidence that polymorphic ERAP1 alters protein function predisposing an individual to AS via its influence on the antigen processing pathway. PMID:25422414

  16. Studies of the radioprotective properties of nicotinyl compounds, aspartic acid, glutamic acid and methionine

    International Nuclear Information System (INIS)

    Radioprotective properties of sodium salts of nicotinyl aspartic acid, nicotinyl methionyl aspartic acid and nicotinyl glutamic acid were tested in mice (NMRI). Experimental animals were irradiated by rayage (9,5 Gy). Parameters were: survival rate, peritoneal fluid cell count, weight and DNA concentration of spleen, hepatic DNA polymerase activity and rate of protein synthesis, lactate dehydrogenase activity in serum, maltase, sucrase and leucine aminopeptidase activitiy in duodenum and jejunum. Following results were obtained: 1. There was no significant difference in survival rate of treated and untreated animals. In treated animals only a short prolongation of survival time was observed. 2. After irradiation a quick reduction of splenic weight and DNA concentration was measured. 3. A reduction of DNA polymerase activity in liver was observed in treated and untreated mice. The rate of hepatic protein synthesis was similar in all animals. A final decrease was observed. 4. Variable activities of maltase, sucrase and leucine aminopeptidase activity in duodenum and jejunum indicated no radioprotective effect of tested substances. In conclusion of these results the tested substances show no significant radioprotective properties. (orig.)

  17. Matched regulation of gastrointestinal performance in the Burmese python, Python molurus.

    Science.gov (United States)

    Cox, Christian L; Secor, Stephen M

    2008-04-01

    In Burmese pythons fasting and feeding cause dramatic regulation of gastric acid production and intestinal nutrient uptake. Predictably, other components of their gastrointestinal tract are similarly regulated with each meal. We therefore assessed the matched regulation of gastrointestinal performance by comparing the postprandial activities and capacities of gastric (pepsin), pancreatic (amylase and trypsin) and intestinal (aminopeptidase-N and maltase) enzymes, and intestinal nutrient uptake. Tissue samples were collected from pythons fasted and at 0.25, 0.5, 1, 2, 3, 4, 6, 10 and 15 days following their consumption of rodent meals equaling 25% of snake body mass. With feeding, pythons experience no significant change in stomach mass, whereas both the pancreas and small intestine doubled in mass. Feeding also triggered a depletion of gastric mucosal pepsinogen, a respective 5.7- and 20-fold increase in the peak activities of pancreatic trypsin and amylase, and a respective 2.3- and 5.5-fold increase in the peak activities of intestinal maltase and aminopeptidase-N. Enzyme activities peaked between 2 and 4 days postfeeding and returned to fasting levels by day 10. Independent of digestive stage, python intestine exhibited a proximal to distal decline in enzyme activity. For both sugars and proteins, intestinal capacities for enzyme activity were significantly correlated with nutrient uptake capacities. The concomitant postprandial upregulation of tissue morphology, intestinal nutrient transport rates and enzyme activities illustrate, for the python, the matched regulation of their gastrointestinal performance with each meal.

  18. Proteinase-producing halophilic lactic acid bacteria isolated from fish sauce fermentation and their ability to produce volatile compounds.

    Science.gov (United States)

    Udomsil, Natteewan; Rodtong, Sureelak; Tanasupawat, Somboon; Yongsawatdigul, Jirawat

    2010-07-15

    Halophilic lactic acid bacteria were isolated from fish sauce mashes fermented at 1 to 12 months. Seven out of sixty-four isolates were selected according to their proteolytic activity and growth at 25% NaCl for characterization and investigation of volatile compound production. All selected isolates were Gram-positive cocci with pairs/tetrads and grew at 0-25% NaCl, pH 4.5-9.0. Results of 16S rRNA gene sequence analysis showed 99% homology to Tetragenococcus halophilus ATCC 33315. The restriction fragment length polymorphism (RFLP) patterns of all isolates were also similar to those of T. halophilus ATCC 33315. These isolates were, thus, identified as T. halophilus. All isolates hydrolyzed fish protein in the medium containing 25% NaCl. Intracellular aminopeptidase of 7 isolates exhibited the highest activity of 2.85-3.67 U/ml toward Ala-p-nitroanilide (Ala-pNA). T.halophilus strains MS33 and M11 showed the highest alanyl aminopeptidase activity (Phalophilus MS33 and MRC5-5-2 were 1-propanol, 2-methylpropanal, and benzaldehyde, corresponding to major volatile compounds in fish sauce. T.halophilus appeared to play an important role in volatile compound formation during fish sauce fermentation. PMID:20541276

  19. Effect of land use on microbial biomass and enzyme activities in tropical soil

    Science.gov (United States)

    Maharjan, Menuka; Sanaullah, Muhammad; Kuzyakov, Yakov

    2016-04-01

    Land use change especially from forest to intensive agriculture for sustaining livelihood causing severe consequence on soil quality. Soil microbial biomass and enzyme activities are very sensitive to change in environment. The objective was to assess effects of three land uses i.e. forest, organic and conventional farming on microbial biomass C and N and enzymes involved in C-cycle (β-glucosidase), N-cycle (leucine-aminopeptidase), P-cycle (Phosphatase) and S-cycle (Sulphatase) at different depth (0-100 cm with 10 cm in interval) of soil in Chitwan, Nepal. The result showed that both carbon and nitrogen content (%) was significantly higher in organic farming than conventional farming and forest. However, the trend decreased in lower depth. Significantly high microbial biomass C and N (μg C and N g-1 soil) were found in organic farming than conventional farming and forest at 0-10 cm but the trend was inconsistent in lower depth. β-glucosidase, leucine-aminopeptidase and sulphatase (nmol g-1 soil) activities were higher in organic and conventional farming compared to forest at 0-20 cm. Phosphatase activity was higher in conventional farming than forest and organic farming at 0-20cm. The activities were inconsistent below 20 cm. Application of farmyard manure and organic matter from the vegetation contributes the higher microbial biomass and enzyme activities in organic farming.

  20. Histochemical distribution of intestinal enzymes of juvenile pacu (Piaractus mesopotamicus) fed lyophilized bovine colostrum.

    Science.gov (United States)

    Moretti, Débora B; Nordi, Wiolene M; Cruz, Thaline M P; Cyrino, José Eurico P; Machado-Neto, Raul

    2014-10-01

    Enzyme activity was evaluated in the intestine of juvenile pacu, Piaractus mesopotamicus, fed diets containing 0, 10 or 20 % of lyophilized bovine colostrum (LBC) inclusion for either 30 or 60 days. The enzymes intestinal acid and alkaline phosphatase (ACP and ALP, respectively), nonspecific esterase (NSE), lipase (LIP), dipeptidyl aminopeptidase IV (DAP IV) and leucine aminopeptidase (LAP) were studied using histochemistry in four intestinal segments (S1, S2, S3 and rectum). Moderate activity of the DAP IV was detected in the three last intestinal segments, but no differences among the treatments were detected. Enzymes LAP, NSE and LIP were weakly stained in all intestinal segments and the inclusion of 10 or 20 % of LBC in the diet commanded a moderate reaction to NSE in the S3 segment at day 60. ACP activity was detected only in the brush border of the S1 segment of fish fed 0 % LBC for either 30 or 60 days. The activity of ALP was very strong in the first intestinal segment, but a weak reaction was seen in the last segments. The inclusion of 20 % of LBC changed the pattern of staining to the ALP, eliciting moderate staining in S2 at day 30 and S1 at day 60. The consumption of diets containing LBC by juvenile pacu did not have significant implications in intestinal enzymatic activity, which still was not fully stimulated. PMID:24823663

  1. DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

    Science.gov (United States)

    Justa-Schuch, Daniela; Silva-Garcia, Maria; Pilla, Esther; Engelke, Michael; Kilisch, Markus; Lenz, Christof; Möller, Ulrike; Nakamura, Fumihiko; Urlaub, Henning; Geiss-Friedlander, Ruth

    2016-01-01

    The aminopeptidase DPP9 removes dipeptides from N-termini of substrates having a proline or alanine in second position. Although linked to several pathways including cell survival and metabolism, the molecular mechanisms underlying these outcomes are poorly understood. We identified a novel interaction of DPP9 with Filamin A, which recruits DPP9 to Syk, a central kinase in B-cell signalling. Syk signalling can be terminated by degradation, requiring the ubiquitin E3 ligase Cbl. We show that DPP9 cleaves Syk to produce a neo N-terminus with serine in position 1. Pulse-chases combined with mutagenesis studies reveal that Ser1 strongly influences Syk stability. Furthermore, DPP9 silencing reduces Cbl interaction with Syk, suggesting that DPP9 processing is a prerequisite for Syk ubiquitination. Consistently, DPP9 inhibition stabilizes Syk, thereby modulating Syk signalling. Taken together, we demonstrate DPP9 as a negative regulator of Syk and conclude that DPP9 is a novel integral aminopeptidase of the N-end rule pathway. DOI: http://dx.doi.org/10.7554/eLife.16370.001 PMID:27614019

  2. Membrane-bound proteases of the gerbil subfornical organ and choroid plexus: an enzyme histochemical study.

    Science.gov (United States)

    Mitro, A; De Bault, L E

    1994-03-01

    Using enzyme-histochemical methods, the membrane-bound peptidases, gamma-glutamyl transpeptidase (gamma-GTP), microsomal alanyl aminopeptidase (mAAP), glutamyl aminopeptidase (EAP), and dipeptidyl peptidase IV (DPP IV), were studied in microvessels of the gerbil subfornical organ (SFO), choroid plexus adjacent to the SFO, and the ependyma of brain ventricle walls in the vicinity of the SFO. Vessels and microvessels of gerbil SFO and choroid plexus were positive for gamma-GTP, mAAP, and EAP, but negative for DPP IV. Blood-brain barrier (BBB) microvessels in the surrounding brain tissue also showed positive reactions for gamma-GTP, mAAP, and EAP but a negative reaction for DPP IV. Both epithelial cells of the choroid plexus and ependymal cells of the ventricle walls were negative for all four studied enzymes. It is suggested that blood-borne peptide hormones which can be substrates for these membrane-bound proteases can be modulated by gamma-GTP, mAAP, and EAP, but not by DPP IV, when they come in contact with the plasma membrane of the endothelial cells of the vessels in gerbil SFO, choroid plexus, and surrounding brain tissue.

  3. Enzyme histochemical studies of membrane proteases in rat subfornical organ.

    Science.gov (United States)

    De Bault, L E; Mitro, A

    1994-12-01

    Localization of membrane proteases glutamyl aminopeptidase (EAP), microsomal alanyl aminopeptidase (mAAP), dipeptidyl peptidase IV (DPP IV) and gamma-glutamyl transpeptidase (gamma-GTP) were studied in vessels of the rat subfornical organ (SFO), ependyma which cover the surface of the SFO, and adjacent brain structures. Results of enzyme histochemical reactions showed strong activity for EAP, mAAP, and gamma-GTP, but absence of DPP IV in microvessels of SFO. The ependyma which cover the SFO was positive for gamma-GTP, but negative for other studied proteases. Our results showed that the spectrum of enzymes in the majority of the vessels of SFO is similar to that of the microvessels of the adjacent brain tissue which were positive for EAP, mAAP, and gamma-GTP, but negative for DPP IV. The relative intensity of the enzyme reactions in vessels varied from central to lateral locations in the SFO and the adjacent brain tissue. There was also a difference in the relative reaction intensity from one enzyme to the other. The presence and heterogeneous distribution of the enzymes are consistent with the hypothesis that membrane proteases of the microvascular endothelium constitute an enzyme-barrier between blood and parenchyma of the SFO and between blood and brain tissue, and may be involved in metabolism or modulation of various peptides when they contact the plasma membrane of the endothelial cells of the vessels.

  4. A remarkable activity of human leukotriene A4 hydrolase (LTA4H) toward unnatural amino acids.

    Science.gov (United States)

    Byzia, Anna; Haeggström, Jesper Z; Salvesen, Guy S; Drag, Marcin

    2014-05-01

    Leukotriene A4 hydrolase (LTA4H--EC 3.3.2.6) is a bifunctional zinc metalloenzyme, which processes LTA4 through an epoxide hydrolase activity and is also able to trim one amino acid at a time from N-terminal peptidic substrates via its aminopeptidase activity. In this report, we have utilized a library of 130 individual proteinogenic and unnatural amino acid fluorogenic substrates to determine the aminopeptidase specificity of this enzyme. We have found that the best proteinogenic amino acid recognized by LTA4H is arginine. However, we have also observed several unnatural amino acids, which were significantly better in terms of cleavage rate (k cat/K m values). Among them, the benzyl ester of aspartic acid exhibited a k cat/K m value that was more than two orders of magnitude higher (1.75 × 10(5) M(-1) s(-1)) as compared to L-Arg (1.5 × 10(3) M(-1) s(-1)). This information can be used for design of potent inhibitors of this enzyme, but may also suggest yet undiscovered functions or specificities of LTA4H.

  5. 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.

  6. Structure of the human histone chaperone FACT Spt16 N-terminal domain

    Energy Technology Data Exchange (ETDEWEB)

    Marcianò, G.; Huang, D. T., E-mail: d.huang@beatson.gla.ac.uk [Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, Scotland (United Kingdom)

    2016-01-22

    The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding.

  7. Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis).

    Science.gov (United States)

    Samac, D; Storey, R

    1981-12-01

    Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling.Jojoba cotyledonary extracts were found to inhibit the enzymic activity of trypsin, chymotrypsin, and pepsin but not the protease from Aspergillus saotoi. The heat-labile trypsin inhibitor substance(s) was found in commercially processed jojoba seed meal and the albumin fraction of seed proteins. Trypsin inhibitor activity decreased with germination.

  8. 棉铃虫中肠氨肽酶 APN4与 Cry1Ac、Cry2Aa 结合能力的比较%Comparison of the binding affinity of the APN4 receptor in Helicoverpa armigera (Hübner) to the Cry1Ac and Cry2Aa insecticidal proteins

    Institute of Scientific and Technical Information of China (English)

    袁向东; 葛朝虹; 肖玉涛; 梁革梅

    2015-01-01

    Objectives] Bt (Bacillus thuringiensis) insecticidal proteins have been widely adopted to control agricultural pests because of their high target specificity. The binding of a Bt insecticidal protein to its specific receptor in the insect midgut plays a key role in the insecticidal action process. Aminopeptidase N (APN) is one of the major Bt protein receptors. To better characterize the molecular mechanism underlying the insecticidal activity of different Bt insecitcidal proteins, and lay the foundation for Bt resistance management and novel Bt insecticidal protein development, we analyzed the binding affinity of aminopeptidase N4 (APN4) to the Cry1Ac and Cry2Aa insecitcidal proteins in Helicoverpa armigera. [Methods] The binding affinity of aminopeptidase N4 (APN4) was assessed using ligand blot analysis and an ELISA binding assay, respectively. [Results] The results show that recombinant APN4 could bind to both Cry1Ac and Cry2A; their respective dissociation constants were 46.7 nmol/L and 26.5 nmol/L. [Conclusion] The results suggest that there was no significant difference in the binding affinity of APN4 to Cry1Ac and Cry2Aa in H. armigera.%【目的】 Bt 杀虫蛋白(Bacillus thuringiensis)具有高度的靶标特异性,已经被广泛用于农业害虫防治。Bt 杀虫蛋白要发挥杀虫活性,必须首先与其受体蛋白结合,氨肽酶 N(Aminopeptidase N)是一类重要的 Bt 受体蛋白。因此,分析该受体与 Bt 杀虫蛋白的结合能力,可为进一步明确不同 Bt 的分子作用机制、Bt 的抗性治理以及新 Bt 的开发应用等提供借鉴。【方法】本文利用 Ligand blot 和 Elisa 方法比较了棉铃虫 Helicoverpa armigera 中肠 APN4(Aminopeptidase N4,APN4)与 Cry1Ac、Cry2Aa 的结合能力。【结果】原核表达的 APN4片段与活化的 Cry1Ac、Cry2Aa 都可以结合,解离常数(Kd)分别是48.59 nmol/L和21.73 nmol/L。【结论】 APN4片段与 Cry1Ac、Cry2Aa 的结合能力

  9. Visualization of enzyme activities inside earthworm biopores by in situ soil zymography

    Science.gov (United States)

    Thu Duyen Hoang, Thi; Razavi, Bahar. S.; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2015-04-01

    Earthworms can strongly activate microorganisms, increase microbial and enzyme activities and consequently the turnover of native soil organic matter. In extremely dynamic microhabitats and hotspots as biopores made by earthworms, the in situ enzyme activities are a footprint of complex biotic interactions. The effect of earthworms on the alteration of enzyme activities inside biopores and the difference between bio-pores and earthworm-free soil was visualized by in situ soil zymography (Spohn and Kuzyakov, 2014). For the first time, we prepared quantitative imaging of enzyme activities in biopores. Furthermore, we developed the zymography technique by direct application of a substrate saturated membrane to the soil to obtain better spatial resolution. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). Simultaneously, maize seed was sown in the soil. Control soil box with maize and without earthworm was prepared in the same way. After two weeks when bio-pore systems were formed by earthworm, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine aminopeptidase) and phosphatase. Followed by non-destructive zymography, biopore samples and control soil were destructively collected to assay enzyme kinetics by fluorogenically labeled substrates method. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. These differences were further confirmed by fluorimetric microplate enzyme assay detected significant difference of Vmax in four above mentioned enzymes. Vmax of β-glucosidase, chitinase, xylanase and phosphatase in biopores is 68%, 108%, 50% and 49% higher than that of control soil. However, no difference in cellobiohydrolase and leucine aminopeptidase kinetics between biopores and control soil were detected. This indicated little effect of earthworms on protein and cellulose transformation in soil

  10. Structural and theoretical studies on rhodium and iridium complexes with 5-nitrosopyrimidines. Effects on the proteolytic regulatory enzymes of the renin-angiotensin system in human tumoral brain cells.

    Science.gov (United States)

    Illán-Cabeza, Nuria A; Jiménez-Pulido, Sonia B; Ramírez-Expósito, María J; García-García, Antonio R; Peña-Ruiz, Tomás; Martínez-Martos, José M; Moreno-Carretero, Miguel N

    2015-02-01

    The reactions of [RhCl(CO)(PPh3)2], [RhCl(CO)2]2 and [IrCl(CO)(PPh3)2] with different 5-nitrosopyrimidines afforded sixteen complexes which have been structurally characterized by elemental analysis, IR and NMR ((1)H and (13)C) spectral methods and luminescence spectroscopy. The crystal and molecular structures of [Rh(III)Cl(VIOH-1)2(PPh3)], [Rh(III)Cl(DVIOH-1)2(PPh3)] and [Rh(II)(DVIOH-1)2(PPh3)2] have been established from single crystal x-ray structure analyses. The three complexes are six-coordinated with both violurato ligands into an equatorial N5,O4-bidentate fashion, but with different mutually arrangements. Theoretical studies were driven on the molecular structure of [Rh(III)Cl(VIOH-1)2(PPh3)] to assess the nature of the metal-ligand interaction as well as the foundations of the cis-trans (3L-2L) isomerism. An assortment of density functional (SOGGA11-X, B1LYP, B3LYP, B3LYP-D3 and wB97XD) has been used, all of them leading to a similar description of the target system. Thus, a topological analysis of the electronic density within AIM scheme and the study of the Mulliken charges yield a metal-ligand link of ionic character. Likewise, it has been proved that the cis-trans isomerism is mainly founded on that metal-ligand interaction with the relativistic effects playing a significant role. Although most of the compounds showed low direct toxicity against the human cell lines NB69 (neuroblastoma) and U373-MG (astroglioma), they differently modify in several ways the renin-angiotensin system (RAS)-regulating proteolytic regulatory enzymes aminopeptidase A (APA), aminopeptidase N (APN) and insulin-regulated aminopeptidase (IRAP). Therefore, these complexes could exert antitumor activity against both brain tumor types, acting through the paracrine regulating system mediated by tissue RAS rather than exerting a direct cytotoxic effect on tumor cells.

  11. Land Cover and Nutrient Loads Explain Changes in Enzymatic Processing of Stream Dissolved Organic Matter

    Science.gov (United States)

    Hosen, J. D.; Febria, C.; McDonough, O.; Palmer, M.

    2012-12-01

    Anthropogenic land use has been shown to alter organic matter composition as well as its processing, export, and retention in headwater streams. Human activities also increase stream nutrient loading and in turn organic matter processing by heterotrophic microbial communities. Using microbial extracellular enzyme activity (EEA) assays combined with dissolved organic matter (DOM) fluorescence spectroscopy, we investigated the interaction between catchment land use, nutrient limitation, heterotrophic microbial communities, and carbon processing in five forested and three urbanized Coastal Plain headwater streams (Maryland, USA). EEA measures microbial production of heterotrophic extracellular enzymes, including aminopeptidase, which facilitates the breakdown of organic nitrogen and phosphatase which facilitates breakdown of organic phosphate. DOM fluorescence spectroscopy enables rapid quantification of different organic matter fluorophores (e.g., amino acid-, humic acid-, and fulvic acid-like). Excitation-emission matrices (EEMs) of DOM fluorescence can be coupled with parallel factor analysis (PARAFAC) for detailed quantitative analysis. Samples were collected quarterly from May 2011 to July 2012 and characterized using both EEA and EEM. We show that significant differences in stream EEA are explained by DOM fluorescence, land cover, and inorganic nutrient inputs. Specifically, urbanized sites were characterized by relatively low ortho-phosphate concentrations, high inorganic nitrogen concentrations, high phosphatase EEA, and greater amino acid-like DOM fluorescence. Aminopeptidase activity increased with increasing amino acid-like DOM fluorescence (i.e., a labile form of DOM for microbes) in forested streams. By contrast aminopeptidase activity did not respond to increasing amino acid-like fluorescence in urbanized streams. This points to a difference in limitation in inorganic nutrients between stream types. Thus, we hypothesize that stream microbial communities

  12. Causal ACTH-Depot Therapy during Pregnancies following Infertility Treatment

    Directory of Open Access Journals (Sweden)

    Rudolf Klimek

    2012-01-01

    Full Text Available The aim of this paper was to confirm the efficacy of adrenocorticotropin depot (ACTH-depot therapy in pregnancies with threatened miscarriage and preterm delivery through the desired stimulation of the adrenal glands controlled by the rest of organism. The activity of hypothalamic-pituitary-adrenal axis plays a key role in pregnancy. Such naturally stimulated endogenous corticosteroid hormones are free from unwanted side effects of their synthetics analogs. Low level of maternal blood ACTH and insufficient increase of induced by hypothalamic hormones oxytocinases (cystine-β-aminopeptidases were indication to ACTH-depot therapy (0.5 mg/week in our consecutive prospective studies. Contrary to antenatal use of synthetic corticosteroids, there are no temporal limits of this therapy, which has to be more often recommended into clinical prevention of fetal morbidity, treatment of premature delivery, and finally elimination of the newborn's mortality caused by the neuroendocrinological gestoses.

  13. Causal ACTH-Depot Therapy during Pregnancies following Infertility Treatment.

    Science.gov (United States)

    Klimek, Rudolf; Klimek, Marek; Gralek, Peter; Jasiczek, Dariusz

    2012-01-01

    The aim of this paper was to confirm the efficacy of adrenocorticotropin depot (ACTH-depot) therapy in pregnancies with threatened miscarriage and preterm delivery through the desired stimulation of the adrenal glands controlled by the rest of organism. The activity of hypothalamic-pituitary-adrenal axis plays a key role in pregnancy. Such naturally stimulated endogenous corticosteroid hormones are free from unwanted side effects of their synthetics analogs. Low level of maternal blood ACTH and insufficient increase of induced by hypothalamic hormones oxytocinases (cystine-β-aminopeptidases) were indication to ACTH-depot therapy (0.5 mg/week) in our consecutive prospective studies. Contrary to antenatal use of synthetic corticosteroids, there are no temporal limits of this therapy, which has to be more often recommended into clinical prevention of fetal morbidity, treatment of premature delivery, and finally elimination of the newborn's mortality caused by the neuroendocrinological gestoses. PMID:22666262

  14. Superoxide dismutase activity as a function of culture aging of B-16 mouse melanoma cells

    Directory of Open Access Journals (Sweden)

    JOVANA B. SIMIC-KRSTIC

    2004-12-01

    Full Text Available The C3 clone of B-16 mouse melanoma was cultured for 1, 6 and 9 days and analysed. The changes which are not directly linked to melanogenesis in the B-16 / C3 cultures during their maturation were characterized. Early (1 day, confluent (6 days and old (9 days cell cultures are distinguished by their leucine aminopeptidase (LAP and a-naphthyl acetate esterase (ANAE isoenzyme patterns. Both quantitative and qualitative changes in LAP and ANAE isoenzyme can be observed during culture maturation. There is an increase in the activity of the enzyme copper, zinc-containing superoxide-dismutase (CuZn SOD. The increaase in the CuZn SOD enzyme activity might be related to B-16/C3 cell melanogenesis and / or to differentiation.

  15. Post-translational suppression of expression of intestinal brush border enzymes by fructose

    DEFF Research Database (Denmark)

    Danielsen, E M

    1989-01-01

    The two major dietary sugars, fructose and sucrose, were found to suppress effectively the biosynthetic renewal of brush border enzymes in the gut. When studied in cultured explants of pig small intestine mucosa, 10-50 mM concentrations of fructose completely prevented the expression of mature...... aminopeptidase N and severely reduced that of sucrase-isomaltase. The instantly occurring and reversible suppressive effect manifested itself as a leupeptin-sensitive degradation of newly synthesized brush border enzymes. The likely mechanism of action of the dietary sugar is by causing an abnormal...... cotranslational glycosylation that in turn triggers a rapid proteolytic breakdown. Our findings suggest that renewal of digestive brush border enzymes is transiently suppressed during intake of fructose- or sucrose-rich meals....

  16. Proteomic Analysis of Responsive Proteins Induced in Japanese Birch Plantlet Treated with Salicylic Acid

    Directory of Open Access Journals (Sweden)

    Hiromu Suzuki

    2014-07-01

    Full Text Available The present study was performed to unravel the mechanisms of systemic acquired resistance (SAR establishment and resistance signaling pathways against the canker-rot fungus (Inonotus obliquus strain IO-U1 infection in Japanese birch plantlet No.8. Modulation of protein-profile induced by salicylic acid (SA-administration was analyzed, and SA-responsive proteins were identified. In total, 5 specifically expressed, 3 significantly increased, and 3 significantly decreased protein spots were identified using liquid chromatography/tandem mass spectrometry (LC/MS/MS and the sequence tag method. These proteins were malate dehydrogenase, succinate dehydrogenase, phosphoglycerate kinase, diaminopimalate decarboxylase, arginase, chorismate mutase, cyclophilin, aminopeptidase, and unknown function proteins. These proteins are considered to be involved in SAR-establishment mechanisms in the Japanese birch plantlet No 8.

  17. [Lysosomal proteinasen and peptidasen in serum of children with inflammatory diseases (author's transl)].

    Science.gov (United States)

    Appel, W; Huth, E; Herrmann, H

    1976-08-01

    In the serum of 43 children the activities of proteinases and peptidases by mean of 41 substrates have been determined in order to get knowledge of overall activities and differentiation of lysosomal proteolytic enzymes. Proteinases, cathepsins A, B, C and D, aminopeptidases, carboxypeptidases, dipeptidases, tripeptidases and aminoacidarylamidases have been checked. The enzyme pattern of the serum of a collective of 15 healthy children or those without serious clinical signs is demonstrated, also the alterations and differentiations in the serum of children with leucemia, pneumonia, inflammatory diseases of the respiratory tract, other inflammatory diseases and common diseases. Leucyl-glycyl-glycyltripeptidase, glycyl-glycyl-glycyltripeptidase, a proteosterase, carboxypeptidase A, a neutrale proteinase and basic proteinase (cathepsin B) and cathepsin C are increased. A distinct elevation has been found only in children with leucemia and pneumonia.

  18. Folding of intestinal brush border enzymes. Evidence that high-mannose glycosylation is an essential early event

    DEFF Research Database (Denmark)

    Danielsen, E M

    1992-01-01

    enzymes. In pulse-labeled mucosal explants, complete synthesis of the polypeptide chains of aminopeptidase N and sucrase-isomaltase required about 2 and 4 min, respectively, whereas maximal antiserum precipitation was acquired with half-times of 4-5 and 8 min, respectively. Fructose, which induces a...... posttranslational process. In the presence of fructose, not only the malglycosylated forms but also the electrophoretically normal, high-mannose-glycosylated form of the brush border enzymes were retained in the endoplasmic reticulum and proteolytically degraded. The results obtained demonstrate an intimate...... interrelationship between glycosylation and polypeptide folding in the synthesis of membrane glycoproteins and, more specifically, indicate that the timing of these two early biosynthetic events is essential for correct polypeptide folding....

  19. Distribution of three microvillar enzymes along the small intestinal crypt-villus axis

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Niels-Christiansen, L L; Poulsen, M D;

    1994-01-01

    of the axis, whereas ApN is found mainly in the villar and upper crypt enterocytes. All three enzymes are detected in the basolateral membrane at all levels along the crypt-villus axis, although ApN and S-I only occurred at low intensities in the villus region. The microvillar/basolateral labelling...... ratio for the three enzymes increases to a varying degree for the three enzymes along the axis suggesting that the sorting efficiency to the apical membrane improves at least for ApN and S-I as the cells mature. These findings might indicate that the enterocytes change from a transcytotic to a direct......Aminopeptidase N (ApN), dipeptidyl peptidase IV (DPP IV) and sucrose-isomaltase (S-I) are differentially expressed along the pig jejunum crypt-villus axis. Quantitative immunoelectron microscopy and enzyme cytochemistry show that DPP IV and S-I are expressed in enterocytes along the entire length...

  20. Sialic Acid Binding Properties of Soluble Coronavirus Spike (S1 Proteins: Differences between Infectious Bronchitis Virus and Transmissible Gastroenteritis Virus

    Directory of Open Access Journals (Sweden)

    Christine Winter

    2013-07-01

    Full Text Available The spike proteins of a number of coronaviruses are able to bind to sialic acids present on the cell surface. The importance of this sialic acid binding ability during infection is, however, quite different. We compared the spike protein of transmissible gastroenteritis virus (TGEV and the spike protein of infectious bronchitis virus (IBV. Whereas sialic acid is the only receptor determinant known so far for IBV, TGEV requires interaction with its receptor aminopeptidase N to initiate infection of cells. Binding tests with soluble spike proteins carrying an IgG Fc-tag revealed pronounced differences between these two viral proteins. Binding of the IBV spike protein to host cells was in all experiments sialic acid dependent, whereas the soluble TGEV spike showed binding to APN but had no detectable sialic acid binding activity. Our results underline the different ways in which binding to sialoglycoconjugates is mediated by coronavirus spike proteins.

  1. Oxytocinase-immunohistochemical demonstration in the immature and term human placenta.

    Science.gov (United States)

    Small, C W; Watkins, W B

    1975-10-27

    Oxytocinase (cystine aminopeptidase) was purified from human retroplacental serum by a combination of fractional precipitation, hydroxylapatite chromatography and gel exlusion chromatography on Sephadex G-200. The purified enzyme possessed a specific activity of 980 mIU/mg using L-cystine-di-p-nitroanilide as substrate. This represented a 3200 fold concentration from the starting material in an overall yield of 12%. Antibodies against oxytocinase were raised in rabbits and the gamma-globulins fraction labelled with fluorescein isothiocyanate prior to its use in the immunofluorescence histochemical localization of the enzyme in human placental tissue. Oxytocinase was confined to the syncytiotrophoblastic cells of normal term, and immature placentas as well as in placentas from patients suffering from severe toxaemia. Specific immunofluorescence was also present in the outer margins of the chorion and to a lesser extent in the amnion.

  2. Functional Genomics and Its Bench-to-Bedside Translation Pertaining to the Identified Susceptibility Alleles and Loci in Ankylosing Spondylitis.

    Science.gov (United States)

    Kenna, Tony J; Hanson, Aimee; Costello, Mary-Ellen; Brown, Matthew A

    2016-10-01

    Ankylosing spondylitis (AS) is a highly heritable disease for which there is a great unmet need for improved therapies. Genetics research has identified several major pathways involved in the disease, from which treatments have either now entered clinical practice or are in development. In particular, therapies targeting the IL-23 pathway were repositioned for use in AS following the discovery of multiple genes in the pathway as determinants of AS risk. Discovery of the association of aminopeptidase genes with AS, and subsequently with psoriasis, inflammatory bowel disease and other conditions, has triggered research into therapies targeting this pathway. The AS-genetic associations point to involvement of gut mucosal immunity in driving disease, and metagenomic studies have provided strong support that AS is a disease driven by interaction between the gut microbiome and host immune system, providing a rationale for the exploration of gut-targeted therapies for the disease. PMID:27641916

  3. Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 1b of Escherichia coli

    International Nuclear Information System (INIS)

    This paper reports the sequence of the active site peptide of penicillin-binding protein 1b from Escherichia coli. Purified penicillin-binding protein 1b was labeled with [14C]penicillin G, digested with trypsin, and partially purified by gel filtration. Upon further purification by high-pressure liquid chromatography, two radioactive peaks were observed, and the major peak, representing over 75% of the applied radioactivity, was submitted to amino acid analysis and sequencing. The sequence Ser-Ile-Gly-Ser-Leu-Ala-Lys was obtained. The active site nucleophile was identified by digesting the purified peptide with aminopeptidase M and separating the radioactive products on high-pressure liquid chromatography. Amino acid analysis confirmed that the serine residue in the middle of the sequence was covalently bonded to the [14C]penicilloyl moiety. A comparison of this sequence to active site sequences of other penicillin-binding proteins and beta-lactamases is presented

  4. A importância de ERAP1 e PPARץ para o desenvolvimento de artrite

    OpenAIRE

    Freire, Patrícia Colchete

    2013-01-01

    A inflamação das articulações, conhecida por artrite, é uma doença que afecta mais de 175 milhões de pessoas no mundo e, além de ter um grande impacto na qualidade de vida dos doentes, constitui uma carga monetária elevada. Mutações em ERAP1 – uma aminopeptidase comummente encontrada no RE e responsável pela apresentação de péptidos ao sistema imunitário – e PPARγ – um receptor nuclear mais frequentemente associado com o processamento de ácidos gordos e glucose – foram previamente associadas ...

  5. ERAP1-ERAP2 dimerization increases peptide-trimming efficiency.

    Science.gov (United States)

    Evnouchidou, Irini; Weimershaus, Mirjana; Saveanu, Loredana; van Endert, Peter

    2014-07-15

    The endoplasmic reticulum aminopeptidases (ERAP)1 and ERAP2 play a critical role in the production of final epitopes presented by MHC class I molecules. Formation of heterodimers by ERAP1 and ERAP2 has been proposed to facilitate trimming of epitope precursor peptides, but the effects of dimerization on ERAP function remain unknown. In this study, we produced stabilized ERAP1-ERAP2 heterodimers and found that they produced several mature epitopes more efficiently than a mix of the two enzymes unable to dimerize. Physical interaction with ERAP2 changes basic enzymatic parameters of ERAP1 and improves its substrate-binding affinity. Thus, by bringing the two enzymes in proximity and by producing allosteric effects on ERAP1, dimerization of ERAP1/2 creates complexes with superior peptide-trimming efficacy. Such complexes are likely to enhance Ag presentation by cells displaying coordinated expression of the two enzymes. PMID:24928998

  6. Isoenzymatic polymorphism in Citrus spp. and Poncirus trifoliata (L. Raf. (Rutaceae

    Directory of Open Access Journals (Sweden)

    Novelli Valdenice Moreira

    2000-01-01

    Full Text Available Isoenzymatic polymorphism analysis was used to determine genetic variability among species and hybrids of Citrus spp. and one accession of Poncirus trifoliata (L. Raf. Ten enzymatic systems aspartate aminotransferase (AAT, acid phosphatase (ACP, leucine aminopeptidase (LAP, 6-phosphogluconate dehydrogenase (6-PGD, isocitrate dehydrogenase (IDH, phosphoglucoisomerase (PGI, phosphoglucomutase (PGM, diaphorase (DIA, shikimate dehydrogenase (SKD and peroxidase (PRX were analyzed. Twenty loci and 48 alleles were identified. Sweet orange cultivars (C. sinensis (L. Osbeck showed the highest polymorphism with the largest number of heterozygous loci, although the alleles of those loci were the same in all cultivars, with the exception of Westin and Lima graúda. Mandarins (C. reticulata Blanco exhibited diverse patterns, whereas Poncirus trifoliata (L. Raf. showed high variability with all Citrus species and hybrids. Exclusive phenotypes were observed in some enzymatic systems, and similar patterns were found among interspecific hybrids and their putative parents.

  7. Comparison among Different Gilthead Sea Bream (Sparus aurata Farming Systems: Activity of Intestinal and Hepatic Enzymes and 13C-NMR Analysis of Lipids

    Directory of Open Access Journals (Sweden)

    Vincenzo Zonno

    2009-12-01

    Full Text Available In order to evaluate differences in general health and nutritional values of gilthead sea bream (Sparus aurata, the effects of semi-intensive, land-based tanks and sea-cages intensive rearing systems were investigated, and results compared with captured wild fish. The physiological state was determined by measuring the activity of three different intestinal digestive enzymes: alkaline phosphatase (ALP, leucine aminopeptidase (LAP and maltase; and the activity of the hepatic ALP. Also, the hepatic content in protein, cholesterol, and lipid were assessed. 13C-NMR analysis for qualitative and quantitative characterization of the lipid fraction extracted from fish muscles for semiintensive and land based tanks intensive systems was performed. The lipid fraction composition showed small but significant differences in the monounsaturated/saturated fatty acid ratio, with the semi-intensive characterized by higher monounsaturated and lower saturated fatty acid content with respect to land based tanks intensive rearing system.

  8. Presence of membranous vesicles in cat seminal plasma: ultrastructural characteristics, protein profile and enzymatic activity.

    Science.gov (United States)

    Polisca, A; Troisi, A; Minelli, A; Bellezza, I; Fontbonne, A; Zelli, R

    2015-02-01

    This study sought to verify the presence of membranous vesicles in cat seminal plasma by means of transmission electron microscopy and to identify protein profile and some of the enzymatic activities associated with these particles. The transmission electron microscopy observations showed the existence of different sized vesicular membranous structures of more or less spherical shape. These vesicles were surrounded by single-, double- or multiple-layered laminar membranes. The vesicle diameters ranged from 16.3 to 387.4 nm, with a mean of 116.5 ± 70.7 nm. Enzyme activity determinations showed the presence of dipeptilpeptidase IV, aminopeptidase, alkaline and acid phosphatase. To our knowledge, this is the first report that identifies and characterizes the membranous vesicles in cat seminal plasma. However, further studies are necessary to identify the exact site of production of these membranous vesicles in the cat male genital tract and to determine their specific roles in the reproductive events of this species.

  9. Characterisation and partial purification of proteolytic enzymes from sardine by-products to obtain concentrated hydrolysates.

    Science.gov (United States)

    Castro-Ceseña, Ana Bertha; del Pilar Sánchez-Saavedra, M; Márquez-Rocha, Facundo J

    2012-11-15

    A procedure to recover proteases and lipases from the by-products of Monterey sardine (Sardinops sagax caerulea) has been developed, comprising 2 steps: a centrifugation at low temperature to eliminate more than 90% of the initial fat content, and an acetone precipitation step. After this treatment, enzymatic activity increased by 33.8% for lipase, 15.5% for trypsin, 14.8% for chymotrypsin, 93.4% for aminopeptidase, and 19.7% for pepsin. The extents of hydrolysis of fish by-product proteins by endogenous enzyme by-product extract, viscera concentrate extract, and commercial Alcalase® were 62%, 85%, and 28%, respectively. The two extract preparations from sardine by-product (viscera and by-product concentrate extracts) produced 3-fold greater hydrolysis than with the commercial enzyme. The recovery of enzyme concentrates from sardine waste has both ecological and economical advantages for the fish industry. PMID:22868132

  10. Carbohydrate maldigestion induces necrotizing enterocolitis in preterm pigs

    DEFF Research Database (Denmark)

    Thymann, Thomas; Møller, Hanne; Stoll, Barbara;

    2009-01-01

    , and aminopeptidase; reduced villus height; transiently reduced in vivo aldohexose uptake; and reduced ex vivo aldohexose uptake capacity in the middle region of the small intestine. Bacterial diversity was low for both diets, but alterations in bacterial composition and luminal concentrations of short-chain fatty......Thymann T, Moller HK, Stoll B, Stoy AC, Buddington RK, Bering SB, Jensen BB, Olutoye OO, Siggers RH, Molbak L, Sangild PT, Burrin DG. Carbohydrate maldigestion induces necrotizing enterocolitis in preterm pigs. Am J Physiol Gastrointest Liver Physiol 297: G1115-G1125, 2009. First published October...... 1, 2009; doi: 10.1152/ajpgi.00261.2009. Necrotizing enterocolitis (NEC) remains the most severe gastrointestinal disorder in preterm infants. It is associated with the initiation of enteral nutrition and may be related to immature carbohydrate digestive capacity. We tested the hypothesis...

  11. Activity and stability of recombinant human superoxide dismutase in buffer solutions and hypothermic perfusates.

    Directory of Open Access Journals (Sweden)

    Senoo,Yoshimasa

    1988-06-01

    Full Text Available The stability of recombinant human superoxide dismutase (r-hSOD in buffer solutions was studied in solutions at various pH and temperatures. Additionally, we studied the effects of incubation with proteases, serum and two types of hypothermic perfusates. R-hSOD was stable in the pH range of 6-11 and at temperatures up to 80 degrees C for 30 min. R-hSOD activity was not affected by incubation with trypsin, aminopeptidase M or serum for 2 h. R-hSOD activity determined at various temperatures (4-37 degrees C did not vary remarkably. R-hSOD in hypothermic perfusates was stable at 4-37 degrees C for 24 h.

  12. Pepsin Egg White Hydrolysate Ameliorates Obesity-Related Oxidative Stress, Inflammation and Steatosis in Zucker Fatty Rats.

    Directory of Open Access Journals (Sweden)

    M Garcés-Rimón

    Full Text Available The aim of this work was to evaluate the effect of the administration of egg white hydrolysates on obesity-related disorders, with a focus on lipid metabolism, inflammation and oxidative stress, in Zucker fatty rats. Obese Zucker rats received water, pepsin egg white hydrolysate (750 mg/kg/day or Rhizopus aminopeptidase egg white hydrolysate (750 mg/kg/day for 12 weeks. Lean Zucker rats received water. Body weight, solid and liquid intakes were weekly measured. At the end of the study, urine, faeces, different organs and blood samples were collected. The consumption of egg white hydrolysed with pepsin significantly decreased the epididymal adipose tissue, improved hepatic steatosis, and lowered plasmatic concentration of free fatty acids in the obese animals. It also decreased plasma levels of tumor necrosis factor-alpha and reduced oxidative stress. Pepsin egg white hydrolysate could be used as a tool to improve obesity-related complications.

  13. Isoenzyme analysis of Arthrobotrys, a nematode-trapping fungus

    Directory of Open Access Journals (Sweden)

    J.V. Araújo

    1997-10-01

    Full Text Available Extraction and isoenzyme analysis of four isolates of Arthrobotrys including A. musiformis, A. robusta and A. conoides were conducted. Among the 14 enzymes studied by starch gel electrophoresis, using morpholine-citrate as gel/electrode buffer, the following nine enzymes showed interpretable banding patterns: a-esterase, fumarase, hexokinase, isocitrate dehydrogenase, leucine aminopeptidase, malate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase and phosphoglucoisomerase. All isolates studied displayed typical isoenzyme phenotypes for each species. Two isolates of A. conoides differed in their a-isoesterase banding patterns, but no differences were observed for the other enzymes. The assay was satisfactory for enzyme extraction and resolution of Arthrobotrys and could be used in future taxonomic and genetic studies of this organism

  14. Fasciola hepatica: Avances en el empleo de candidatos vacunales (Fasciola hepatica: Advance of vaccine candidates’ employ

    Directory of Open Access Journals (Sweden)

    Naranjo, Feliciano, Dany;

    2008-04-01

    Full Text Available ResumenDiferentes estudios sugieren que la vacunación con determinados antígenos de Fasciola hepatica puede ser un medio eficaz para el control de esta enfermedad. Este artículo revisa los resultados de varios ensayos vacunales recientes con los más importantes candidatos vacunales: proteínas de unión a ácidos grasos (FABP, cisteíno proteasas (catepsinas, hemoglobina, leucin aminopeptidasa (LAP y proteínas del tipo saponina.SummarySeveral studies suggest the vaccination with determined Fasciola hepatica’s antigens can be an efficient mean for the control of this disease. This paper reviews the results of several recent vaccine trials with the major vaccine candidates: fatty acid binding protein (FABP, cysteine (cathepsins proteases, haemoglobulin, leucine aminopeptidase (LAP, and a saposin-like protein.

  15. Identification of ABCC2 as a binding protein of Cry1Ac on brush border membrane vesicles from Helicoverpa armigera by an improved pull-down assay.

    Science.gov (United States)

    Zhou, Zishan; Wang, Zeyu; Liu, Yuxiao; Liang, Gemei; Shu, Changlong; Song, Fuping; Zhou, Xueping; Bravo, Alejandra; Soberón, Mario; Zhang, Jie

    2016-08-01

    Cry1Ac toxin-binding proteins from Helicoverpa armigera brush border membrane vesicles were identified by an improved pull-down method that involves coupling Cry1Ac to CNBr agarose combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). According to the LC-MS/MS results, Cry1Ac toxin could bind to six classes of aminopeptidase-N, alkaline phosphatase, cadherin-like protein, ATP-binding cassette transporter subfamily C protein (ABCC2), actin, ATPase, polycalin, and some other proteins not previously characterized as Cry toxin-binding molecules such as dipeptidyl peptidase or carboxyl/choline esterase and some serine proteases. This is the first report that suggests the direct binding of Cry1Ac toxin to ABCC2 in H. armigera. PMID:27037552

  16. Deep-apical tubules: dynamic lipid-raft microdomains in the brush-border region of enterocytes

    DEFF Research Database (Denmark)

    Hansen, Gert H; Pedersen, Jens; Niels-Christiansen, Lise-Lotte;

    2003-01-01

    microdomains. Deep-apical tubules were positioned close to the actin rootlets of adjacent microvilli in the terminal web region, which had a diameter of 50-100 nm, and penetrated up to 1 microm into the cytoplasm. Markers for transcytosis, IgA and the polymeric immunoglobulin receptor, as well as the resident...... lipid raft-containing compartments, but little is otherwise known about these raft microdomains. We therefore studied in closer detail apical lipid-raft compartments in enterocytes by immunogold electron microscopy and biochemical analyses. Novel membrane structures, deep-apical tubules, were visualized...... brush-border enzyme aminopeptidase N, were present in these deep-apical tubules. We propose that deep-apical tubules are a specialized lipid-raft microdomain in the brush-border region functioning as a hub in membrane trafficking at the brush border. In addition, the sensitivity to cholesterol depletion...

  17. Expressed sequence tags from larval gut of the European corn borer (Ostrinia nubilalis: Exploring candidate genes potentially involved in Bacillus thuringiensis toxicity and resistance

    Directory of Open Access Journals (Sweden)

    Crespo Andre LB

    2009-06-01

    Full Text Available Abstract Background Lepidoptera represents more than 160,000 insect species which include some of the most devastating pests of crops, forests, and stored products. However, the genomic information on lepidopteran insects is very limited. Only a few studies have focused on developing expressed sequence tag (EST libraries from the guts of lepidopteran larvae. Knowledge of the genes that are expressed in the insect gut are crucial for understanding basic physiology of food digestion, their interactions with Bacillus thuringiensis (Bt toxins, and for discovering new targets for novel toxins for use in pest management. This study analyzed the ESTs generated from the larval gut of the European corn borer (ECB, Ostrinia nubilalis, one of the most destructive pests of corn in North America and the western world. Our goals were to establish an ECB larval gut-specific EST database as a genomic resource for future research and to explore candidate genes potentially involved in insect-Bt interactions and Bt resistance in ECB. Results We constructed two cDNA libraries from the guts of the fifth-instar larvae of ECB and sequenced a total of 15,000 ESTs from these libraries. A total of 12,519 ESTs (83.4% appeared to be high quality with an average length of 656 bp. These ESTs represented 2,895 unique sequences, including 1,738 singletons and 1,157 contigs. Among the unique sequences, 62.7% encoded putative proteins that shared significant sequence similarities (E-value ≤ 10-3with the sequences available in GenBank. Our EST analysis revealed 52 candidate genes that potentially have roles in Bt toxicity and resistance. These genes encode 18 trypsin-like proteases, 18 chymotrypsin-like proteases, 13 aminopeptidases, 2 alkaline phosphatases and 1 cadherin-like protein. Comparisons of expression profiles of 41 selected candidate genes between Cry1Ab-susceptible and resistant strains of ECB by RT-PCR showed apparently decreased expressions in 2 trypsin-like and 2

  18. Reliable five-minute test strip method for identification of Streptococcus pyogenes.

    Science.gov (United States)

    Dealler, S F; Campbell, L; Kerr, K G; McGoldrick, J; Flannigan, K A; Hawkey, P M

    1989-04-01

    A novel and rapid method (Strep Strip, Lab M) for identification of Streptococcus pyogenes was evaluated. The method combines the established test for pyroglutamyl aminopeptidase (PYR) with a rapid chromogenic test for beta-glucosidase on a paper strip. The test was evaluated with 274 clinical isolates and 237 culture collection isolates of beta-haemolytic streptococci. Streptococcus pyogenes was identified with 100% specificity. Six isolates identified as Lancefield group A and which might therefore be assumed to be Streptococcus pyogenes were shown by the test strip method not to be this species. The beta-glucosidase test on the test strip allowed differentiation between enterococci and Streptococcus pyogenes (both PYR positive) in all cases. The test strip method represents an economical and accurate method for identification of Streptococcus pyogenes in clinical material. PMID:2565813

  19. Time course proteomic profiling of cellular responses to immunological challenge in the sea urchin, Heliocidaris erythrogramma.

    Science.gov (United States)

    Dheilly, Nolwenn M; Haynes, Paul A; Raftos, David A; Nair, Sham V

    2012-06-01

    Genome sequences and high diversity cDNA arrays have provided a detailed molecular understanding of immune responses in a number of invertebrates, including sea urchins. However, complementary analyses have not been undertaken at the level of proteins. Here, we use shotgun proteomics to describe changes in the abundance of proteins from coelomocytes of sea urchins after immunological challenge and wounding. The relative abundance of 345 reproducibly identified proteins were measured 6, 24 and 48 h after injection. Significant changes in the relative abundance of 188 proteins were detected. These included pathogen-binding proteins, such as the complement component C3 and scavenger receptor cysteine rich proteins, as well as proteins responsible for cytoskeletal remodeling, endocytosis and intracellular signaling. An initial systemic reaction to wounding was followed by a more specific response to immunological challenge involving proteins such as apolipophorin, dual oxidase, fibrocystin L, aminopeptidase N and α-2-macroglobulin.

  20. Galectin-4 and small intestinal brush border enzymes form clusters

    DEFF Research Database (Denmark)

    Danielsen, E M; van Deurs, B

    1997-01-01

    Detergent-insoluble complexes prepared from pig small intestine are highly enriched in several transmembrane brush border enzymes including aminopeptidase N and sucrase-isomaltase, indicating that they reside in a glycolipid-rich environment in vivo. In the present work galectin-4, an animal lectin...... lacking a N-terminal signal peptide for membrane translocation, was discovered in these complexes as well, and in gradient centrifugation brush border enzymes and galectin-4 formed distinct soluble high molecular weight clusters. Immunoperoxidase cytochemistry and immunogold electron microscopy showed...... by a nonclassical pathway, and the brush border enzymes represent a novel class of natural ligands for a member of the galectin family. Newly synthesized galectin-4 is rapidly "trapped" by association with intracellular structures prior to its apical secretion, but once externalized, association with brush border...

  1. Enzyme Activities in Waste Water and Activated Sludge

    DEFF Research Database (Denmark)

    Nybroe, Ole; Jørgensen, Per Elberg; Henze, Mogens

    1992-01-01

    The purpose of the present study was to evaluate the potential of selected enzyme activity assays to determine microbial abundance and heterotrophic activity in waste water and activated sludge. In waste water, esterase and dehydrogenase activities were found to correlate with microbial abundance...... measured as colony forming units of heterotrophic bacteria. A panel of four enzyme activity assays, α-glucosidase, alanine-aminopeptidase, esterase and dehydrogenase were used to characterize activated sludge and anaerobic hydrolysis sludge from a pilot scale plant. The enzymatic activity profiles were...... distinctly different, suggesting that microbial populations were different, or had different physiological properties, in the two types of sludge. Enzyme activity profiles in activated sludge from four full-scale plants seemed to be highly influenced by the composition of the inlet. Addition of hydrolysed...

  2. Allium sativum aqueous extract prevents potassium dichromate-induced nephrotoxicity and lipid oxidation in rats

    Directory of Open Access Journals (Sweden)

    Sergio L. Becerra-Torres

    2014-04-01

    Full Text Available Context: The potassium dichromate (K2Cr2O7 induces nephrotoxicity by oxidative stress mechanisms. Aims: To study the potential protection of an aqueous extract of Allium sativum against the K2Cr2O7-induced nephrotoxicity and lipid oxidation in rats. Methods: Twenty four hours after treatment, biomarkers such as proteinuria, creatinine clearance, malondialdehyde production, specific enzyme activity of gamma glutamyl transpeptidase and alanine aminopeptidase, and renal clearance of para-aminohippuric acid and inulin were measured. Results: The K2Cr2O7 caused significant renal dysfunction, but A. sativum extract prevented this condition by improving all measured biomarkers. Conclusions: A single injection of K2Cr2O7 induced nephrotoxicity in rats, but the supply of an Allium sativum aqueous extract prevented the disorders caused by this metal.

  3. Characterization of the proteases in the midgut of the xylophagous larvae of Oemona hirta (Coleoptera:Cerambycidae)

    Institute of Scientific and Technical Information of China (English)

    Brian David Shaw; John Tane Christeller

    2009-01-01

    The protein digestive capability oftbe larvae of the longhorn beetle (Oemona hirta,Coleoptera:Cerambycidae,Fabricius,1775) was investigated.This species feeds only on wood where there is a high proportion of vascular tissue.The pH of the midgut,the major digestive organ,was alkaline and protein hydrolysis was maximal at alkaline pH.Use of specific synthetic peptide substrates showed that the major protease activities were the endopeptidases,trypsin and chymotrypsin-like activity,and the exopeptidase,leucine aminopeptidase and the pH curves corresponded to that with protein substrate.Studies using a range ofsefine protease inhibitors as well as specific inhibitors ofmetalloproteases,cysteine proteases and aspartate proteases confirmed a serine protease-based digestive system similar to earlier reports of sapwood-feeding Cerambycids.Control of these insect pests using protease inhibitors is discussed.

  4. Dipeptidyl peptidase IV and its inhibitors: therapeutics for type 2 diabetes and what else?

    Science.gov (United States)

    Juillerat-Jeanneret, Lucienne

    2014-03-27

    The proline-specific dipeptidyl aminopeptidase IV (DPP IV, DPP-4, CD26), widely expressed in mammalians, releases X-Pro/Ala dipeptides from the N-terminus of peptides. DPP IV is responsible of the degradation of the incretin peptide hormones regulating blood glucose levels. Several families of DPP IV inhibitors have been synthesized and evaluated. Their positive effects on the degradation of the incretins and the control of blood glucose levels have been demonstrated in biological models and in clinical trials. Presently, several DPP IV inhibitors, the "gliptins", are approved for type 2 diabetes or are under clinical evaluation. However, the gliptins may also be of therapeutic interest for other diseases beyond the inhibition of incretin degradation. In this Perspective, the biological functions and potential substrates of DPP IV enzymes are reviewed and the characteristics of the DPP IV inhibitors are discussed in view of type 2 diabetes and further therapeutic interest. PMID:24099035

  5. 新生血管靶向肽NGR与肿瘤靶向治疗%Relationship between neovasculature homing motif NGR and tumor targeted therapy

    Institute of Scientific and Technical Information of China (English)

    冯飞雪; 夏海滨

    2011-01-01

    NGR (Asn-Gly-Arg) motif exploited by phage display can selectively recognize neovasculature by its binding to an endothelium-associated form of aminopeptidase N (CD13).NGR peptides can be used for ligand-directed targeted delivery of various drugs and viral vectors to tumors or to other tissues with an angiogenesis process.NGR can convert to isoaspartateglycine-arginine (isoDGR) by asparagine deamidation, isoDGR is a ligand for αvβ3 integrin, which could be used as a new targeting peptide of tumor neovasculature for the study of tumor targeted therapy.The paper reviews the structural and functional properties of the NGR motif and its application in tumor targeted therapy.%NGR(Asn-Gly-Arg)是通过噬菌体展示技术筛选出来的能够和肿瘤新生血管特异结合的三肽模体,可以通过内皮细胞上的氨肽酶N(aminopeptidase N,亦称CD13)与新生血管发生特异性的结合.NGR多肽可以将多种药物分子和病毒载体靶向运输到肿瘤或者进行血管再生的组织中.NGR模体上的天冬酰胺脱酰胺后生成异天冬氨酸-甘氨酸-精氨酸异构体(iso DGR).isoDGR是整联蛋αvβ3的配体,可以作为一种新的肿瘤新生血管靶向肽用于肿瘤靶向治疗的研究.本文主要对NGR模体的结构和功能以及其在肿瘤靶向治疗中的应用作一综述.

  6. Aurelia aurita ephyrae reshape a coastal microbial community

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    Luca eZoccarato

    2016-05-01

    Full Text Available Over the last two decades, increasing attention has been paid to the impact of jellyfish blooms on marine communities. Aurelia aurita is one of the most studied of the Scyphozoans, and several studies have been carried out to describe its role as a top-down controller within the classical food web. However, little data are available to define the effects of these jellyfish on microbial communities. The aims of this study were to describe the predation impact of A. aurita ephyrae on a natural microplanktonic assemblage, and to determine any reshaping effects on the prokaryote community composition and functioning. Surface coastal water was used to set up a 24-h grazing experiment in microcosms. Samples were collected to determine the variations in prey biomass, heterotrophic carbon production, extracellular leucine aminopeptidase activity, and grazing pressure. A next-generation sequencing technique was used to investigate biodiversity shifts within the prokaryote and protist communities through the small subunit rRNA tag approach. This study shows that A. aurita ephyrae were responsible for large decreases in the abundances of the more motile microplankton groups, such as tintinnids, Dinophyceae, and aloricate ciliates. Bacillariophyceae and Mediophyceae showed smaller reductions. No evidence of selective predation emerged in the analysis of the community diversity down to the family level. The heterotrophic prokaryote biomass increased significantly (by up to 45%, in parallel with increases in heterotrophic carbon production and leucine aminopeptidase activity (40%. Significant modifications were detected in prokaryotic community composition. Some classes of Gammaproteobacteria and Flavobacteriia showed higher relative abundances when exposed to A. aurita ephyrae, while there was a net decrease for Alphaproteobacteria. Overall, this study provides new insight into the effects of A. aurita on microbial communities, underlining their selective

  7. Aurelia aurita Ephyrae Reshape a Coastal Microbial Community.

    Science.gov (United States)

    Zoccarato, Luca; Celussi, Mauro; Pallavicini, Alberto; Fonda Umani, Serena

    2016-01-01

    Over the last two decades, increasing attention has been paid to the impact of jellyfish blooms on marine communities. Aurelia aurita is one of the most studied of the Scyphozoans, and several studies have been carried out to describe its role as a top-down controller within the classical food web. However, little data are available to define the effects of these jellyfish on microbial communities. The aims of this study were to describe the predation impact of A. aurita ephyrae on a natural microplanktonic assemblage, and to determine any reshaping effects on the prokaryote community composition and functioning. Surface coastal water was used to set up a 24-h grazing experiment in microcosms. Samples were collected to determine the variations in prey biomass, heterotrophic carbon production (HCP), extracellular leucine aminopeptidase activity, and grazing pressure. A next-generation sequencing technique was used to investigate biodiversity shifts within the prokaryote and protist communities through the small subunit rRNA tag approach. This study shows that A. aurita ephyrae were responsible for large decreases in the abundances of the more motile microplankton groups, such as tintinnids, Dinophyceae, and aloricate ciliates. Bacillariophyceae and Mediophyceae showed smaller reductions. No evidence of selective predation emerged in the analysis of the community diversity down to the family level. The heterotrophic prokaryote biomass increased significantly (by up to 45%), in parallel with increases in HCP and leucine aminopeptidase activity (40%). Significant modifications were detected in prokaryotic community composition. Some classes of Gammaproteobacteria and Flavobacteriia showed higher relative abundances when exposed to A. aurita ephyrae, while there was a net decrease for Alphaproteobacteria. Overall, this study provides new insight into the effects of A. aurita on microbial communities, underlining their selective predation toward the more motile groups of

  8. Harmful effects of silver nanoparticles on a complex detrital model system.

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    Tlili, Ahmed; Cornut, Julien; Behra, Renata; Gil-Allué, Carmen; Gessner, Mark O

    2016-08-01

    The rapid proliferation of silver nanoparticles (AgNP) in industry and the environment requires realistic toxicity assessments based on approaches that consider the biological complexity of ecosystems. Here we assessed the acute toxicity of carbonate-coated AgNP and, for comparison, AgNO3 (Ag(+)) by using a model system consisting of decomposing plant litter and the associated fungal and bacterial decomposers as central players in the functioning of stream ecosystems. Little variation in size and surface charge during the experiment indicated that the AgNP used were essentially stable. AgNP disrupted bacterial growth (≤83% reduction in protein biosynthesis, EC50 = 0.3 μM), clearly affected fungal growth (≤61% reduction in ergosterol synthesis, EC50 = 47 μM) with both endpoints more sensitive to AgNP than to Ag(+). Fungal reproduction, in contrast, was stimulated by AgNP, but not Ag(+), at concentrations up to 25 μM. Both AgNP and Ag(+ )also stimulated extracellular alkaline phosphatase but reduced leucine aminopeptidase, whereas β-glucosidase was stimulated by AgNP and reduced by Ag(+). Importantly, the provision of cysteine, a chelating ligand that complexes free Ag(+), failed to alleviate AgNP toxicity to microbial growth, clearly demonstrating particle-mediated toxicity independent of the presence of ionic silver. This contrasts with the observed inhibition of leucine aminopeptidase by Ag(+), which accounted for 2-6% of the total silver in treatments receiving AgNP. These results show that although outcomes of AgNP and Ag(+ )exposure assessed by different functional endpoints vary widely, AgNP strongly interferes with bacterial growth and a range of other microbial processes, resulting in severe consequences for natural microbial communities and ecosystem functioning. PMID:26634870

  9. Subacute effects of carbofuran on enzyme functions in rat small intestine.

    Science.gov (United States)

    Gera, Nidhi; Kiran, Ravi; Mahmood, Akhtar

    2009-02-01

    The effect of carbofuran administration to rats has been studied on enzymes functions in rat intestine. Carbofuran was administrated 4.0 mg/kg body weight for 7 days or 2.8 mg/kg body weight for 30 days daily by Ryle's tube. Animals given carbofuran for 30 days exhibited retarded growth compared to control group. The activities of sucrase (56%), alkaline phosphatase (62%), leucine aminopeptidase (56%), and gamma-glutamyl trans peptidase (84%) were enhanced in animals given carbofuran for 7 days. Enhancement in the activities of alkaline phosphatase and leucine amino peptidase (92-96%) was also observed in animals exposed to carbofuran for 30 days, but the activities of sucrase (28%) and gamma-glutamyl transpeptidase (49%) were reduced under these conditions. There was no change in activities of maltase, lactase, and trehalase in pesticide-treated animals for 7 or 30 days. The activity of lactate dehydrogenase was enhanced (p carbofuran toxicity. The activities of glucose-6-phosphatase and glutamate pyruvate transaminase were also enhanced (p carbofuran exposure. The activity of glutamate oxaloacetate transaminase was unaltered in carbofuran toxicity. Kinetic analysis of brush border enzymes revealed a change in V(max) with no change in apparent Km. Western blot analysis of brush border sucrase, alkaline phosphatase, and leucine aminopeptidase corroborated the enzyme activity data. Intestinal histological revealed distruption of the villi, and comet assay showed disintegration of DNA in enterocytes of animals exposed to carbofuran for 30 days. These findings suggest that carbofuran toxicity may modulate digestive functions in rat intestine.

  10. Stabilizing effect of biochar on soil extracellular enzymes after a denaturing stress.

    Science.gov (United States)

    Elzobair, Khalid A; Stromberger, Mary E; Ippolito, James A

    2016-01-01

    Stabilizing extracellular enzymes may maintain enzymatic activity while protecting enzymes from proteolysis and denaturation. A study determined whether a fast pyrolysis hardwood biochar (CQuest™) would reduce evaporative losses, subsequently stabilizing soil extracellular enzymes and prohibiting potential enzymatic activity loss following a denaturing stress (microwaving). Soil was incubated in the presence of biochar (0%, 1%, 2%, 5%, or 10% by wt.) for 36 days and then exposed to microwave energies (0, 400, 800, 1600, or 3200 J g(-1) soil). Soil enzymes (β-glucosidase, β-d-cellobiosidase, N-acetyl-β-glucosaminidase, phosphatase, leucine aminopeptidase, β-xylosidase) were analyzed by fluorescence-based assays. Biochar amendment reduced leucine aminopeptidase and β-xylosidase potential activity after the incubation period and prior to stress exposure. The 10% biochar rate reduced soil water loss at the lowest stress level (400 J microwave energy g(-1) soil). Enzyme stabilization was demonstrated for β-xylosidase; intermediate biochar application rates prevented a complete loss of this enzyme's potential activity after soil was exposed to 400 (1% biochar treatment) or 1600 (5% biochar treatment) J microwave energy g(-1) soil. Remaining enzyme potential activities were not affected by biochar, and activities decreased with increasing stress levels. We concluded that biochar has the potential to reduce evaporative soil water losses and stabilize certain extracellular enzymes where activity is maintained after a denaturing stress; this effect was biochar rate and enzyme dependent. While biochar may reduce the potential activity of certain soil extracellular enzymes, this phenomenon was not universal as the majority of enzymes assayed in this study were unaffected by exposure to biochar.

  11. The Synthesis of L-Alanyl and β-Alanyl Derivatives of 2-Aminoacridone and Their Application in the Detection of Clinically-Important Microorganisms.

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    Marie Cellier

    Full Text Available In clinical microbiology the speed with which pathogenic microorganisms may be detected has a direct impact on patient health. One important strategy used in the laboratory is the growth of cultures in the presence of an enzymatic substrate which, once transformed by the appropriate microbial enzyme, generates a detectable colour or fluorescence output. Such substrates have previously been prepared by our group and others and are available as commercial diagnostic kits, however they all suffer from some degree of diffusion when used in a solid growth medium. This diffusion complicates the detection and differentiation of species in polymicrobial cultures and so we sought to improve on our previous work. In this work we have prepared and evaluated a series of novel fluorogenic enzyme substrates based on N-substituted-2-aminoacridones. All of the prepared substrates were found to be suitable for the detection and differentiation of certain microorganisms, however those based on the 2-amino-10-benzylacridone core in particular showed no apparent diffusion when incorporated into solid growth media. On transformation these substrates generated brightly fluorescent colonies that are clearly contrasted with the background medium due to the difference in emission wavelength (λem 445-450 nm for the substrate, λem 550 nm for the product. Here we have shown that our L-alanyl aminopeptidase substrate, 2-(N-L-alanylamino-10-benzylacridone, is particularly suited to the detection of Gram-negative bacteria, and our β-alanyl aminopeptidase substrate, 2-(N- β-alanylamino-10-benzylacridone, to the detection of Pseudomonas aeruginosa and Serratia marcescens when grown on solid media incorporating these substrates. The resulting fluorophore shows no apparent diffusion from the colonies of interest, and the enhanced sensitivity offered by fluorescent emission may allow for the detection of these organisms as microcolonies using automated fluorescence microscopy.

  12. Balancing selection maintains a form of ERAP2 that undergoes nonsense-mediated decay and affects antigen presentation.

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    Aida M Andrés

    2010-10-01

    Full Text Available A remarkable characteristic of the human major histocompatibility complex (MHC is its extreme genetic diversity, which is maintained by balancing selection. In fact, the MHC complex remains one of the best-known examples of natural selection in humans, with well-established genetic signatures and biological mechanisms for the action of selection. Here, we present genetic and functional evidence that another gene with a fundamental role in MHC class I presentation, endoplasmic reticulum aminopeptidase 2 (ERAP2, has also evolved under balancing selection and contains a variant that affects antigen presentation. Specifically, genetic analyses of six human populations revealed strong and consistent signatures of balancing selection affecting ERAP2. This selection maintains two highly differentiated haplotypes (Haplotype A and Haplotype B, with frequencies 0.44 and 0.56, respectively. We found that ERAP2 expressed from Haplotype B undergoes differential splicing and encodes a truncated protein, leading to nonsense-mediated decay of the mRNA. To investigate the consequences of ERAP2 deficiency on MHC presentation, we correlated surface MHC class I expression with ERAP2 genotypes in primary lymphocytes. Haplotype B homozygotes had lower levels of MHC class I expressed on the surface of B cells, suggesting that naturally occurring ERAP2 deficiency affects MHC presentation and immune response. Interestingly, an ERAP2 paralog, endoplasmic reticulum aminopeptidase 1 (ERAP1, also shows genetic signatures of balancing selection. Together, our findings link the genetic signatures of selection with an effect on splicing and a cellular phenotype. Although the precise selective pressure that maintains polymorphism is unknown, the demonstrated differences between the ERAP2 splice forms provide important insights into the potential mechanism for the action of selection.

  13. Characterization of the mechanism of action of the genetically modified Cry1AbMod toxin that is active against Cry1Ab-resistant insects.

    Science.gov (United States)

    Muñóz-Garay, Carlos; Portugal, Leivi; Pardo-López, Liliana; Jiménez-Juárez, Nuria; Arenas, Ivan; Gómez, Isabel; Sánchez-López, Rosana; Arroyo, Raquel; Holzenburg, Andreas; Savva, Christos G; Soberón, Mario; Bravo, Alejandra

    2009-10-01

    Bacillus thuringiensis Cry toxins are used in the control of insect pests. They are pore-forming toxins with a complex mechanism that involves the sequential interaction with receptors. They are produced as protoxins, which are activated by midgut proteases. Activated toxin binds to cadherin receptor, inducing an extra cleavage including helix alpha-1, facilitating the formation of a pre-pore oligomer. The toxin oligomer binds to secondary receptors such as aminopeptidase and inserts into lipid rafts forming pores and causing larval death. The primary threat to efficacy of Bt-toxins is the evolution of insect resistance. Engineered Cry1AMod toxins, devoid of helix alpha-1, could be used for the control of resistance in lepidopterans by bypassing the altered cadherin receptor, killing resistant insects affected in this receptor. Here we analyzed the mechanism of action of Cry1AbMod. We found that alkaline pH and the presence of membrane lipids facilitates the oligomerization of Cry1AbMod. In addition, tryptophan fluorescence emission spectra, ELISA binding to pure aminopeptidase receptor, calcein release assay and analysis of ionic-conductance in planar lipid bilayers, indicated that the secondary steps in mode of action that take place after interaction with cadherin receptor such as oligomerization, receptor binding and pore formation are similar in the Cry1AbMod and in the wild type Cry1Ab. Finally, the membrane-associated structure of Cry1AbMod oligomer was analyzed by electron crystallography showing that it forms a complex with a trimeric organization. PMID:19559004

  14. Supplementing formula-fed piglets with a low molecular weight fraction of bovine colostrum whey results in an improved intestinal barrier.

    Science.gov (United States)

    De Vos, M; Huygelen, V; Van Raemdonck, G; Willemen, S; Fransen, E; Van Ostade, X; Casteleyn, C; Van Cruchten, S; Van Ginneken, C

    2014-08-01

    To test the hypothesis that a low molecular weight fraction of colostral whey could affect the morphology and barrier function of the small intestine, 30 3-d-old piglets (normal or low birth weight) were suckled (n = 5), artificially fed with milk formula (n = 5), or artificially fed with milk formula with a low molecular weight fraction of colostral whey (n = 5) until 10 d of age. The small intestine was sampled for histology (haematoxylin and eosin stain; anti-KI67 immunohistochemistry) and enzyme activities (aminopeptidase A, aminopeptidase N, dipeptidylpeptidase IV, lactase, maltase, and sucrase). In addition, intestinal permeability was evaluated via a dual sugar absorption test and via the measurement of occludin abundance. Artificially feeding of piglets reduced final BW (P < 0.001), villus height (P < 0.001), lactase (P < 0.001), and dipeptidylpeptidase IV activities (P < 0.07), whereas crypt depth (P < 0.001) was increased. No difference was observed with regard to the permeability measurements when comparing artificially fed with naturally suckling piglets. Supplementing piglets with the colostral whey fraction did not affect BW, enzyme activities, or the outcome of the dual sugar absorption test. On the contrary, the small intestines of supplemented piglets had even shorter villi (P = 0.001) than unsupplemented piglets and contained more occludin (P = 0.002). In conclusion, at 10 d of age, no differences regarding intestinal morphology and permeability measurements were observed between the 2 BW categories. In both weight categories, the colostral whey fraction affected the morphology of the small intestine but did not improve the growth performances or the in vivo permeability. These findings should be acknowledged when developing formulated milk for neonatal animals with the aim of improving the performance of low birth weight piglets. PMID:25012977

  15. Changes in digestive enzyme activities during larval development of Chinese loach Paramisgurnus dabryanus (Dabry de Thiersant, 1872).

    Science.gov (United States)

    Zhang, Yun-Long; Wu, Qiao-Wan; Hu, Wei-Hua; Wang, Fan; Zhao, Zhong-Bo; He, Hui; Shao, Wei-Han; Fan, Qi-Xue

    2015-12-01

    The digestive physiology of Chinese loach (Paramisgurnus dabryanus) was studied by assessing the specific and total activities of different pancreatic (trypsin, chymotrypsin, amylase and lipase), gastric (pepsin) and intestinal (alkaline phosphatase and leucine-aminopeptidase) enzymes from hatching to 40 days after hatching (DAH). Larvae were reared at 24.4 ± 0.4 °C and fed with rotifers from mouth opening (4 DAH) to 15 DAH, from 10 to 35 DAH with Cladocera and from 30 to 40 DAH with compound diet. Enzyme activities for trypsin, chymotrypsin, amylase and lipase were detected before the onset of exogenous feeding, indicating that these enzymes were genetically pre-programmed. Most of the pancreatic enzyme specific activities increased until 20 DAH and decreased thereafter. The pepsin activity of Chinese loach was firstly detected at 30 DAH, indicating the appearance of functional gastric gland. Alkaline phosphatase specific activity was detected from hatching onward, showed marked increase and reached the second peak at 20 DAH, while a gradual increase in specific leucine-aminopeptidase activity was observed until the end of the experiment. Accordingly, the larvae of Chinese loach possess a functional digestive system before the onset of exogenous feeding and the digestive capacity gradually increases as development progresses. The abrupt increase in intestinal enzyme activities between 10 and 20 DAH demonstrates onset of juvenile-like digestive mode in Chinese loach larvae. The increase in pepsin activity after 30 DAH indicates the shift from alkaline to acidic digestion in Chinese loach larvae, which may be considered as the onset of weaning. PMID:26232086

  16. Resistance to Bacillus thuringiensis Toxin Cry2Ab in Trichoplusia ni Is Conferred by a Novel Genetic Mechanism.

    Science.gov (United States)

    Song, Xiaozhao; Kain, Wendy; Cassidy, Douglas; Wang, Ping

    2015-08-01

    The resistance to the Bacillus thuringiensis (Bt) toxin Cry2Ab in a greenhouse-originated Trichoplusia ni strain resistant to both Bt toxins Cry1Ac and Cry2Ab was characterized. Biological assays determined that the Cry2Ab resistance in the T. ni strain was a monogenic recessive trait independent of Cry1Ac resistance, and there existed no significant cross-resistance between Cry1Ac and Cry2Ab in T. ni. From the dual-toxin-resistant T. ni strain, a strain resistant to Cry2Ab only was isolated, and the Cry2Ab resistance trait was introgressed into a susceptible laboratory strain to facilitate comparative analysis of the Cry2Ab resistance with the susceptible T. ni strain. Results from biochemical analysis showed no significant difference between the Cry2Ab-resistant and -susceptible T. ni larvae in midgut proteases, including caseinolytic proteolytic activity and zymogram profile and serine protease activities, in midgut aminopeptidase and alkaline phosphatase activity, and in midgut esterases and hemolymph plasma melanization activity. For analysis of genetic linkage of Cry2Ab resistance with potential Cry toxin receptor genes, molecular markers for the midgut cadherin, alkaline phosphatase (ALP), and aminopeptidase N (APN) genes were identified between the original greenhouse-derived dual-toxin-resistant and the susceptible laboratory T. ni strains. Genetic linkage analysis showed that the Cry2Ab resistance in T. ni was not genetically associated with the midgut genes coding for the cadherin, ALP, and 6 APNs (APN1 to APN6) nor associated with the ABC transporter gene ABCC2. Therefore, the Cry2Ab resistance in T. ni is conferred by a novel but unknown genetic mechanism. PMID:26025894

  17. Deep sequencing of the transcriptomes of soybean aphid and associated endosymbionts.

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    Sijun Liu

    Full Text Available BACKGROUND: The soybean aphid has significantly impacted soybean production in the U.S. Transcriptomic analyses were conducted for further insight into leads for potential novel management strategies. METHODOLOGY/PRINCIPAL FINDINGS: Transcriptomic data were generated from whole aphids and from 2,000 aphid guts using an Illumina GAII sequencer. The sequence data were assembled de novo using the Velvet assembler. In addition to providing a general overview, we demonstrate (i the use of the Multiple-k/Multiple-C method for de novo assembly of short read sequences, followed by BLAST annotation of contigs for increased transcript identification: From 400,000 contigs analyzed, 16,257 non-redundant BLAST hits were identified; (ii analysis of species distributions of top non-redundant hits: 80% of BLAST hits (minimum e-value of 1.0-E3 were to the pea aphid or other aphid species, representing about half of the pea aphid genes; (iii comparison of relative depth of sequence coverage to relative transcript abundance for genes with high (membrane alanyl aminopeptidase N or low transcript abundance; (iv analysis of the Buchnera transcriptome: Transcripts from 57.6% of the genes from Buchnera aphidicola were identified; (v identification of Arsenophonus and Wolbachia as potential secondary endosymbionts; (vi alignment of full length sequences from RNA-seq data for the putative salivary gland protein C002, the silencing of which has potential for aphid management, and the putative Bacillus thuringiensis Cry toxin receptors, aminopeptidase N and alkaline phosphatase. CONCLUSIONS/SIGNIFICANCE: THIS STUDY PROVIDES THE MOST COMPREHENSIVE DATA SET TO DATE FOR SOYBEAN APHID GENE EXPRESSION: This work also illustrates the utility of short-read transcriptome sequencing and the Multiple-k/Multiple-C method followed by BLAST annotation for rapid identification of target genes for organisms for which reference genome sequences are not available, and extends the utility

  18. An Investigation into the Gastrointestinal Stability of Exenatide in the Presence of Pure Enzymes, Everted Intestinal Rings and Intestinal Homogenates.

    Science.gov (United States)

    Sun, Yanan; Wang, Mengshu; Sun, Bingxue; Li, Feng; Liu, Shubo; Zhang, Yong; Zhou, Yan; Chen, Yan; Kong, Wei

    2016-01-01

    The purpose of this study was to investigate the gastrointestinal stability of exenatide to determine the key factor(s) contributing to peptide degradation during the oral delivery process. The effects of pH and various digestive enzymes on the degradation kinetics of exenatide were determined. Moreover, the degradation clearances of peptide were also examined using rat everted intestinal rings and intestinal homogenates from various intestinal locations. Exenatide was comparatively stable within a pH range of 1.2-8. However, obvious degradation was observed in the presence of digestive enzymes. The order of enzymes, in terms of ability to degradate exenatide, was chymotrypsin>aminopeptidase N>carboxypeptidase A>trypsin>pepsin. Chymotrypsin showed the greatest ability to degrade exenatide (half-life t1/2, 5.784×10(-2) h), whereas aminopeptidase N and carboxylpeptidase A gave t1/2 values of 3.53 and 10.16 h, respectively. The degradation of exenatide was found to be peptide concentration- and intestinal site-dependent, with a lower clearance in the upper part of the duodenum and the lower part of the ileum. When using intestinal homogenates as enzyme sources, the order, in terms of peptide degradation ability, was ileum>jejunum>duodenum. However, no significant difference was observed in the remaining peptide concentrations throughout 2 h of incubation, which may be due to the involvement of cytosolic enzymes. These results revealed key factors contributing to peptide degradation, and suggest that the inhibition of chymotrypsin and site-specific delivery of exenatide might be advantageous in overcoming metabolic obstacles during its oral delivery.

  19. Electroosmotic push-pull perfusion: description and application to qualitative analysis of the hydrolysis of exogenous galanin in organotypic hippocampal slice cultures.

    Science.gov (United States)

    Rupert, Amy E; Ou, Y; Sandberg, M; Weber, S G

    2013-05-15

    We demonstrate here a method that perfuses a small region of an organotypic hippocampal culture with a solution containing an enzyme substrate, a neuropeptide. Perfusate containing hydrolysis products is continually collected and subsequently analyzed for the products of the enzymatic degradation of the peptide substrate. The driving force for perfusion is an electric field. The fused silica capillaries used as "push" and "pull" or "source" and "collection" capillaries have a ζ-potential that is negative and greater in magnitude than the tissue's ζ-potential. Thus, depending on the magnitudes of particular dimensions, the electroosmotic flow in the capillaries augments the fluid velocity in the tissue. The flow rate is not directly measured; however, we determine it using a finite-element approach. We have determined the collection efficiency of the system using an all d-amino acid internal standard. The flow rates are low, in the nL/min range, and adjustable by controlling the current or voltage in the system. The collection efficiency of the d-amino acid peptide internal standard is variable, increasing with increased current and thus electroosmotic flow rate. The collection efficiency can be rationalized in the context of a Peclet number. Electroosmotic push-pull perfusion of the neuropeptide galanin (gal1-29) through the extracellular space of an organotypic hippocampal culture results in its hydrolysis by ectopeptidase reactions occurring in the extracellular space. The products of hydrolysis were identified by MALDI-MS. Experiments at two levels of current (8-12 μA and 19-40 μA) show that the probability of seeing hydrolysis products (apparently from aminopeptidases) is greater in the Cornu Ammonis area 3 (CA3) than in the Cornu Ammonis area 1 (CA1) in the higher current experiments. In the lower current experiments, shorter peptide products of aminopeptidases (gal13-29 to gal20-19) are seen with greater frequency in CA3 than in CA1 but there is no

  20. 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

  1. Effects of human activities on the ecological processes of river biofilms in a highly urbanized river

    Science.gov (United States)

    Hung, R.; Li, M.

    2013-12-01

    Many anthropogenic disturbances and their effects of aquatic ecosystem are difficult to quantify in urbanized rivers. In past, specific taxa analysis of community structure was a common approach in river health monitoring studies. However, it is still difficult to understand stream ecosystem integrity without considering ecosystem processes. The complex species composition and metabolism of a river biofilm have the capacity to interact and/or modulate their surrounding environment. Because of their short life cycles, species richness, and worldwide distribution, structure and function of river biofilm communities are sensitive to change in environmental conditions. Therefore, biofilms are widely used as early warning systems of water pollution for water quality monitoring studies. In this study, we used river biofilms as a bioindicator by examining their extracellular enzyme activities and photosynthesis efficiency to understand human activities on the ecological processes of river ecosystem in a highly urbanized river. We sampled four sites along the Keelung River, Taiwan, based on different intensities of anthropogenic disturbances including water pollution index, population densities, land use types and types of stream habitats. Two study sites are heavily influenced by human activities and the others are not. The activities of extracellular enzymes within the biofilm play an important function for organic matter decomposition and nutrient cycling. We measured seven extracellular enzyme activities (β-d-glucosidase, phosphatase, leucine-aminopeptidase, sulfatase, peroxidase, polyphenol oxidase, and esterase) to examine specific enzyme activity changes at four study sites monthly. In addition, relative proportion of each extracellular enzyme activity on total enzyme activities was calculated in order to examine the relationship between functional biofilm profiles and different urban intensities. Among four study sites, leucine-aminopeptidase and esterase

  2. Hot experience for cold-adapted microorganisms: temperature sensitivity of soil enzymes

    Science.gov (United States)

    Liu, Shibin; Razavidezfuly, Baharsadat; Kuzyakov, Yakov

    2016-04-01

    The temperature sensitivity of enzymes responsible for organic matter decomposition in cold environment soil, where warming is expected to be greatest is crucial. Based on Michaelis-Menten kinetics and Arrhenius function, we hypothesized that cold-adapted microorganisms will produce high efficient enzymes at cold temperatures (enzymes with lower apparent activation energy (Ea) at cold temperature ranges). To test our hypothesis, 30 g soil of Tibetan Plateau (4100 m a.s.l., annual temperature 2.4 °C) in 4 replicates were incubated for one month over a temperature range of 0-40 °C (with 5 °C steps) and determined the kinetic parameters of six enzymes involved in decomposing organics: cellobiohydrolase and β-glucosidase, which are commonly measured as enzymes responsible for consecutive stages of cellulose degradation; xylanase, which is responsible for breaking down hemicelluloses; acid phosphatase, which mineralizes organic P to phosphate by hydrolyzing phosphoric (mono) ester bonds under acidic conditions. Activities of leucine aminopeptidase and tyrosine aminopeptidase were analyzed to assess the hydrolysis of L-peptide bonds. The apparent activation energy varied between enzymes from 42 (phosphatase) to 54 (cellobiohydrolase) kJ mol-1 corresponding to the Q10 values of the enzyme reactions of 1.8-2.3. The increase of substrate affinity (Km) with temperature was gradual for most tested enzymes from 0-20 °C (enzymes involved in C cycle), (proteases) and 0-40 °C (phosphatase). However, within a high range of temperatures (25-40 °C) the hydrolytic activity was governed by enzymes with nearly constant substrate affinity. Overall, for enzymes involved in C cycle and proteases, a strong increase (30-40%) in Km at high temperatures (25 °C) reflects an expression of multiple isoenzymes each with different temperature optima and probable shift of microbial community. The general trend of catalytic efficiency (Vmax/Km) demonstrated a gradual increase with

  3. Antiviral therapy effects upon hepatitis C cholestatic syndrome.

    Science.gov (United States)

    Vere, C C; Gofiţă, Eliza; Forţofoiu, C; Streba, Letiţia Adela Maria; Genunche, Amelia

    2007-01-01

    Cholestasis includes, as a syndrome, all clinical and biological manifestations caused by the deficient or simply absent biliar secretion or caused by the obstruction of the biliary ducts. The hepatic cholestasis from the chronic hepatitis C (HC VHC) is a result of the altered interlobular biliary canalicules, caused by the modified cellular transport mechanisms and it is associated with a medium to severe degree of fibrosis. The aim of this study was to evaluate the efficiency of antiviral therapy in HC VHC patients. The study included a number of 37 HC VHC patients admitted at the Medical Department no. 1 of the Emergency County Hospital of Craiova; they were treated with Pegasys, 180 microg/week and Copegus, 1000 or 1200 mg/day, taking in consideration their weight, for 48 weeks and they were monitored for 24 weeks after the treatment. The following parameters were analyzed: direct bilirubine, total cholesterol, alkaline phosphatase, gamma-glutamiltranspeptidase and leucin-aminopeptidase. Under treatment, the clinical status caused by the cholestasis (pruritus, icteric syndrome, hemoragipary syndrome) was improved in six of the given cases (16.22%). Before therapy, the hepatic cholestasis was present in 20 patients (54.05%), and after treatment in 14 patients (37.83%). During therapy, the average values for all the monitored parameters decreased: direct bilirubine (0.38 +/- 0.18 mg/dl vs. 0.34 +/- 0.24 mg/dl, p = 0.0867), total cholesterol (198.53 md/dl vs. 183.16 mg/dl, p = 0.0808), alkaline phosphatase (236.99 +/- 79.09 iu/l vs. 227.82 +/- 87.59 iu/l, p = 0.0845), gamma-glutamiltranspeptidase (47 +/- 32.89 iu/l vs. 43.91 +/- 29.66 iu/l, p = 0.1509), and leucin-aminopeptidase (32.33 +/- 13.22 iu/l vs. 28.95 +/- 14.22 iu/l, p = 0.0038). Under antiviral treatment there was noticed an improvement of the cholestasis clinical status in a small number of cases. Antiviral therapy favorably influenced the liver cholestasis associated in patients with chronic hepatitis

  4. A high-throughput, in-vitro assay for Bacillus thuringiensis insecticidal proteins.

    Science.gov (United States)

    Izumi Willcoxon, Michi; Dennis, Jaclyn R; Lau, Sabina I; Xie, Weiping; You, You; Leng, Song; Fong, Ryan C; Yamamoto, Takashi

    2016-01-10

    A high-throughput, in-vitro assay for Bacillus thuringiensis (Bt) insecticidal proteins designated as Cry was developed and evaluated for screening a large number of Cry protein variants produced by DNA shuffling. This automation-amenable assay exploits an insect cell line expressing a single receptor of Bt Cry proteins. The Cry toxin used to develop this assay is a variant of the Cry1Ab protein called IP1-88, which was produced previously by DNA shuffling. Cell mortality caused by the activated Bt Cry toxin was determined by chemical cell viability assay in 96/384-well microtiter plates utilizing CellTiter 96(®) obtained from Promega. A widely-accepted mode-of-action theory of certain Bt Cry proteins suggests that the activated toxin binds to one or more receptors and forms a pore through the insect gut epithelial cell apical membrane. A number of insect proteins such as cadherin-like protein (Cad), aminopeptidase-N (APN), alkaline phosphatase (ALP) and ABC transporter (ABCC) have been identified as the receptors of Bt Cry toxins. In this study, Bt Cry toxin receptors Ostrinia nubilalis (European corn borer) cadherin-like protein (On-Cad) and aminopeptidase-N 1 and 3 (On-APN1, On-APN3) and Spodoptera frugiperda (fall armyworm) cadherin-like protein (Sf-Cad) were cloned in an insect cell line, Sf21, and a mammalian cell line, Expi293F. It was observed by ligand blotting and immunofluorescence microscopy that trypsin-activated IP1-88 bound to On-Cad and On-APN1, but not Sf-Cad or On-APN3. In contrast, IP1-88 bound only to APN1 in BBMV (Brush Border Membrane Vesicles) prepared from the third and fourth-instar O. nubilalis larval midgut. The sensitivity of the recombinant cells to the toxin was then tested. IP1-88 showed no toxicity to non-recombinant Sf21 and Expi293F. Toxicity was observed only when the On-Cad gene was cloned and expressed. Sf-Cad and On-APN1 were not able to make those cells sensitive to the toxin. Since the expression of On-Cad alone was

  5. In vivo inhibition of dipeptidyl peptidase IV activity by pro-pro-diphenyl-phosphonate (Prodipine).

    Science.gov (United States)

    De Meester, I; Belyaev, A; Lambeir, A M; De Meyer, G R; Van Osselaer, N; Haemers, A; Scharpé, S

    1997-07-01

    Dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5), also known as CD26, is a membrane-bound serine protease that cleaves off aminoterminal dipeptides from peptides with a penultimate proline (or, at a much slower rate, a penultimate alanine). Recently, we synthesized and characterized a number of dipeptide-derived diphenylphosphonates. Out of the resulting series of slow-binding irreversible inhibitors of DPP IV, diphenyl 1-(S)-prolylpyrrolidine-2(R,S)-phosphonate hydrochloride (Pro-Pro-diphenylphosphonate or Prodipine) was selected for further study. We investigated the in vivo applicability of Prodipine. Male rabbits weighing 3-4 kg received a single intravenous injection with 10 mg Prodipine or saline. After 1 hr, plasma DPP IV activity had decreased to less than 20% of the preinjection value and remained unchanged during a 24-hr observation period. In a next step, we aimed to study (i) the dose dependency and (ii) the duration of the effect after a single intravenous dose of Prodipine. A profound and long-lasting inhibition of plasma DPP IV activity was observed in the treated animals (1, 5 or 10 mg). It took 5 to 8 days to reach half of the pretreatment DPP IV activity and generally more than 20 days for a complete recovery. Systemic treatment with Prodipine not only led to inhibition of plasma DPP IV activity but also decreased tissue DPP IV activity in circulating mononuclear cells, kidney cortex, thymus, spleen, lung, and liver. No differences in activities of the related peptidases aminopeptidase P (APP, EC 3.4.11.9), prolyl oligopeptidase (PO, EC 3.4.21.26), or aminopeptidase M (mAAP, EC 3.4.11.2) were detected between Prodipine-treated and control rabbits. The in vivo applicability of this chemically stable, irreversible inhibitor of DPP IV opens new possibilities, not only to further unravel the biological functions of this intriguing ectopeptidase, but also to explore this enzyme as a new target in various fields of pharmacological research.

  6. Elastinolytic and proteolytic enzymes.

    Science.gov (United States)

    Kessler, Efrat; Safrin, Mary

    2014-01-01

    Pseudomonas aeruginosa secretes into its environment at least seven extracellular proteases: pseudolysin (LasB protease; elastase), aeruginolysin (alkaline proteinase), staphylolysin (staphylolytic endopeptidase; LasA protease), lysyl endopeptidase (protease IV; PrpL), PASP (P. aeruginosa small protease), LepA (Large ExoProtease A), and an aminopeptidase. Their action on host proteins, both individually and synergistically, plays important roles in pathogenesis of P. aeruginosa infections. Methods to measure/detect their activities are fundamental for understanding their physiological functions, roles in pathogenesis, mechanisms of action, regulation, and secretion. Most assays for determination/detection of proteolytic activity employ modified/non-modified casein or gelatin as substrates. In the quantitative assay, fragments generated from azocasein are separated from undigested substrate by trichloroacetic acid precipitation and their absorbance is measured. In non-quantitative assays, proteolytic activity is detected as clearing zones around bacterial growth or samples of culture supernatants on casein containing solid media formed due to local casein degradation. In zymography, individual proteases are detected as clear bands in gelatin/casein containing gels after SDS-PAGE separation, renaturation and protein staining. The elastinolytic capacity of P. aeruginosa is reflected by clearing zones on nutrient agar plates containing insoluble elastin instead of casein. Mueller-Hinton agar plates on which S. aureus cells are grown as a lawn are used to assess the susceptibility of S. aureus isolates to staphylolysin. A clear zone around a staphylolysin-containing sample indicates inhibition of S. aureus growth. Methods for measuring the activity of individual proteases are based on their cleavage specificity. These include assays of elastinolytic activity of pseudolysin and/or staphylolysin using elastin-Congo red as a substrate, a method for determination of

  7. Evolution of proteolytic and physico-chemical characteristics of Norwegian dry-cured ham during its processing.

    Science.gov (United States)

    Petrova, Inna; Tolstorebrov, Ignat; Mora, Leticia; Toldrá, Fidel; Eikevik, Trygve Magne

    2016-11-01

    Proteolytic activity and physico-chemical characteristics were studied for Norwegian dry-cured ham at four different times of processing: raw hams, post-salted hams (3 months of processing), hams selected in the middle of the production (12 months of processing) and hams at the end of the processing (24 months). Cathepsin H activity decreased until negligible values after 3 months of processing, whereas cathepsins B and B+L were inactive at 12 months. AAP was the most active aminopeptidase whereas RAP and MAP were active just during the first 12 months of processing. Proteolysis index reached a value of 4.56±1.03 % with non-significant differences between 12 and 24 months of ripening. Peptide identification by LC-MS/MS was done and two peptides (GVEEPPKGHKGNKK and QAISNNKDQGSY) showing a linear response with the time of processing were found. Unfreezable water content and glass transition temperature were investigated using differential scanning calorimetry (DSC) technique with non-significant differences in the temperature of glass transition for 12 and 24 months of processing. PMID:27371871

  8. The temporal analysis of yeast exponential phase using shotgun proteomics as a fermentation monitoring technique

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Eric L. [McGill University, Montreal, Quebec; Orsat, Valerie [McGill University; Shah, Manesh B [ORNL; Herndon, Elizabeth M [ORNL; Hettich, Robert {Bob} L [ORNL; Verberkmoes, Nathan C [ORNL; Lefsrud, Mark G [McGill University, Montreal, Quebec

    2012-01-01

    System biology and bioprocess technology can be better understood using shotgun proteomics as a monitoring system during the fermentation. We demonstrated a shotgun proteomic method to monitor the temporal yeast proteome in early, middle and late exponential phases. Our study identified a total of 1389 proteins combining all 2D-LC-MS/MS runs. The temporal Saccharomyces cerevisiae proteome was enriched with proteolysis, radical detoxification, translation, one-carbon metabolism, glycolysis and TCA cycle. Heat shock proteins and proteins associated with oxidative stress response were found throughout the exponential phase. The most abundant proteins observed were translation elongation factors, ribosomal proteins, chaperones and glycolytic enzymes. The high abundance of the H-protein of the glycine decarboxylase complex (Gcv3p) indicated the availability of glycine in the environment. We observed differentially expressed proteins and the induced proteins at mid-exponential phase were involved in ribosome biogenesis, mitochondria DNA binding/replication and transcriptional activator. Induction of tryptophan synthase (Trp5p) indicated the abundance of tryptophan during the fermentation. As fermentation progressed toward late exponential phase, a decrease in cell proliferation was implied from the repression of ribosomal proteins, transcription coactivators, methionine aminopeptidase and translation-associated proteins.

  9. Isolated tumor endothelial cells maintain specific character during long-term culture

    Energy Technology Data Exchange (ETDEWEB)

    Matsuda, Kohei [Vascular Biology, Hokkaido University Graduate School of Dental Medicine, N13 W7, Kita-ku, Sapporo 060-8586 (Japan); Oral Pathology and Biology, Hokkaido University Graduate School of Dental Medicine, N13 W7, Kita-ku, Sapporo 060-8586 (Japan); Oral and Maxillofacial Surgery, Hokkaido University Graduate School of Dental Medicine, N13 W7, Kita-ku, Sapporo 060-8586 (Japan); Ohga, Noritaka [Vascular Biology, Hokkaido University Graduate School of Dental Medicine, N13 W7, Kita-ku, Sapporo 060-8586 (Japan); Hida, Yasuhiro [Surgical Oncology, Hokkaido University Graduate School of Medicine, N13 W7, Kita-ku, Sapporo 060-8586 (Japan); Muraki, Chikara [Oral Pathology and Biology, Hokkaido University Graduate School of Dental Medicine, N13 W7, Kita-ku, Sapporo 060-8586 (Japan); Oral and Maxillofacial Surgery, Hokkaido University Graduate School of Dental Medicine, N13 W7, Kita-ku, Sapporo 060-8586 (Japan); Tsuchiya, Kunihiko [Vascular Biology, Hokkaido University Graduate School of Dental Medicine, N13 W7, Kita-ku, Sapporo 060-8586 (Japan); Renal and Genitourinary Surgery, Hokkaido University Graduate School of Medicine, N13 W7, Kita-ku, Sapporo 060-8586 (Japan); Kurosu, Takuro [Vascular Biology, Hokkaido University Graduate School of Dental Medicine, N13 W7, Kita-ku, Sapporo 060-8586 (Japan); Oral Pathology and Biology, Hokkaido University Graduate School of Dental Medicine, N13 W7, Kita-ku, Sapporo 060-8586 (Japan); Oral and Maxillofacial Surgery, Hokkaido University Graduate School of Dental Medicine, N13 W7, Kita-ku, Sapporo 060-8586 (Japan); Akino, Tomoshige [Vascular Biology, Hokkaido University Graduate School of Dental Medicine, N13 W7, Kita-ku, Sapporo 060-8586 (Japan); Renal and Genitourinary Surgery, Hokkaido University Graduate School of Medicine, N13 W7, Kita-ku, Sapporo 060-8586 (Japan); Shih, Shou-Ching [Pathology, Beth Israel Deaconess Medical Center, 330 Brookline Ave, Boston, MA 02215 (United States); and others

    2010-04-16

    Tumor angiogenesis is necessary for solid tumor progression and metastasis. Increasing evidence indicates that tumor endothelial cells (TECs) are more relevant to the study of tumor angiogenesis than normal endothelial cells (NECs) because their morphologies and gene expression are different from NECs. However, it is challenging to isolate and culture large numbers of pure ECs from tumor tissue since the percentage of ECs is only about 1-2% and tumor cells and fibroblasts easily overgrow them. In addition, there has been concern that isolated TECs may lose their special phenotype once they are dissociated from tumor cells. In this study, we have successfully purified murine TECs from four different human tumor xenografts and NECs from murine dermal tissue. Isolated ECs expressed endothelial markers, such as CD31, VE-cadherin (CD144), and endoglin (CD105), for more than 3 months after isolation. TECs maintained tumor endothelial-specific markers, such as tumor endothelial marker 8 (TEM8) and aminopeptidase N (APN), as in tumor blood vessels in vivo. In addition, TECs were more proliferative and motile than NECs. TECs showed a higher response to VEGF and higher expression of VEGF receptors-1 and -2 than NECs did. Stem cell antigen-1 was up-regulated in all four TECs, suggesting that they have a kind of stemness. Cultured TECs maintain distinct biological differences from NECs as in vivo. In conclusion, it was suggested that TECs are relevant material for tumor angiogenesis research.

  10. Cell-associated proteolytic enzymes from marine phytoplankton

    Energy Technology Data Exchange (ETDEWEB)

    Berges, J.A.; Falkowski, P.G. [Brookhaven National Lab., Upton, NY (United States)

    1996-08-01

    Despite their central importance in cell metabolism, little is known about proteases in marine phytoplankton. Caseinolytic and leucine aminopeptidase (LAP) activities was surveyed in log-phase cultures of the chlorophyte Dunaliella tertiolecta Butcher, the diatom Thalassiosira weissflogii Fryxell et Hasle, the chrysophyte Isochrysis galbana Parke, the coccolithophorid Emiliania huxleyi Hay et Mohler, and the cyanobacterium Synechococcus sp. LAP activity was very low at pH < 6 and peaked between pH 7.5 and 8.5 in all species, whereas caseinolytic activity in most species showed only minor peaks in the pH 4-5 range and broad maxima above pH 8. Acidic vacuolar proteases apparently represented only a small fraction of total protease activity. Attempts to classify protease using selective inhibitors were inconclusive. Caserinolytic activities were remarkably stable. Casein zymograms were used to identify >200-and <20-kDa proteases in homogenates of log-phase T. weissflogii; only the smaller protease was found in D. tertiolecta. Antibodies in the ATPase subunit (C) of the conserved, chloroplastic Clp protease from Pisum cross-reacted with proteins in Synechococcus, D. tertiolecta, and I. galbana, but no cross-reactions were found for any species with antibodies against the ClpP subunit from either E. coli or Nicotiana. Our results show that phytoplankton contain a diverse complement of proteases with novel characteristics. 46 refs., 6 figs., 1 tab.

  11. Identification and Validation of Expressed Sequence Tags from Pigeonpea (Cajanus cajan L.) Root.

    Science.gov (United States)

    Kumar, Ravi Ranjan; Yadav, Shailesh; Joshi, Shourabh; Bhandare, Prithviraj P; Patil, Vinod Kumar; Kulkarni, Pramod B; Sonkawade, Swati; Naik, G R

    2014-01-01

    Pigeonpea (Cajanus cajan (L) Millsp.) is an important food legume crop of rain fed agriculture in the arid and semiarid tropics of the world. It has deep and extensive root system which serves a number of important physiological and metabolic functions in plant development and growth. In order to identify genes associated with pigeonpea root, ESTs were generated from the root tissues of pigeonpea (GRG-295 genotype) by normalized cDNA library. A total of 105 high quality ESTs were generated by sequencing of 250 random clones which resulted in 72 unigenes comprising 25 contigs and 47 singlets. The ESTs were assigned to 9 functional categories on the basis of their putative function. In order to validate the possible expression of transcripts, four genes, namely, S-adenosylmethionine synthetase, phosphoglycerate kinase, serine carboxypeptidase, and methionine aminopeptidase, were further analyzed by reverse transcriptase PCR. The possible role of the identified transcripts and their functions associated with root will also be a valuable resource for the functional genomics study in legume crop. PMID:24895494

  12. Sex Difference in Oxytocin-Induced Anti-Hyperalgesia at the Spinal Level in Rats with Intraplantar Carrageenan-Induced Inflammation.

    Science.gov (United States)

    Chow, Lok-Hi; Chen, Yuan-Hao; Wu, Wan-Chuan; Chang, En-Pei; Huang, Eagle Yi-Kung

    2016-01-01

    Previously, we demonstrated intrathecal administration of oxytocin strongly induced anti-hyperalgesia in male rats. By using an oxytocin-receptor antagonist (atosiban), the descending oxytocinergic pathway was found to regulate inflammatory hyperalgesia in our previous study using male rats. The activity of this neural pathway is elevated during hyperalgesia, but whether this effect differs in a sex-dependent manner remains unknown. We conducted plantar tests on adult male and female virgin rats in which paw inflammation was induced using carrageenan. Exogenous (i.t.) application of oxytocin exerted no anti-hyperalgesic effect in female rats, except at an extremely high dose. Female rats exhibited similar extent of hyperalgesia to male rats did when the animals received the same dose of carrageenan. When atosiban was administered alone, the severity of hyperalgesia was not increased in female rats. Moreover, insulin-regulated aminopeptidase (IRAP) was expressed at higher levels in the spinal cords of female rats compared with those of male rats. Oxytocin-induced anti-hyperalgesia exhibits a sex-dependent difference in rats. This difference can partially result from the higher expression of IRAP in the spinal cords of female rats, because IRAP functions as an enzyme that degrades oxytocin. Our study confirms the existence of a sex difference in oxytocin-induced anti-hyperalgesia at the spinal level in rats. PMID:27606886

  13. Electrochemical Characterization of Protein Adsorption onto YNGRT-Au and VLGXE-Au Surfaces

    Directory of Open Access Journals (Sweden)

    Hanna Trzeciakiewicz

    2015-08-01

    Full Text Available The adsorption of the proteins CD13, mucin and bovine serum albumin on VLGXE-Au and YNGRT-Au interfaces was monitored by electrochemical impedance spectroscopy in the presence of [Fe(CN6]3−/4−. The hydrophobicity of the Au surface was tailored using specific peptides, blocking agents and diluents. The combination of blocking agents (ethanolamine or n-butylamine and diluents (hexanethiol or 2-mercaptoethanol was used to prepare various peptide-modified Au surfaces. Protein adsorption onto the peptide-Au surfaces modified with the combination of n-butylamine and hexanethiol produced a dramatic decrease in the charge transfer resistance, Rct, for all three proteins. In contrast, polar peptide-surfaces induced a minimal change in Rct for all three proteins. Furthermore, an increase in Rct was observed with CD13 (an aminopeptidase overexpressed in certain cancers in comparison to the other proteins when the VLGXE-Au surface was modified with n-butylamine as a blocking agent. The electrochemical data indicated that protein adsorption may be modulated by tailoring the peptide sequence on Au surfaces and that blocking agents and diluents play a key role in promoting or preventing protein adsorption. The peptide-Au platform may also be used for targeting cancer biomarkers with designer peptides.

  14. Electrochemical Characterization of Protein Adsorption onto YNGRT-Au and VLGXE-Au Surfaces.

    Science.gov (United States)

    Trzeciakiewicz, Hanna; Esteves-Villanueva, Jose; Soudy, Rania; Kaur, Kamaljit; Martic-Milne, Sanela

    2015-01-01

    The adsorption of the proteins CD13, mucin and bovine serum albumin on VLGXE-Au and YNGRT-Au interfaces was monitored by electrochemical impedance spectroscopy in the presence of [Fe(CN)6](3-/4-). The hydrophobicity of the Au surface was tailored using specific peptides, blocking agents and diluents. The combination of blocking agents (ethanolamine or n-butylamine) and diluents (hexanethiol or 2-mercaptoethanol) was used to prepare various peptide-modified Au surfaces. Protein adsorption onto the peptide-Au surfaces modified with the combination of n-butylamine and hexanethiol produced a dramatic decrease in the charge transfer resistance, Rct, for all three proteins. In contrast, polar peptide-surfaces induced a minimal change in Rct for all three proteins. Furthermore, an increase in Rct was observed with CD13 (an aminopeptidase overexpressed in certain cancers) in comparison to the other proteins when the VLGXE-Au surface was modified with n-butylamine as a blocking agent. The electrochemical data indicated that protein adsorption may be modulated by tailoring the peptide sequence on Au surfaces and that blocking agents and diluents play a key role in promoting or preventing protein adsorption. The peptide-Au platform may also be used for targeting cancer biomarkers with designer peptides. PMID:26262621

  15. Differential sensitivity of bat cells to infection by enveloped RNA viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses.

    Directory of Open Access Journals (Sweden)

    Markus Hoffmann

    Full Text Available Bats (Chiroptera host major human pathogenic viruses including corona-, paramyxo, rhabdo- and filoviruses. We analyzed six different cell lines from either Yinpterochiroptera (including African flying foxes and a rhinolophid bat or Yangochiroptera (genera Carollia and Tadarida for susceptibility to infection by different enveloped RNA viruses. None of the cells were sensitive to infection by transmissible gastroenteritis virus (TGEV, a porcine coronavirus, or to infection mediated by the Spike (S protein of SARS-coronavirus (SARS-CoV incorporated into pseudotypes based on vesicular stomatitis virus (VSV. The resistance to infection was overcome if cells were transfected to express the respective cellular receptor, porcine aminopeptidase N for TGEV or angiotensin-converting enzyme 2 for SARS-CoV. VSV pseudotypes containing the S proteins of two bat SARS-related CoV (Bg08 and Rp3 were unable to infect any of the six tested bat cell lines. By contrast, viral pseudotypes containing the surface protein GP of Marburg virus from the family Filoviridae infected all six cell lines though at different efficiency. Notably, all cells were sensitive to infection by two paramyxoviruses (Sendai virus and bovine respiratory syncytial virus and three influenza viruses from different subtypes. These results indicate that bat cells are more resistant to infection by coronaviruses than to infection by paramyxoviruses, filoviruses and influenza viruses. Furthermore, these results show a receptor-dependent restriction of the infection of bat cells by CoV. The implications for the isolation of coronaviruses from bats are discussed.

  16. Proteomic amino-termini profiling reveals targeting information for protein import into complex plastids.

    Directory of Open Access Journals (Sweden)

    Pitter F Huesgen

    Full Text Available In organisms with complex plastids acquired by secondary endosymbiosis from a photosynthetic eukaryote, the majority of plastid proteins are nuclear-encoded, translated on cytoplasmic ribosomes, and guided across four membranes by a bipartite targeting sequence. In-depth understanding of this vital import process has been impeded by a lack of information about the transit peptide part of this sequence, which mediates transport across the inner three membranes. We determined the mature N-termini of hundreds of proteins from the model diatom Thalassiosira pseudonana, revealing extensive N-terminal modification by acetylation and proteolytic processing in both cytosol and plastid. We identified 63 mature N-termini of nucleus-encoded plastid proteins, deduced their complete transit peptide sequences, determined a consensus motif for their cleavage by the stromal processing peptidase, and found evidence for subsequent processing by a plastid methionine aminopeptidase. The cleavage motif differs from that of higher plants, but is shared with other eukaryotes with complex plastids.

  17. PhAP protease from Pseudoalteromonas haloplanktis TAC125: Gene cloning, recombinant production in E. coli and enzyme characterization

    Science.gov (United States)

    de Pascale, D.; Giuliani, M.; De Santi, C.; Bergamasco, N.; Amoresano, A.; Carpentieri, A.; Parrilli, E.; Tutino, M. L.

    2010-08-01

    Cold-adapted proteases have been found to be the dominant activity throughout the cold marine environment, indicating their importance in bacterial acquisition of nitrogen-rich complex organic compounds. However, few extracellular proteases from marine organisms have been characterized so far, and the mechanisms that enable their activity in situ are still largely unknown. Aside from their ecological importance and use as model enzyme for structure/function investigations, cold-active proteolytic enzymes offer great potential for biotechnological applications. Our studies on cold adapted proteases were performed on exo-enzyme produced by the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125. By applying a proteomic approach, we identified several proteolytic activities from its culture supernatant. PhAP protease was selected for further investigations. The encoding gene was cloned and the protein was recombinantly produced in E. coli cells. The homogeneous product was biochemically characterised and it turned out that the enzyme is a Zn-dependent aminopeptidase, with an activity dependence from assay temperature typical of psychrophilic enzymes.

  18. Isolated tumor endothelial cells maintain specific character during long-term culture

    International Nuclear Information System (INIS)

    Tumor angiogenesis is necessary for solid tumor progression and metastasis. Increasing evidence indicates that tumor endothelial cells (TECs) are more relevant to the study of tumor angiogenesis than normal endothelial cells (NECs) because their morphologies and gene expression are different from NECs. However, it is challenging to isolate and culture large numbers of pure ECs from tumor tissue since the percentage of ECs is only about 1-2% and tumor cells and fibroblasts easily overgrow them. In addition, there has been concern that isolated TECs may lose their special phenotype once they are dissociated from tumor cells. In this study, we have successfully purified murine TECs from four different human tumor xenografts and NECs from murine dermal tissue. Isolated ECs expressed endothelial markers, such as CD31, VE-cadherin (CD144), and endoglin (CD105), for more than 3 months after isolation. TECs maintained tumor endothelial-specific markers, such as tumor endothelial marker 8 (TEM8) and aminopeptidase N (APN), as in tumor blood vessels in vivo. In addition, TECs were more proliferative and motile than NECs. TECs showed a higher response to VEGF and higher expression of VEGF receptors-1 and -2 than NECs did. Stem cell antigen-1 was up-regulated in all four TECs, suggesting that they have a kind of stemness. Cultured TECs maintain distinct biological differences from NECs as in vivo. In conclusion, it was suggested that TECs are relevant material for tumor angiogenesis research.

  19. Major effect of hydrogen peroxide on bacterioplankton metabolism in the Northeast Atlantic.

    Directory of Open Access Journals (Sweden)

    Federico Baltar

    Full Text Available Reactive oxygen species such as hydrogen peroxide have the potential to alter metabolic rates of marine prokaryotes, ultimately impacting the cycling and bioavailability of nutrients and carbon. We studied the influence of H2O2 on prokaryotic heterotrophic production (PHP and extracellular enzymatic activities (i.e., β-glucosidase [BGase], leucine aminopeptidase [LAPase] and alkaline phosphatase [APase] in the subtropical Atlantic. With increasing concentrations of H2O2 in the range of 100-1000 nM, LAPase, APase and BGase were reduced by up to 11, 23 and 62%, respectively, in the different water layers. Incubation experiments with subsurface waters revealed a strong inhibition of all measured enzymatic activities upon H2O2 amendments in the range of 10-500 nM after 24 h. H2O2 additions also reduced prokaryotic heterotrophic production by 36-100% compared to the rapid increases in production rates occurring in the unamended controls. Our results indicate that oxidative stress caused by H2O2 affects prokaryotic growth and hydrolysis of specific components of the organic matter pool. Thus, we suggest that oxidative stress may have important consequences on marine carbon and energy fluxes.

  20. The death enzyme CP14 is a unique papain-like cysteine proteinase with a pronounced S2 subsite selectivity.

    Science.gov (United States)

    Paireder, Melanie; Mehofer, Ulrich; Tholen, Stefan; Porodko, Andreas; Schähs, Philipp; Maresch, Daniel; Biniossek, Martin L; van der Hoorn, Renier A L; Lenarcic, Brigita; Novinec, Marko; Schilling, Oliver; Mach, Lukas

    2016-08-01

    The cysteine protease CP14 has been identified as a central component of a molecular module regulating programmed cell death in plant embryos. CP14 belongs to a distinct subfamily of papain-like cysteine proteinases of which no representative has been characterized thoroughly to date. However, it has been proposed that CP14 is a cathepsin H-like protease. We have now produced recombinant Nicotiana benthamiana CP14 (NbCP14) lacking the C-terminal granulin domain. As typical for papain-like cysteine proteinases, NbCP14 undergoes rapid autocatalytic activation when incubated at low pH. The mature protease is capable of hydrolysing several synthetic endopeptidase substrates, but cathepsin H-like aminopeptidase activity could not be detected. NbCP14 displays a strong preference for aliphatic over aromatic amino acids in the specificity-determining P2 position. This subsite selectivity was also observed upon digestion of proteome-derived peptide libraries. Notably, the specificity profile of NbCP14 differs from that of aleurain-like protease, the N. benthamiana orthologue of cathepsin H. We conclude that CP14 is a papain-like cysteine proteinase with unusual enzymatic properties which may prove of central importance for the execution of programmed cell death during plant development. PMID:27246477

  1. Oral Insulin Stimulates Intestinal Department and Enzyme Maturation in Newborn Pigs

    Institute of Scientific and Technical Information of China (English)

    WANG Tian; XU Ruo-jun; HUO Yong-jiu

    2004-01-01

    The effects of oral insulin on intestinal tissue growth and brush border enzyme activities in newborn pigs were examined in this study. Newborn unsuckled pigs were bottle-fed for3 days with artificial milk(M),milk supplemented with 60mIUmL-1 of insulin(IH)or hydrolyzed milk(HM). Compared with newborn unsuckled pigs,piglets bottle-fed for 3 days all gained in intestinal weight and length significantly despite a mild loss in body weight during the experimental period. The activities of lactase and alkaline phosphatase(AKP)in the small intestinal mucosa declined markedly in pigs fed with M,but the activity of maltase increased significantly during the experimental period. Dietary protein pre-hydrolysis had no significant effect on intestinal tissue mass or length,but it moderated the decline of intestinal lactase and AKP activities. Dietary supplementation of insulin significantly increased mucosal protein content and brush border activities of lactase,maltase,AKP and aminopeptidase(AP)in the small intestine.The effect. of insulin treatment was particularly obvious at the distal region of the small intestine. These results demonstrate that oral insulin can stimulate intestinal digestive enzyme activities in newborn pigs. The finding supports the hypothesis that milk-borne insulin plays a role in regulating postnatal gut development in the suckling young.

  2. Diagnosis of. Hepatocellular Carcinoma in Patients with Liver Cir­rhosis Using Liver Function Assays

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    Itoshima,Tatsuya

    1984-04-01

    Full Text Available Sex, age and 21 routine liver function assays were analyzed by stepwise selection and the best-of-all-possible-combinations method to identify a small group of assays valuable in establishing which liver cirrhosis (LC patients have a high risk of hepatocellular carcinoma (HCC, when alpha-fetoprotein (AFP is not elevated. Data was obtained from 115 HCC and 122 LC patients on admission. Tumor size correlated with AFP (0.73, alkaline phosphatase (ALP, 0.47, leucine aminopeptidase (LAP, 0.42, lactic dehydrogenase (LDH, 0.42, and the glutamic oxaloacetic transaminase (GOT/glutamic pyruvic transaminase (GPT ratio (GOT/GPT, 0.41. The mean of the correct diagnosis rates (CDR of HCC and LC utilizing AFP as the sole parameter (89% was markedly higher than those of the other parameters. The best-of-all-possible-combinations method presented a more powerful combination than stepwise selection. The best combination of 7 parameters (LAP, GOT/GPT, choline esterase, one-hour erythrocyte sedimentation rate, age, albumin/globulin ratio, and total bilirubin presented a mean CDR of 80%, HCC CDR of 77%, and false positive rate of 18%. LC patients statistically diagnosed as having HCC by these 7 parameters are proposed as high risk patients. Fourteen (78% of 18 HCC patients who were AFP-negative were statistically diagnosed. This analysis can be applied to LC patients to distinguish those that should be followed closely by imaging diagnostic techniques.

  3. Radiation hygienization of poultry processing wastes

    International Nuclear Information System (INIS)

    Radiation hygienization has been standardized as an essential step in the preparation of value added products such as protein hydrolysates using proteolytic enzymes from chicken intestine. The total bacterial count and coliform count of the intestine were 1± 0.54 X 107 and 9.7 ± 2.36 X 106 cfu/g tissue respectively. A dose dependent decrease in bacterial count was observed when chicken intestine was exposed to gamma radiation. The total bacterial count was reduced by 4 log cycles and 6 log cycles at 5 kGy and 10 kGy respectively. During post irradiation storage for 20 days at 4 degC, the total bacterial count was found to increase by 2 log cycles in these samples. A radiation dose of 25 kGy rendered the samples sterile, which remained so during the entire period of storage. Exposure to ionizing radiation did not cause any significant change in the activities of tissue proteases. Nearly 90% of the activities of cathepsin D and aminopeptidase were retained in samples exposed to 25 kGy even after storage for 20 days. (author)

  4. Altered binding of the Cry1Ac toxin to larval membranes but not to the toxin-binding protein in Plodia interpunctella selected for resistance to different Bacillus thuringiensis isolates.

    Science.gov (United States)

    Mohammed, S I; Johnson, D E; Aronson, A I

    1996-11-01

    Immunoblotting and cytochemical procedures were used to determine whether toxin binding was altered in strains of the Indianmeal moth, Plodia interpunctella, selected for resistance to various strains of Bacillus thuringiensis. Each of these B. thuringiensis subspecies produces a mixture of protoxins, primarily Cry1 types, and the greatest insect resistance is to the Cry1A protoxins. In several cases, however, there was also resistance to toxins not present in the B. thuringiensis strains used for selection. The Cry1Ab and Cry1Ac toxins bound equally well over a range of toxin concentrations and times of incubation to a single protein of ca. 80-kDa in immunoblots of larval membrane extracts from all of the colonies. This binding protein is essential for toxicity since a mutant Cry1Ac toxin known to be defective in binding and thus less toxic bound poorly to the 80-kDa protein. This binding protein differed in size from the major aminopeptidase N antigens implicated in toxin binding in other insects. Binding of fluorescently labeled Cry1Ac or Cry1Ab toxin to larval sections was found at the tips of the brush border membrane prepared from the susceptible but not from any of the resistant P. interpunctella. Accessibility of a major Cry1A-binding protein appears to be altered in resistant larvae and could account for their broad resistance to several B. thuringiensis toxins.

  5. Pseudomonas aeruginosa vesicles associate with and are internalized by human lung epithelial cells

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    Kuehn Meta J

    2009-02-01

    Full Text Available Abstract Background Pseudomonas aeruginosa is the major pathogen associated with chronic and ultimately fatal lung infections in patients with cystic fibrosis (CF. To investigate how P. aeruginosa-derived vesicles may contribute to lung disease, we explored their ability to associate with human lung cells. Results Purified vesicles associated with lung cells and were internalized in a time- and dose-dependent manner. Vesicles from a CF isolate exhibited a 3- to 4-fold greater association with lung cells than vesicles from the lab strain PAO1. Vesicle internalization was temperature-dependent and was inhibited by hypertonic sucrose and cyclodextrins. Surface-bound vesicles rarely colocalized with clathrin. Internalized vesicles colocalized with the endoplasmic reticulum (ER marker, TRAPα, as well as with ER-localized pools of cholera toxin and transferrin. CF isolates of P. aeruginosa abundantly secrete PaAP (PA2939, an aminopeptidase that associates with the surface of vesicles. Vesicles from a PaAP knockout strain exhibited a 40% decrease in cell association. Likewise, vesicles from PAO1 overexpressing PaAP displayed a significant increase in cell association. Conclusion These data reveal that PaAP promotes the association of vesicles with lung cells. Taken together, these results suggest that P. aeruginosa vesicles can interact with and be internalized by lung epithelial cells and contribute to the inflammatory response during infection.

  6. Immunoproteasomes edit tumors, which then escapes immune recognition.

    Science.gov (United States)

    Joyce, Sebastian

    2015-12-01

    In 1985, John Monaco--the discoverer of LMP-2 and -7, the inducible components of the immunoproteasome--asked his advanced immunology class as to why the MHC region contained not only structural genes, but several others as well, whose functions were then unknown. As we drew a blank, he quipped: perchance because many of the MHC genes are induced by IFN-γ! The ensuing three decades have witnessed the unveiling of the profound fundamental and clinical implications of that classroom tête-à-tête. Amongst its multitudinous effects, IFN-γ induces genes enhancing antigen processing and presentation to T cells; such as those encoding cellular proteases and activators of proteases. In this issue, Keller et al. [Eur. J. Immunol. 2015. 45: 3257-3268] demonstrate that the limited success of MART-1/Melan-A-targeted immunotherapy in melanoma patients could be due to inefficient MART-1(26-35) presentation, owing to the proteolytic activities of IFN-γ-inducible β2i/MECL-1, proteasome activator 28 (PA28), and endoplasmic reticulum-associated aminopeptidase-associated with antigen processing (ERAP). Specifically, whilst β2i and PA28 impede MART-1(26-35) liberation from its precursor protein, ERAP-1 degrades this epitope. Hence, critical to effective cancer immunotherapy is deep knowledge of T-cell-targeted tumor antigens and how cellular proteases generate protective epitope(s) from them, or destroy them. PMID:26527367

  7. Investigation of natural biofilms formed during the production of drinking water from surface water embankment filtration.

    Science.gov (United States)

    Emtiazi, Farahnaz; Schwartz, Thomas; Marten, Silke Mareike; Krolla-Sidenstein, Peter; Obst, Ursula

    2004-03-01

    Populations of bacteria in biofilms from different sites of a drinking water production system were analysed. Polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analyses revealed changing DNA band patterns, suggesting a population shift during bank filtration and processing at the waterworks. In addition, common DNA bands that were attributed to ubiquitous bacteria were found. Biofilms even developed directly after UV disinfection (1-2m distance). Their DNA band patterns only partly agreed with those of the biofilms from the downstream distribution system. Opportunistic pathogenic bacteria in biofilms were analysed using PCR and Southern blot hybridisation (SBH). Surface water appeared to have a direct influence on the composition of biofilms in the drinking water distribution system. In spite of preceding filtration and UV disinfection, opportunistic pathogens such as atypical mycobacteria and Legionella spp. were found in biofilms of drinking water, and Pseudomonas aeruginosa was detected sporadically. Enterococci were not found in any biofilm. Bacterial cell counts in the biofilms from surface water to drinking water dropped significantly, and esterase and alanine-aminopeptidase activity decreased. beta-glucosidase activity was not found in the biofilms. Contrary to the results for planktonic bacteria, inhibitory effects were not observed in biofilms. This suggested an increased tolerance of biofilm bacteria against toxic compounds.

  8. Response of bacterioplankton activity in an Arctic fjord system to elevated pCO2: results from a mesocosm perturbation study

    Directory of Open Access Journals (Sweden)

    U. Riebesell

    2013-01-01

    Full Text Available The effect of elevated seawater carbon dioxide (CO2 on the activity of a natural bacterioplankton community in an Arctic fjord system was investigated by a mesocosm perturbation study in the frame of the European Project on Ocean Acidification (EPOCA. A pCO2 range of 175–1085 μatm was set up in nine mesocosms deployed in the Kongsfjorden (Svalbard. The activity of natural extracellular enzyme assemblages increased in response to acidification. Rates of β-glucosidase and leucine-aminopeptidase increased along the gradient of mesocosm pCO2. A decrease in seawater pH of 0.5 units almost doubled rates of both enzymes. Heterotrophic bacterial activity was closely coupled to phytoplankton productivity in this experiment. The bacterioplankton community responded to rising chlorophyll a concentrations after a lag phase of only a few days with increasing protein production and extracellular enzyme activity. Time-integrated primary production and bacterial protein production were positively correlated, strongly suggesting that higher amounts of phytoplankton-derived organic matter were assimilated by heterotrophic bacteria at increased primary production. Primary production increased under high pCO2 in this study, and it can be suggested that the efficient heterotrophic carbon utilisation had the potential to counteract the enhanced autotrophic CO2 fixation. However, our results also show that beneficial pCO2-related effects on bacterial activity can be mitigated by the top-down control of bacterial abundances in natural microbial communities.

  9. Cell-free extracellular enzymatic activity is linked to seasonal temperature changes: a case study in the Baltic Sea

    Science.gov (United States)

    Baltar, Federico; Legrand, Catherine; Pinhassi, Jarone

    2016-05-01

    Extracellular enzymatic activities (EEAs) are a crucial step in the degradation of organic matter. Dissolved (cell-free) extracellular enzymes in seawater can make up a significant contribution of the bulk EEA. However, the factors controlling the proportion of dissolved EEA in the marine environment remain unknown. Here we studied the seasonal changes in the proportion of dissolved relative to total EEA (of alkaline phosphatase (APase), β-glucosidase (BGase), and leucine aminopeptidase (LAPase)), in the Baltic Sea for 18 months. The proportion of dissolved EEA ranged between 37 and 100, 0 and 100, and 34 and 100 % for APase, BGase, and LAPase, respectively. A consistent seasonal pattern in the proportion of dissolved EEA was found among all the studied enzymes, with values up to 100 % during winter and hydrolysis rates from microbial dynamics in cold waters. This implies that under cold conditions, cell-free enzymes can contribute to substrate availability at large distances from the producing cell, increasing the dissociation between the hydrolysis of organic compounds and the actual microbes producing the enzymes. This might also suggest a potential effect of global warming on the hydrolysis of organic matter via a reduction of the contribution of cell-free enzymes to the bulk hydrolytic activity.

  10. INSULIN AND INSULIN RESISTANCE: NEW MOLECULE MARKERS AND TARGET MOLECULE FOR THE DIAGNOSIS AND THERAPY OF DISEASES OF THE CENTRAL NERVOUS SYSTEM

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    A. B. Salmina

    2013-01-01

    Full Text Available The review summarizes current data on the role of insulin in the regulation of t glucose metabolism in the central nervous system at physiologic and pathologic conditions. For many years, the brain has been considered as an insulin-independent organ which utilizes glucose without insulin activity. However, it is become clear now that insulin not only regulates glucose transport and metabolism, but also has modulatory efftects in impact on excitability, proliferation and differentiation of brain progenitor cells, synaptic plasticity and memory formation, secretion of neurotransmitters, apoptosis. We have critically reviewed literature information and our own data on the role of insulin and insulin resistance in neuron-glia metabolic coupling, regulation of NAD+ metabolism and action of NAdependent enzymes, neurogenesis, brain development in (pathophysiological conditions. The paper clarifies interrelations between alterations in glucose homeostasis, development of insulin resistance and development of neurodegeneration (Alzheimer's disease and Parkinson's disease, autism, stroke, and depression. We discuss the application of novel molecular markers of insulin resistance (adipokines, α-hydroxybutyrate, BDNF, insulin-regulated aminopeptidase, provasopressin and molecular targets for diagnostics and treatment of brain disorders associated with insulin resistance.

  11. Microbial exoenzymes as bioindicators of acid rock drainage impacts in the Finniss River

    International Nuclear Information System (INIS)

    Sediment samples were collected from several sites along the East Branch of the Finniss River during the dry season (June, 1999), when the East Branch is drying into a series of ponds. The sites included those upstream from the Rum Jungle mine site (EB8A, EB8B, FCA, FCB), a site receiving acid leachate from the waste rock (WO), sites downstream from the mine that are impacted by acid and metal contamination (EB6, TCP, EB5D, EB4U, EB2) and reference sites not subject to acid rock drainage (HS, EB4S, LFRB). Exoenzyme activities were measured with a spectrofluorometric technique that involved measuring the increase in fluorescence when an artificial fluorogenic substrate (that mimics the natural substrate) is hydrolysed to a highly fluorescent product. The present findings indicate that the acid rock drainage impacted sediments contain acidophilic, heterotrophic microorganisms, bacteria and/or fungi, producing extracellular enzymes adapted to the acid conditions. This study has demonstrated that measurements of extracellular enzyme activities in river sediments provide a rapid, sensitive technique for determining microbial activity and productivity. In aquatic ecosystems some exoenzymes, particularly leucine-aminopeptidase, could be used as bioindicators of pollution from acid rock drainage

  12. Evaluation of Genetic Variations in Maize Seedlings Exposed to Electric Field Based on Protein and DNA Markers

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    Asma A. AL-Huqail

    2015-01-01

    Full Text Available The current study analyzed proteins and nuclear DNA of electric fields (ELF exposed and nonexposed maize seedlings for different exposure periods using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, isozymes, random amplified polymorphic DNA (RAPD, and comet assay, respectively. SDS-PAGE analysis revealed total of 46 polypeptides bands with different molecular weights ranging from 186.20 to 36.00 KDa. It generated distinctive polymorphism value of 84.62%. Leucine-aminopeptidase, peroxidase, and catalase isozymes showed the highest values of polymorphism (100% based on zymograms number, relative front (Rf, and optical intensity while esterase isozyme generated polymorphism value of 83.33%. Amino acids were analyzed using high-performance liquid chromatography, which revealed the presence of 17 amino acids of variable contents ranging from 22.65% to 28.09%. RAPD revealed that 78 amplified DNA products had highly polymorphism value (95.08% based on band numbers, with variable sizes ranging from 120 to 992 base pairs and band intensity. Comet assay recorded the highest extent of nuclear DNA damage as percentage of tailed DNA (2.38% and tail moment unit (5.36 at ELF exposure of maize nuclei for 5 days. The current study concluded that the longer ELF exposing periods had genotoxic stress on macromolecules of maize cells and biomarkers used should be augmented for reliable estimates of genotoxicity after exposure of economic plants to ELF stressors.

  13. Effects of an extracted lectin from Citrullus colocynthis L. (Cucurbitaceae on survival, digestion and energy reserves of Ectomyelois ceratoniae Zeller (Lepidoptera: Pyralidae

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    Samar eRamzi

    2013-11-01

    Full Text Available Lectins are the widespread and heterogeneous proteins of plants that serve as storage proteins in defensive mechanisms against herbivores. A lectin was extracted and purified from seeds of Citrullus colocynthis by Sepharose 4B-Galactose and DEAE-cellulose fast flow chromatographies. Different concentrations of the lectin were added to artificial diet of Ectomyelois ceratoniae to find out its effect on some biological parameters, digestive physiology and amount of storage macromolecules. It was found that CCA increased life span from 23.44 days in control to 28.59 days in the individuals fed on 2% of CCA. Larval survivals on control and CCA diets were 93.3 and 66.6%, respectively. Different concentrations of CCA significantly affected α-amylase and general proteolytic activities in larvae but TAG-lipase activity had no significant changes. Activities of all specific proteases decreased when larvae were fed on different concentrations of CCA except for aminopeptidase. Meanwhile, amount of storage macromolecules in the larvae fed on different concentrations of CCA statistically decreased versus control. These results demonstrated that CCA could intervene in physiology of E. ceratoniae and survival of larvae. Therefore, it can be taken into consideration in IPM of the pest through plant breeding programs.

  14. Chronic irradiation as an ecological factor affecting genetic population structure

    International Nuclear Information System (INIS)

    Genetic structure of two Centaurea scabiosa L. populations was studied by frequency distribution of leucine aminopeptidase (LAP) locus genotypes. The experimental population has been growing under conditions of chronic irradiation, with the dose per generation amounting to 1.2 to 25.5 Gy. In it, mutational variants are observed with a frequency of 5.4.10(-3)-4.5.10(-2) per generation (as compared to control population frequency at 5.4.10(-4)). Indexes for heterozygosity, mean number of genotypes, and effective number of alleles were higher in the experimental population. Segregation analysis revealed no differences in viability in the control population, and all genotypic combinations were found to be nearly neutral. In the experimental population, however, significant differences in relative viability of the genotypes were disclosed. The relative viability of heterozygotes for mutant allele C' was nearly maximum, while heterozygotes for other mutant alleles showed minimum viability. We reach the conclusion that the differences in genetic structure of the populations under investigation can be explained by the chronic irradiation factor that brought out differences in adaptability of both normal and mutant genotypes. The suggestion is that intra-locus interactions of the C' allele with normal alleles determine plant resistance to a wide range of unfavorable environmental conditions

  15. Irradiation Injury Temporarily Induces Enhancement of APN/CD13 Peptidase Activity on Aorta-gonads-mesonephros (AGM)-derived Stromal Cells

    Institute of Scientific and Technical Information of China (English)

    ZHU Yan; HUANG Lifang; LUO Xiaohua; SUN Hanying; RAN Dan; ZHANG Kejie; ZHENG Miao; ZHOU Kun; LIU Wenli

    2007-01-01

    This study was designed to investigate the expression of aminopeptidase N (APN)/CD13 on intraembryonic AGM stromal cells, and the change of its enzymatic activity after irradiation injury.The expression of APN/CD13 on AGM stromal cells was assayed by RT-PCR and immunihistochemistry. After the stromal cells in AGM region were irradiated with 8.0 Gy of 60Co γ-rays, APN/CD13 enzymatic activity was measured by spectrophotometer at different time points. The result showed that AGM stromal cells strongly expressed APN/CD13. The enzymatic activity of APN/CD13 decreased temporarily after irradiation injury, then increased to higher level 4 h after irradiation, and it returned to the pre-irradiation level 24 to 48 h after the irradiation. The enzymatic activity of APN/CD13 was temporarily enhanced after irradiation injury, which might be one of the compensatory mechanisms that promote the hematopoietic recovery after irradiation.

  16. Intestinal adaptation to diet in the young domestic and wild turkey (Meleagris gallopavo).

    Science.gov (United States)

    Foye, O T; Black, B L

    2006-02-01

    The short-term effects of diet on jejunal growth, alanine transport rate, and leucine aminopeptidase activity (LAP) were compared in the domestic and wild turkey poult. One-day-old poults of each strain were fed diets of high vs., low protein, with carbohydrate varied to maintain isocaloric conditions. Prior to feeding, relative jejunal mass and alanine transport rates were not significantly different in the two turkey strains, whereas LAP activity was 270% higher in wild poults. After feeding for 72 h, relative jejunal mass doubled in both turkey strains. In domestic turkeys, alanine transport rate and LAP activity were reduced by approximately 42% and 25%, respectively, in poults fed a 24% protein-69% carbohydrate diet vs. a 49% protein-35% carbohydrate diet. Analysis of the combined data from feeding experiments revealed that alanine transport rate was not correlated with total food, protein or lipid intake, but was negatively correlated with carbohydrate consumption (Pwild turkeys, neither alanine transport rate nor LAP activity were altered by diet. These results reveal that domestic turkey hatchlings can modulate protein digestive and absorptive functions as protein/carbohydrate composition of the diet changes and suggest that high dietary carbohydrate down-regulates the intestinal alanine transporter.

  17. Persistence of bacterial proteolytic enzymes in lake ecosystems.

    Science.gov (United States)

    Kiersztyn, Bartosz; Siuda, Waldemar; Chróst, Ryszard J

    2012-04-01

    This study analyzes proteolytic enzyme persistence and the role of dead (or metabolically inactive) aquatic bacteria in organic matter cycling. Samples from four lakes of different trophic status were used. Irrespective of the trophic status of the examined lakes, bacterial aminopeptidases remained active even 72 h after the death of the bacteria that produced them. The total pool of proteolytic enzymes in natural lake water samples was also stable. We found that the rates of amino acid enzymatic release from proteinaceous matter added to preserved lake water sample were constant for at least 96 h (r(2)  = 0.99, n = 17, P ≤ 0.0001, V(max)  = 84.6 nM h(-1) ). We also observed that proteases built into bacterial cell debris fragments remained active for a long time, even after the total destruction of cells. Moreover, during 24 h of incubation time, about 20% of these enzymatically active fragments adsorbed onto natural seston particles, becoming a part of the 'attached enzymes system' that is regarded as the 'hot-spot' of protein degradation in aquatic ecosystems. PMID:22150269

  18. Proteomic analysis of urinary exosomes from patients of early IgA nephropathy and thin basement membrane nephropathy.

    Science.gov (United States)

    Moon, Pyong-Gon; Lee, Jeong-Eun; You, Sungyong; Kim, Taek-Kyun; Cho, Ji-Hoon; Kim, In-San; Kwon, Tae-Hwan; Kim, Chan-Duck; Park, Sun-Hee; Hwang, Daehee; Kim, Yong-Lim; Baek, Moon-Chang

    2011-06-01

    To identify biomarker candidates associated with early IgA nephropathy (IgAN) and thin basement membrane nephropathy (TBMN), the most common causes presenting isolated hematuria in childhood, a proteomic approach of urinary exosomes from early IgAN and TBMN patients was introduced. The proteomic results from the patients were compared with a normal group to understand the pathophysiological processes associated with these diseases at the protein level. The urinary exosomes, which reflect pathophysiological processes, collected from three groups of young adults (early IgAN, TBMN, and normal) were trypsin-digested using a gel-assisted protocol, and quantified by label-free LC-MS/MS, using an MS(E) mode. A total of 1877 urinary exosome proteins, including cytoplasmic, membrane, and vesicle trafficking proteins, were identified. Among the differentially expressed proteins, four proteins (aminopeptidase N, vasorin precursor, α-1-antitrypsin, and ceruloplasmin) were selected as biomarker candidates to differentiate early IgAN from TBMN. We confirmed the protein levels of the four biomarker candidates by semi-quantitative immunoblot analysis in urinary exosomes independently prepared from other patients, including older adult groups. Further clinical studies are needed to investigate the diagnostic and prognostic value of these urinary markers for early IgAN and TBMN. Taken together, this study showed the possibility of identifying biomarker candidates for human urinary diseases using urinary exosomes and might help to understand the pathophysiology of early IgAN and TBMN at the protein level.

  19. Identification and characterization of potentially algal-lytic marine bacteria strongly associated with the toxic dinoflagellate Alexandrium catenella.

    Science.gov (United States)

    Amaro, Ana M; Fuentes, María S; Ogalde, Sandra R; Venegas, Juan A; Suárez-Isla, Benjamín A

    2005-01-01

    The toxic dinoflagellate Alexandrium catenella isolated from fjords in Southern Chile produces several analogues of saxitoxin and has been associated with outbreaks of paralytic shellfish poisoning. Three bacterial strains, which remained in close association with this dinoflagellate in culture, were isolated by inoculating the dinoflagellate onto marine agar. The phenotypically different cultivable bacterial colonies were purified. Their genetic identification was done by polymerase chain reaction amplification of the 16S rRNA genes. Partial sequence analysis suggested that the most probable affiliations were to two bacterial phyla: Proteobacteria and the Cytophaga group. The molecular identification was complemented by morphological data and biochemical profiling. The three bacterial species, when grown separately from phytoplankton cells in high-nutrient media, released algal-lytic compounds together with aminopeptidase, lipase, glucosaminidase, and alkaline phosphatase. When the same bacteria, free of organic nutrients, were added back to the algal culture they displayed no detrimental effects on the dinoflagellate cells and recovered their symbiotic characteristics. This observation is consistent with phylogenetic analysis that reveals that these bacteria correspond to species distinct from other bacterial strains previously classified as algicidal bacteria. Thus, bacterial-derived lytic activities are expressed only in the presence of high-nutrient culture media and it is likely that in situ environmental conditions may modulate their expression. PMID:15926994

  20. The Zymovars of Vibrio cholerae: Multilocus Enzyme Electrophoresis of Vibrio cholerae

    Directory of Open Access Journals (Sweden)

    Fernanda S Freitas

    2002-06-01

    Full Text Available Zymovars analysis also known as multilocus enzyme electrophoresis is applied here to investigate the genetic variation of Vibrio cholerae strains and characterise strains or group of strains of medical and epidemiological interest. Fourteen loci were analyzed in 171 strains of non-O1 non-O139, 32 classical and 61 El Tor from America, Africa, Europe and Asia. The mean genetic diversity was 0.339. It is shown that the same O antigen (both O1 and non-O1 may be present in several geneticaly diverse (different zymovars strains. Conversely the same zymovar may contain more than one serogroup. It is confirmed that the South American epidemic strain differs from the 7th pandemic El Tor strain in locus LAP (leucyl leucyl aminopeptidase. Here it is shown that this rare allele is present in 1 V. mimicus and 4 non-O1 V. cholerae. Non toxigenic O1 strains from South India epidemic share zymovar 14A with the epidemic El Tor from the 7th pandemic, while another group have diverse zymovars. The sucrose negative epidemic strains isolated in French Guiana and Brazil have the same zymovar of the current American epidemic V. cholerae.

  1. Simple enzymatic procedure for L-carnosine synthesis: whole-cell biocatalysis and efficient biocatalyst recycling.

    Science.gov (United States)

    Heyland, Jan; Antweiler, Nicolai; Lutz, Jochen; Heck, Tobias; Geueke, Birgit; Kohler, Hans-Peter E; Blank, Lars M; Schmid, Andreas

    2010-01-01

    β-Peptides and their derivates are usually stable to proteolysis and have an increased half-life compared with α-peptides. Recently, β-aminopeptidases were described as a new enzyme class that enabled the enzymatic degradation and formation of β-peptides. As an alternative to the existing chemical synthesis routes, the aim of the present work was to develop a whole-cell biocatalyst for the synthesis and production of β-peptides using this enzymatic activity. For the optimization of the reaction system we chose the commercially relevant β,α-dipeptide L-carnosine (β-alanine-L-histidine) as model product. We were able to show that different recombinant yeast and bacteria strains, which overexpress a β-peptidase, could be used directly as whole-cell biocatalysts for the synthesis of L-carnosine. By optimizing relevant reaction conditions for the best-performing recombinant Escherichia coli strain, such as pH and substrate concentrations, we obtained high l-carnosine yields of up to 71%. Long-time as well as biocatalyst recycling experiments indicated a high stability of the developed biocatalyst for at least five repeated batches. Application of the recombinant E. coli in a fed-batch process enabled the accumulation of l-carnosine to a concentration of 3.7 g l(-1). PMID:21255308

  2. Larval midgut modifications associated with Bti resistance in the yellow fever mosquito using proteomic and transcriptomic approaches

    Directory of Open Access Journals (Sweden)

    Tetreau Guillaume

    2012-06-01

    Full Text Available Abstract Background Bacillus thuringiensis var. israelensis (Bti is a natural larval mosquito pathogen producing pore-forming toxins targeting the midgut of Diptera larvae. It is used worldwide for mosquito control. Resistance mechanisms of an Aedes aegypti laboratory strain selected for 30 generations with field-collected leaf litter containing Bti toxins were investigated in larval midguts at two levels: 1. gene transcription using DNA microarray and RT-qPCR and 2. differential expression of brush border membrane proteins using DIGE (Differential In Gel Electrophoresis. Results Several Bti Cry toxin receptors including alkaline phosphatases and N-aminopeptidases and toxin-binding V-ATPases exhibited altered expression levels in the resistant strain. The under-expression of putative Bti-receptors is consistent with Bt-resistance mechanisms previously described in Lepidoptera. Four soluble metalloproteinases were found under-transcribed together with a drastic decrease of metalloproteinases activity in the resistant strain, suggesting a role in resistance by decreasing the amount of activated Cry toxins in the larval midgut. Conclusions By combining transcriptomic and proteomic approaches, we detected expression changes at nearly each step of the ingestion-to-infection process, providing a short list of genes and proteins potentially involved in Bti-resistance whose implication needs to be validated. Collectively, these results open the way to further functional analyses to better characterize Bti-resistance mechanisms in mosquitoes.

  3. Responses of absolute and specific soil enzyme activities to long term additions of organic and mineral fertilizer.

    Science.gov (United States)

    Zhang, Xinyu; Dong, Wenyi; Dai, Xiaoqin; Schaeffer, Sean; Yang, Fengting; Radosevich, Mark; Xu, Lili; Liu, Xiyu; Sun, Xiaomin

    2015-12-01

    Long-term phosphorus (P) and nitrogen (N) applications may seriously affect soil microbial activity. A long-term field fertilizer application trial was established on reddish paddy soils in the subtropical region of southern China in 1998. We assessed the effects of swine manure and seven different rates or ratios of NPK fertilizer treatments on (1) the absolute and specific enzyme activities per unit of soil organic carbon (SOC) or microbial biomass carbon (MBC) involved in C, N, and P transformations and (2) their relationships with soil environmental factors and soil microbial community structures. The results showed that manure applications led to increases in the absolute and specific activities of soil β-1,4-glucosidase(βG), β-1,4-N-acetylglucosaminidase (NAG), and leucine aminopeptidase (LAP). The absolute and specific acid phosphatase (AP) activities decreased as mineral P fertilizer application rates and ratios increased. Redundancy analysis (RDA) showed that there were negative correlations between absolute and specific AP activities, pH, and total P contents, while there were positive correlations between soil absolute and specific βG, NAG, and LAP enzyme activities, and SOC and total N contents. RDA showed that the contents of actinomycete and Gram-positive bacterium PLFA biomarkers are more closely related to the absolute and specific enzyme activities than the other PLFA biomarkers (Pfertilizer application rates to subtropical paddy soils should not exceed 44 kg P ha(-1) year(-1). PMID:26196069

  4. Quantitative role of shrimp fecal bacteria in organic matter fluxes in a recirculating shrimp aquaculture system.

    Science.gov (United States)

    Beardsley, Christine; Moss, Shaun; Malfatti, Francesca; Azam, Farooq

    2011-07-01

    Microorganisms play integral roles in the cycling of carbon (C) and nitrogen (N) in recirculating aquaculture systems (RAS) for fish and shellfish production. We quantified the pathways of shrimp fecal bacterial activities and their role in C- and N-flux partitioning relevant to culturing Pacific white shrimp, Penaeus (Litopenaeus) vannamei, in RAS. Freshly produced feces from P. vannamei contained 0.6-7 × 10(10) bacteria g(-1) dry wt belonging to Bacteroidetes (7%), Alphaproteobacteria (4%), and, within the Gammaproteobacteria, almost exclusively to the genus Vibrio (61%). Because of partial disintegration of the feces (up to 27% within 12 h), the experimental seawater became inoculated with fecal bacteria. Bacteria grew rapidly in the feces and in the seawater, and exhibited high levels of aminopeptidase, chitinase, chitobiase, alkaline phosphatase, α- and β-glucosidase, and lipase activities. Moreover, fecal bacteria enriched the protein content of the feces within 12 h, potentially enriching the feces for the coprophagous shrimp. The bacterial turnover time was much faster in feces (1-10 h) than in mature RAS water (350 h). Thus, shrimp fecal bacteria not only inoculate RAS water but also contribute to bacterial abundance and productivity, and regulate system processes important for shrimp health. PMID:21426366

  5. Brain met-enkephalin immunostaining after subacute and subchronic exposure to benzene

    Energy Technology Data Exchange (ETDEWEB)

    Gandarias, J.M. de; Echevarria, E.; Martinez-Millan, L.; Casis, L. [Univ. of the Basque Country, Bilbao (Spain); Martinez-Garcia, F. [Univ. of Valencia (Spain)

    1994-01-01

    Benzene is used in a wide variety of domestic and occupational activities, and due to its lipophilic nature, it accumulates in lipid-rich tissues like the brain. In this sense, neurotoxic action has long been associated with organic solvent exposure and it has been shown that benzene, injected in a single dose or during a prolongued administration, modifies the content of dopamine, noradrenaline, serotonin and its main metabolite 5-hydroxy indolacetic acid, in several brain regions of the rat, then revealing a stimulating action on brain monoamine synthesis and turnover. However, information concerning neurotoxic action of benzene exposure in vivo on peptidergic neuromodulatory systems is still lacking. Nevertheless, it has been recently described that subacute benzene exposure in rats generates regional changes in brain aminopeptidase activity. These proteolytic enzymes have been widely associated with metabolic control of neuropeptides and it has been suggested that they could play a role in benzene neurotoxic mechanism by hypothetically changing regional neuropeptide levels. This being the case, we focused on analyzing met-enkephalin immunostaining in different brain regions of the rat after subacute and subchronic administration of benzene. 12 refs., 3 figs.

  6. Caracterização isoenzimática de oito acessos de Erva-de-bicho

    Directory of Open Access Journals (Sweden)

    Lopes Reginalda C.

    2003-01-01

    Full Text Available Na caracterização de oito acessos de Polygonum punctatum Ell., utilizou-se a técnica de eletroforese horizontal em gel de amido a 12%. Determinaram-se os fenótipos isoenzimáticos da malato desidrogenase (MDH, fosfatase ácida (ACP, peroxidase (PO, glutamato desidrogenase (GDH, leucina aminopeptidase (LAP, esterase (EST, álcool desidrogenase (ADH, glutamato oxaloacetato transaminase (GOT, isocitrato desidrogenase (IDH e chiquimato desidrogenase (SKDH, utilizando extratos de folhas jovens de cada acesso. A eletroforese foi conduzida em géis de amido de milho e amido hidrolisado. O amido de milho mostrou resolução satisfatória na separação das bandas isoenzimáticas da IDH, ADH e MDH. No entanto, para os demais sistemas, o amido hidrolisado apresentou resolução superior à do amido de milho. Todos os sistemas enzimáticos estudados, com exceção do sistema LAP, apresentaram elevado polimorfismo, permitindo estabelecer padrões individuais em todos os acessos. Isso demonstrou o potencial das isoenzimas como marcadores genéticos no gênero Polygonum.

  7. Use of Whole Exome Sequencing for the Identification of Ito-Based Arrhythmia Mechanism and Therapy

    Science.gov (United States)

    Sturm, Amy C; Kline, Crystal F; Glynn, Patric; Johnson, Benjamin L; Curran, Jerry; Kilic, Ahmet; Higgins, Robert S D; Binkley, Philip F; Janssen, Paul M L; Weiss, Raul; Raman, Subha V; Fowler, Steven J; Priori, Silvia G; Hund, Thomas J; Carnes, Cynthia A; Mohler, Peter J

    2015-01-01

    Background Identified genetic variants are insufficient to explain all cases of inherited arrhythmia. We tested whether the integration of whole exome sequencing with well-established clinical, translational, and basic science platforms could provide rapid and novel insight into human arrhythmia pathophysiology and disease treatment. Methods and Results We report a proband with recurrent ventricular fibrillation, resistant to standard therapeutic interventions. Using whole-exome sequencing, we identified a variant in a previously unidentified exon of the dipeptidyl aminopeptidase-like protein-6 (DPP6) gene. This variant is the first identified coding mutation in DPP6 and augments cardiac repolarizing current (Ito) causing pathological changes in Ito and action potential morphology. We designed a therapeutic regimen incorporating dalfampridine to target Ito. Dalfampridine, approved for multiple sclerosis, normalized the ECG and reduced arrhythmia burden in the proband by >90-fold. This was combined with cilostazol to accelerate the heart rate to minimize the reverse-rate dependence of augmented Ito. Conclusions We describe a novel arrhythmia mechanism and therapeutic approach to ameliorate the disease. Specifically, we identify the first coding variant of DPP6 in human ventricular fibrillation. These findings illustrate the power of genetic approaches for the elucidation and treatment of disease when carefully integrated with clinical and basic/translational research teams. PMID:26015324

  8. Effects of an extracted lectin from Citrullus colocynthis L. (Cucurbitaceae) on survival, digestion and energy reserves of Ectomyelois ceratoniae Zeller (Lepidoptera: Pyralidae).

    Science.gov (United States)

    Ramzi, Samar; Sahragard, Ahad; Sendi, Jalal J; Aalami, Ali

    2013-01-01

    Lectins are the heterogeneous proteins in plants that serve as storage proteins via defensive mechanisms against herbivores. In the current study, a lectin was extracted and purified from seeds of Citrullus colocynthis by Sepharose 4B-Galactose and DEAE-cellulose fast flow chromatographies. Different concentrations of the lectin were added to artificial diet of Ectomyelois ceratoniae larvae finding out its effect on some biological parameters, digestive physiology and amount of storage macromolecules. It was found that CCA (C. colocynthis Agglutinin) increased life span from 23.44 days in control to 28.59 days in the treated individuals. Survival of larvae on control and CCA diets were 93.3 and 66.6%, respectively. Different concentrations of CCA significantly affected α-amylase and general proteolytic activities except for TAG-lipase activity. Activities of all specific proteases decreased when larvae were fed on different concentrations of CCA except for aminopeptidase. Meanwhile, amount of storage macromolecules in the larvae fed on different concentrations of CCA statistically decreased vs. control. These results demonstrated that CCA could intervene in physiology of E. ceratoniae and survival of larvae. Therefore, it can be taken into consideration in IPM of the pest through plant breeding programs.

  9. Nosema ceranae escapes fumagillin control in honey bees.

    Directory of Open Access Journals (Sweden)

    Wei-Fone Huang

    2013-03-01

    Full Text Available Fumagillin is the only antibiotic approved for control of nosema disease in honey bees and has been extensively used in United States apiculture for more than 50 years for control of Nosema apis. It is toxic to mammals and must be applied seasonally and with caution to avoid residues in honey. Fumagillin degrades or is diluted in hives over the foraging season, exposing bees and the microsporidia to declining concentrations of the drug. We showed that spore production by Nosema ceranae, an emerging microsporidian pathogen in honey bees, increased in response to declining fumagillin concentrations, up to 100% higher than that of infected bees that have not been exposed to fumagillin. N. apis spore production was also higher, although not significantly so. Fumagillin inhibits the enzyme methionine aminopeptidase2 (MetAP2 in eukaryotic cells and interferes with protein modifications necessary for normal cell function. We sequenced the MetAP2 gene for apid Nosema species and determined that, although susceptibility to fumagillin differs among species, there are no apparent differences in fumagillin binding sites. Protein assays of uninfected bees showed that fumagillin altered structural and metabolic proteins in honey bee midgut tissues at concentrations that do not suppress microsporidia reproduction. The microsporidia, particularly N. ceranae, are apparently released from the suppressive effects of fumagillin at concentrations that continue to impact honey bee physiology. The current application protocol for fumagillin may exacerbate N. ceranae infection rather than suppress it.

  10. Use of activity-based probes to develop high throughput screening assays that can be performed in complex cell extracts.

    Directory of Open Access Journals (Sweden)

    Edgar Deu

    Full Text Available BACKGROUND: High throughput screening (HTS is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities. METHODOLOGY AND PRINCIPAL FINDINGS: Here, we described a method to use activity-based probes (ABPs to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z'>0.8 that are suitable for use in screening large collections of small molecules (i.e >300,000 for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates. CONCLUSIONS: We believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic.

  11. Improved QM/MM Linear-Interaction Energy Model for Substrate Recognition in Zinc-Containing Metalloenzymes.

    Science.gov (United States)

    Miranda, Williams E; Ngo, Van A; Valiente, Pedro A; Noskov, Sergei Yu

    2016-08-18

    One of the essential challenges in the description of receptor-drug interactions in the presence of various polyvalent cations (such as zinc, magnesium, or iron) is the accurate assessment of the electronic effects due to cofactor binding. The effects can range from partial electronic polarization of the proximal atoms in a receptor and bound substrate to long-range effects related to partial charge transfer and electronic delocalization effects between the cofactor and the drug. Here, we examine the role of the explicit account for electronic effects for a panel of small-molecule inhibitors binding to the zinc-aminopeptidase PfA-M1, an essential target for antimalarial drug development. Our study on PfA-M1:inhibitor interactions at the QM level reveals that the partial charge and proton transfer due to bound zinc ion are important mechanisms in the inhibitors' recognition and catalysis. The combination of classical MD simulations with a posteriori QM/MM corrections with novel DFTB parameters for the zinc cation and the linear-interaction energy (LIE) approach offers by far the most accurate estimates for the PfA-M1:inhibitor binding affinities, opening the door for future inhibitor design. PMID:27448039

  12. Fluorogenic Membrane Overlays to Enumerate Total and Fecal Escherichia coli and Total Vibrionaceae in Shellfish and Seawater

    Directory of Open Access Journals (Sweden)

    Gary P. Richards

    2010-01-01

    Full Text Available Three assays were developed to enumerate total and fecal Escherichia coli and total Vibrionaceae in shellfish, seawater, and other foods and environmental samples. Assays involve membrane overlays of overnight colonies on nonselective agar plates to detect -glucuronidase and lysyl aminopeptidase activities for E. coli and Vibrionaceae, respectively. Cellulose membranes containing the substrates 4-methylumbeferyl--D-glucuronide (MUG produced a bright blue fluorescence when overlaid onto E. coli, while L-lysyl-7-amino-4-trifluoromethylcoumarin produced green fluorescent foci when overlaid onto Vibrionaceae family members. A multiplex assay was also developed for simultaneously enumerating total E. coli and total Vibrionaceae in oysters and seawater. Overall, 65% of overlaid E. coli (non-O157:H7 were MUG-positive, compared with 62% as determined by the most-probable-number-MUG assay. The overlays are rapid, simple, and cost effective for quantification purposes. This research provides practical alternatives for monitoring bacterial indicators and potential pathogens in complex samples, including molluscan shellfish.

  13. Measurements of Microbial Community Activities in Individual Soil Macroaggregates

    Energy Technology Data Exchange (ETDEWEB)

    Bailey, Vanessa L.; Bilskis, Christina L.; Fansler, Sarah J.; McCue, Lee Ann; Smith, Jeff L.; Konopka, Allan

    2012-05-01

    The functional potential of single soil aggregates may provide insights into the localized distribution of microbial activities better than traditional assays conducted on bulk quantities of soil. Thus, we scaled down enzyme assays for {beta}-glucosidase, N-acetyl-{beta}-D-glucosaminidase, lipase, and leucine aminopeptidase to measure of the enzyme potential of individual aggregates (250-1000 {mu}m diameter). Across all enzymes, the smallest aggregates had the greatest activity and the range of enzyme activities observed in all aggregates supports the hypothesis that functional potential in soil may be distributed in a patchy fashion. Paired analyses of ATP as a surrogate for active microbial biomass and {beta}-glucosidase on the same aggregates suggest the presence of both extracellular {beta}-glucosidase functioning in aggregates with no detectable ATP and also of relatively active microbial communities (high ATP) that have low {beta}-glucosidase potentials. Studying function at a scale more consistent with microbial habitat presents greater opportunity to link microbial community structure to microbial community function.

  14. ERAP1 variants are associated with ankylosing spondylitis in East Asian population: a new Chinese case-control study and meta-analysis of published series.

    Science.gov (United States)

    Chen, C; Zhang, X

    2015-06-01

    Endoplasmic reticulum aminopeptidase 1 (ERAP1) has been confirmed to be associated with ankylosing spondylitis (AS) in Caucasian. However, whether they are associated with AS in East Asian population remains unidentified. We investigated this relationship by a new Chinese case-control study and a meta-analysis of published series. 368 cases and 460 controls were recruited in the Chinese case-control study. Genotyping was completed using the chip-based matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Allelic associations were analysed using contingency tables. In the meta-analysis, up to 2748 cases and 2774 controls from seven different studies and the new Chinese study were combined using Review Manager software version 5.1.1. Mantel-Haenszel or Inverse Variance test was used to calculate fixed or random-effects pooled ORs. In the new Chinese study, strong association with AS was observed for marker rs10050860, rs27434 and rs1065407 at P value of ERAP1 variants are associated with AS in East Asian population, indicating a common pathogenic mechanism for AS in East Asians and Caucasians. PMID:25817437

  15. ERAP1 structure, function and pathogenetic role in ankylosing spondylitis and other MHC-associated diseases.

    Science.gov (United States)

    Alvarez-Navarro, Carlos; López de Castro, José A

    2014-01-01

    The endoplasmic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in the final processing of Major Histocompatibility Complex class I (MHC-I) ligands and with a significant influence in the stability and immunological properties of MHC-I proteins. ERAP1 polymorphism is associated with ankylosing spondylitis among HLA-B27-positive individuals and the altered enzymatic activity of natural variants has significant effects on the HLA-B27 peptidome, suggesting a critical pathogenetic role of peptides in this disease. Likewise, the association of ERAP1 with other MHC-I associated disorders and its epistasis with their susceptibility MHC alleles point out to a general role of the MHC-I peptidome in these diseases. The functional interaction between ERAP1 and HLA-B27 or other MHC-I molecules may be related to the processing of specific epitopes, or to a more general peptide-dependent influence on other biological features of the MHC-I proteins. In addition, from a consideration of the reported functions of ERAP1, including its involvement in angiogenesis and macrophage activation, a more complex and multi-level influence in the inflammatory and immune pathways operating in these diseases cannot be ruled out. PMID:23916068

  16. ERAP1 in the pathogenesis of ankylosing spondylitis.

    Science.gov (United States)

    Reeves, Emma; Elliott, Tim; James, Edward; Edwards, Christopher J

    2014-12-01

    The endoplasmic reticulum aminopeptidase 1 (ERAP1) performs a major role in antigen processing, trimming N-terminally extended peptides to the final epitope for presentation by major histocompatibility complex class I molecules. Recent genome-wide association studies have identified single nucleotide polymorphisms (SNPs) within ERAP1 as being associated with disease, in particular ankylosing spondylitis (AS). AS is a polygenic chronic inflammatory disease with a strong genetic link to HLA-B27 known for over 40 years. The association of ERAP1 SNPs with AS susceptibility is only observed in HLA-B27-positive individuals, which intersect on the antigen processing pathway. Recent evidence examining the trimming activity of polymorphic ERAP1 highlights its role in generating peptides for loading onto and stabilizing HLA-B27, and the consequent alterations in the interaction of specific NK cell receptors, and the activation of the unfolded protein response as important in the mechanism of disease pathogenesis. Here, we discuss the recent genetic association findings linking ERAP1 SNPs with AS disease susceptibility and the effect of these variants on ERAP1 function, highlighting mechanisms by which AS may arise. The identification of these functional variants of ERAP1 may lead to better stratification of AS patients by providing a diagnostic tool and a potential therapeutic target. PMID:25434650

  17. ERAP1 regulates natural killer cell function by controlling the engagement of inhibitory receptors.

    Science.gov (United States)

    Cifaldi, Loredana; Romania, Paolo; Falco, Michela; Lorenzi, Silvia; Meazza, Raffaella; Petrini, Stefania; Andreani, Marco; Pende, Daniela; Locatelli, Franco; Fruci, Doriana

    2015-03-01

    The endoplasmic reticulum aminopeptidase ERAP1 regulates innate and adaptive immune responses by trimming peptides for presentation by MHC class I (MHC-I) molecules. Herein, we demonstrate that genetic or pharmacological inhibition of ERAP1 on human tumor cell lines perturbs their ability to engage several classes of inhibitory receptors by their specific ligands, including killer cell Ig-like receptors (KIR) by classical MHC-I-peptide (pMHC-I) complexes and the lectin-like receptor CD94-NKG2A by nonclassical pMHC-I complexes, in each case leading to natural killer (NK) cell killing. The protective effect of pMHC-I complexes could be restored in ERAP1-deficient settings by the addition of known high-affinity peptides, suggesting that ERAP1 was needed to positively modify the affinity of natural ligands. Notably, ERAP1 inhibition enhanced the ability of NK cells to kill freshly established human lymphoblastoid cell lines from autologous or allogeneic sources, thereby promoting NK cytotoxic activity against target cells that would not be expected because of KIR-KIR ligand matching. Overall, our results identify ERAP1 as a modifier to leverage immune functions that may improve the efficacy of NK cell-based approaches for cancer immunotherapy. PMID:25592150

  18. ERp44 Exerts Redox-Dependent Control of Blood Pressure at the ER.

    Science.gov (United States)

    Hisatsune, Chihiro; Ebisui, Etsuko; Usui, Masaya; Ogawa, Naoko; Suzuki, Akio; Mataga, Nobuko; Takahashi-Iwanaga, Hiromi; Mikoshiba, Katsuhiko

    2015-06-18

    Blood pressure maintenance is vital for systemic homeostasis, and angiotensin II is a critical regulator. The upstream mechanisms that regulate angiotensin II are not completely understood. Here, we show that angiotensin II is regulated by ERp44, a factor involved in disulfide bond formation in the ER. In mice, genetic loss of ERp44 destabilizes angiotensin II and causes hypotension. We show that ERp44 forms a mixed disulfide bond with ERAP1, an aminopeptidase that cleaves angiotensin II. ERp44 controls the release of ERAP1 in a redox-dependent manner to control blood pressure. Additionally, we found that systemic inflammation triggers ERAP1 retention in the ER to inhibit hypotension. These findings suggest that the ER redox state calibrates serum angiotensin II levels via regulation of the ERp44-ERAP1 complex. Our results reveal a link between ER function and normotension and implicate the ER redox state as a potential risk factor in the development of cardiovascular disease. PMID:25959394

  19. Immune surveillance for ERAAP dysfunction.

    Science.gov (United States)

    Nagarajan, Niranjana A; Shastri, Nilabh

    2013-09-01

    The ER aminopeptidase associated with antigen processing, ERAAP (or ERAP1), is essential for trimming peptides that are presented by MHC class I molecules. ERAP1 is inhibited by human cytomegalovirus, and ERAP1 polymorphisms are associated with autoimmune diseases. How the immune system detects ERAAP dysfunction, however, is unknown. We have shown previously that ERAAP-deficient cells present an immunogenic pMHC I repertoire, that elicits CD8+ T cell response in WT mice. Additionally, we discovered that the WT CD8+ T cells recognized novel peptides presented by non-classical, or MHC class Ib, molecules on ERAAP-deficient cells. The MHC Ib restricted WT CD8 T cells eliminated ERAAP-deficient cells in vitro and in vivo. We identified the FL9 peptide, presented by Qa-1(b), a MHC class Ib molecule exclusively on ERAAP-deficient cells. Remarkably, T cells specific for the FL9-Qa-1(b) complex were frequent in naïve WT mice, and had an antigen-experienced phenotype. Thus, novel non-classical pQa-1(b) complexes direct cytotoxic T cells to target cells with defective peptide processing in the endoplasmic reticulum. Here, we discuss the implications of our findings, and the possible roles of pMHC Ib-specific T cells in immune surveillance for ERAAP dysfunction. PMID:23433779

  20. Pathogenesis of Behçet's disease: autoinflammatory features and beyond.

    Science.gov (United States)

    Gül, Ahmet

    2015-07-01

    Behçet's disease (BD) is an inflammatory disorder of unknown aetiology characterised by recurrent attacks affecting the mucocutaneous tissues, eyes, joints, blood vessels, brain and gastrointestinal tract. It is a multifactorial disease classified as a variable vessel vasculitis, and several environmental triggers may induce inflammatory episodes in genetically susceptible individuals. BD has several autoinflammatory features including recurrent self-limited clinical manifestations overlapping with monogenic autoinflammatory disorders, significant host predisposition and abnormally increased inflammatory response, with a robust innate component. Human leukocyte antigen (HLA)-B*51 is the strongest susceptibility factor described so far affecting the disease risk and typical phenotype. Non-HLA genetic associations such as endoplasmic reticulum aminopeptidase 1 (ERAP1), interleukin 23 receptor (IL23R) and IL10 variations suggest that BD shares susceptibility genes and inflammatory pathways with spondyloarthritis. Although genomewide association studies revealed an increased risk associated with recessively inherited ERAP1 variations in HLA-B*51 positive patients, it is not clear yet whether certain peptide-HLA allele combinations result in an adaptive response by a self-antigen-directed cytotoxic response or an innate response by modulating an NK cell activity or causing an unfolded protein response. Understanding of major histocompatibility complex (MHC) Class I-driven inflammatory response is expected to provide insights for the development of better treatment and remission-induction options in BD as well as in ankylosing spondylitis (AS) and psoriasis. PMID:26068404

  1. ERAP1 functions override the intrinsic selection of specific antigens as immunodominant peptides, thereby altering the potency of antigen-specific cytolytic and effector memory T-cell responses.

    Science.gov (United States)

    Rastall, David P W; Aldhamen, Yasser A; Seregin, Sergey S; Godbehere, Sarah; Amalfitano, Andrea

    2014-12-01

    Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a critical component of the adaptive immune system that has been shown to increase or decrease the presentation of specific peptides on MHC class I molecules. Here, we have demonstrated that ERAP1 functions are not only important during the presentation of antigen-derived peptides, but these functions can also completely change which antigen-derived peptides ultimately become selected as immunodominant T-cell epitopes. Our results suggest that ERAP1 may do this by destroying epitopes that would otherwise become immunodominant in the absence of adequate ERAP1 functionality. We further establish that ERAP1-mediated influences on T-cell functions are both qualitative and quantitative, by demonstrating that loss of ERAP1 function redirects CTL killing toward a different set of antigen-derived epitopes and increases the percent of antigen-specific memory T cells elicited by antigen exposure. As a result, our studies suggest that normal ERAP1 activity can act to suppress the numbers of T effector memory cells that respond to a given antigen. This unique finding may shed light on why certain ERAP1 single nucleotide polymorphisms are associated with several autoimmune diseases, for example, by significantly altering the robustness and quality of CD8+ T-cell memory responses to antigen-derived peptides. PMID:25087231

  2. Naturally occurring ERAP1 haplotypes encode functionally distinct alleles with fine substrate specificity.

    Science.gov (United States)

    Reeves, Emma; Edwards, Christopher J; Elliott, Tim; James, Edward

    2013-07-01

    Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims peptides for MHC class I presentation, influencing the degree and specificity of CD8(+) T cell responses. Single-nucleotide polymorphisms within the exons encoding ERAP1 are associated with autoimmune diseases and cervical carcinoma, but it is not known whether they act independently or as disease-associated haplotypes. We sequenced ERAP1 from 20 individuals and show that single-nucleotide polymorphisms occur as distinct haplotypes in the human population and that these haplotypes encode functionally distinct ERAP1 alleles. Using a wide range of substrates, we are able to demonstrate that for any given substrate distinct ERAP1 alleles can be "normal," "hypofunctional," or "hyperfunctional" and that each allele has a trend bias toward one of these three activities. Thus, the repertoire of peptides presented at the cell surface for recognition by CTL is likely to depend on the precise combination of both MHC class I and ERAP1 alleles expressed within an individual, and has important implications for predisposition to disease. PMID:23733883

  3. Revisiting MHC genes in spondyloarthritis.

    Science.gov (United States)

    Breban, Maxime; Costantino, Félicie; André, Claudine; Chiocchia, Gilles; Garchon, Henri-Jean

    2015-06-01

    Spondyloarthritis (SpA) refers to a variety of inflammatory rheumatic disorders with strong heritability. Shared genetic predisposition, as shown by familial aggregation, is largely attributable to the major histocompatibility complex (MHC) locus, which was estimated to account for approximately half of the whole disease heritability. The first predisposing allele identified more than 40 years ago is HLA-B27, which is a major gene predisposing to all forms of SpA. However, despite intensive research, its pathogenesis remains uncertain. Other MHC alleles belonging to the class I and class II regions have been identified to exert additional effect. Candidate-gene approaches and genome-wide studies have recently allowed identification of several new loci residing outside of the MHC region that are involved in the predisposition to SpA. Interestingly, some of those new genes, such as ERAP1, ERAP2, and NPEPPS, code for aminopeptidases that are involved in MHC class I presentation and were shown to interact with HLA-B27. PMID:25903667

  4. A functional variant in ERAP1 predisposes to multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Franca Rosa Guerini

    Full Text Available The ERAP1 gene encodes an aminopeptidase involved in antigen processing. A functional polymorphism in the gene (rs30187, Arg528Lys associates with susceptibility to ankylosying spondylitis (AS, whereas a SNP in the interacting ERAP2 gene increases susceptibility to another inflammatory autoimmune disorder, Crohn's disease (CD. We analysed rs30187 in 572 Italian patients with CD and in 517 subjects suffering from multiple sclerosis (MS; for each cohort, an independent sex- and age-matched control group was genotyped. The frequency of the 528Arg allele was significantly higher in both disease cohorts compared to the respective control population (for CD, OR = 1.20 95%CI: 1.01-1.43, p = 0.036; for RRMS, OR = 1.26; 95%CI: 1.04-1.51, p = 0.01. Meta-analysis with the Wellcome Trust Cases Control Consortium GWAS data confirmed the association with MS (p(meta = 0.005, but not with CD. In AS, the rs30187 variant has a predisposing effect only in an HLA-B27 allelic background. It remains to be evaluated whether interaction between ERAP1 and distinct HLA class I alleles also affects the predisposition to MS, and explains the failure to provide definitive evidence for a role of rs30187 in CD. Results herein support the emerging concept that a subset of master-regulatory genes underlay the pathogenesis of autoimmunity.

  5. Genotypic and technological characterization of Leuconostoc isolates to be used as adjunct starters in Manchego cheese manufacture.

    Science.gov (United States)

    Nieto-Arribas, Pedro; Seseña, Susana; Poveda, Justa M; Palop, Llanos; Cabezas, Lourdes

    2010-02-01

    Twenty-seven Leuconostoc (Ln.) isolates from Manchego cheese were characterized by phenotypic and genotypic methods, and their technological abilities studied in order to test their potential use as dairy starter components. While phenotypic diversity was evaluated by studying the biochemical characteristics of technological interest (i.e. acidifying and aminopeptidase activities), genotypic diversity was evidenced by using Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR). Additional technological abilities such as lipolytic, proteolytic and autolytic activities, salt and pH tolerance and production of dextran, flavour compounds and biogenic amines, were investigated. The marked differences among strains reflected the existing biodiversity in naturally fermented products. After statistically evaluating their performance, strains C0W2, belonging to Ln. lactis, and C16W5 and N2W5, belonging to Ln. mesenteroides subsp. dextranicum, revealed the best properties to be used in mixed dairy starter cultures. This study evidences the fact that natural environments can be considered as a proper source of useful strains, for the dairy industry. PMID:19913697

  6. Antibiotic susceptibility and heterogeneity in technological traits of lactobacilli isolated from Algerian goat's milk.

    Science.gov (United States)

    Bousmaha-Marroki, Leila; Marroki, Ahmed

    2015-08-01

    The objective of this study was to identify and study the heterogeneity of technological traits of lactobacilli from goat's milk of Algeria and to evaluate in vitro their safety aspect. Using API50 CHL system and 16S rDNA sequencing, 51 % of strains were assigned as Lactobacillus plantarum, 34 % as L. pentosus, 7 % as L. rhamnosus and 8 % as L. fermentum. A large variability was noted for the acidifying capacity in skim milk after 6, 12 and 24 h of incubation. All strains expressed aminopeptidase activity against alanine-ρ-NA and leucine-ρ-NA at different levels. All strains were resistant to vancomycin and most of strains showed more susceptibility to β-lactam antibiotic. High susceptibility toward the inhibitors of protein synthesis was also observed. Minimum inhibitory concentrations data obtained revealed that isolates were susceptible to penicillin and chloramphenicol, and resistant to gentamicin and vancomycin. Minimum inhibitory concentrations distribution of other antibiotics showed variability. The analysis of graphical representation of principal component analysis of technological properties of L. plantarum and L. pentosus strains showed diversity among the isolates. Finally, eight L. plantarum (LAM1, LAM3, LAM21, LAM25, LAM35, LF15, LAM34, and LAM35), four L. pentosus (LAM38, LAM39, LF9 and LF16) and two L. rhamnosus (LF3 and LF10) strains, could be good candidates as adjunct culture in dairy product in Algeria.

  7. Adaptive regulation of digestive performance in the genus Python.

    Science.gov (United States)

    Ott, Brian D; Secor, Stephen M

    2007-01-01

    The adaptive interplay between feeding habits and digestive physiology is demonstrated by the Burmese python, which in response to feeding infrequently has evolved the capacity to widely regulate gastrointestinal performance with feeding and fasting. To explore the generality of this physiological trait among pythons, we compared the postprandial responses of metabolism and both intestinal morphology and function among five members of the genus Python: P. brongersmai, P. molurus, P. regius, P. reticulatus and P. sebae. These infrequently feeding pythons inhabit Africa, southeast Asia and Indonesia and vary in body shape from short and stout (P. brongersmai) to long and slender (P. reticulatus). Following the consumption of rodent meals equaling 25% of snake body mass, metabolic rates of pythons peaked at 1.5 days at levels 9.9- to 14.5-fold of standard metabolic rates before returning to prefeeding rates by day 6-8. Specific dynamic action of these meals (317-347 kJ) did not differ among species and equaled 23-27% of the ingested energy. For each species, feeding triggered significant upregulation of intestinal nutrient transport and aminopeptidase-N activity. Concurrently, intestinal mass doubled on average for the five species, in part due to an 85% increase in mucosal thickness, itself a product of 27-59% increases in enterocyte volume. The integrative response of intestinal functional upregulation and tissue hypertrophy enables each of these five python species, regardless of body shape, to modulate intestinal performance to meet the demands of their large infrequent meals.

  8. Cellular FRET-Biosensors to Detect Membrane Targeting Inhibitors of N-Myristoylated Proteins.

    Directory of Open Access Journals (Sweden)

    Arafath Kaja Najumudeen

    Full Text Available Hundreds of eukaryotic signaling proteins require myristoylation to functionally associate with intracellular membranes. N-myristoyl transferases (NMT responsible for this modification are established drug targets in cancer and infectious diseases. Here we describe NANOMS (NANOclustering and Myristoylation Sensors, biosensors that exploit the FRET resulting from plasma membrane nanoclustering of myristoylated membrane targeting sequences of Gαi2, Yes- or Src-kinases fused to fluorescent proteins. When expressed in mammalian cells, NANOMS report on loss of membrane anchorage due to chemical or genetic inhibition of myristoylation e.g. by blocking NMT and methionine-aminopeptidase (Met-AP. We used Yes-NANOMS to assess inhibitors of NMT and a cherry-picked compound library of putative Met-AP inhibitors. Thus we successfully confirmed the activity of DDD85646 and fumagillin in our cellular assay. The developed assay is unique in its ability to identify modulators of signaling protein nanoclustering, and is amenable to high throughput screening for chemical or genetic inhibitors of functional membrane anchorage of myristoylated proteins in mammalian cells.

  9. Automated Solution-Phase Synthesis of Insect Glycans to Probe the Binding Affinity of Pea Enation Mosaic Virus.

    Science.gov (United States)

    Tang, Shu-Lun; Linz, Lucas B; Bonning, Bryony C; Pohl, Nicola L B

    2015-11-01

    Pea enation mosaic virus (PEMV)--a plant RNA virus transmitted exclusively by aphids--causes disease in multiple food crops. However, the aphid-virus interactions required for disease transmission are poorly understood. For virus transmission, PEMV binds to a heavily glycosylated receptor aminopeptidase N in the pea aphid gut and is transcytosed across the gut epithelium into the aphid body cavity prior to release in saliva as the aphid feeds. To investigate the role of glycans in PEMV-aphid interactions and explore the possibility of viral control through blocking a glycan interaction, we synthesized insect N-glycan terminal trimannosides by automated solution-phase synthesis. The route features a mannose building block with C-5 ester enforcing a β-linkage, which also provides a site for subsequent chain extension. The resulting insect N-glycan terminal trimannosides with fluorous tags were used in a fluorous microarray to analyze binding with fluorescein isothiocyanate-labeled PEMV; however, no specific binding between the insect glycan and PEMV was detected. To confirm these microarray results, we removed the fluorous tag from the trimannosides for isothermal titration calorimetry studies with unlabeled PEMV. The ITC studies confirmed the microarray results and suggested that this particular glycan-PEMV interaction is not involved in virus uptake and transport through the aphid. PMID:26457763

  10. Characterization of Lactobacillus from Algerian goat's milk based on phenotypic, 16S rDNA sequencing and their technological properties

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    Ahmed Marroki

    2011-03-01

    Full Text Available Nineteen strains of Lactobacillus isolated from goat's milk from farms in north-west of Algeria were characterized. Isolates were identified by phenotypic, physiological and genotypic methods and some of their important technological properties were studied. Phenotypic characterization was carried out by studying physiological, morphological characteristics and carbohydrate fermentation patterns using API 50 CHL system. Isolates were also characterized by partial 16S rDNA sequencing. Results obtained with phenotypic methods were correlated with the genotypic characterization and 13 isolates were identified as L. plantarum, two isolates as L. rhamnosus and one isolate as L. fermentum. Three isolates identified as L. plantarum by phenotypic characterization were found to be L. pentosus by the genotypic method. A large diversity in technological properties (acid production in skim milk, exopolysaccharide production, aminopeptidase activity, antibacterial activity and antibiotic susceptibility was observed. Based on these results, two strains of L. plantarum (LbMS16 and LbMS21 and one strain of L. rhamnosus (LbMF25 have been tentatively selected for use as starter cultures in the manufacture of artisanal fermented dairy products in Algeria.

  11. Brush border membrane vesicles from dipteran midgut: a tool for studies on nutrient absorption

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    MG Leonardi

    2006-12-01

    Full Text Available Brush border membrane vesicles (BBMV from insects midgut can be successfully used to study several membrane phenomena, including nutrient absorption, ions permeability and insecticides mode of action. Midgut BBMV, purified from Musca domestica whole larvae, were used for the functional characterization of leucine transport. The amino acid uptake was accelerated in the presence of sodium or potassium and increased significantly when the extravesicular pH was 5.0, in agreement with the luminal pH in vivo. Radiolabelled leucine uptake was significantly reduced by an excess of cold leucine, histidine, serine and glycine, suggesting that the amino acid transporter is a broad scope carrier that does not recognize proline, glutamine and the dibasic amino acids lysine and arginine.Midgut BBMV were also obtained from homogenization of M. domestica and Bactrocera oleae adults. The final preparations showed a high enrichment in the specific activity of the BBM marker enzymes aminopeptidase N and γ-glutamyl transpeptidase, and were poorly contaminated by basolateral membranes, as indicated by the low specific activities of their marker enzyme Na+/K+ ATPase. Electron microscopy of B. oleae BBM fraction showed the presence of closed vesicles. Similar SDS-PAGE patterns, with numerous distinct bands, were detected for both B. oleae and M. domestica BBMV.

  12. Role of receptors in Bacillus thuringiensis crystal toxin activity.

    Science.gov (United States)

    Pigott, Craig R; Ellar, David J

    2007-06-01

    Bacillus thuringiensis produces crystalline protein inclusions with insecticidal or nematocidal properties. These crystal (Cry) proteins determine a particular strain's toxicity profile. Transgenic crops expressing one or more recombinant Cry toxins have become agriculturally important. Individual Cry toxins are usually toxic to only a few species within an order, and receptors on midgut epithelial cells have been shown to be critical determinants of Cry specificity. The best characterized of these receptors have been identified for lepidopterans, and two major receptor classes have emerged: the aminopeptidase N (APN) receptors and the cadherin-like receptors. Currently, 38 different APNs have been reported for 12 different lepidopterans. Each APN belongs to one of five groups that have unique structural features and Cry-binding properties. While 17 different APNs have been reported to bind to Cry toxins, only 2 have been shown to mediate toxin susceptibly in vivo. In contrast, several cadherin-like proteins bind to Cry toxins and confer toxin susceptibility in vitro, and disruption of the cadherin gene has been associated with toxin resistance. Nonetheless, only a small subset of the lepidopteran-specific Cry toxins has been shown to interact with cadherin-like proteins. This review analyzes the interactions between Cry toxins and their receptors, focusing on the identification and validation of receptors, the molecular basis for receptor recognition, the role of the receptor in resistant insects, and proposed models to explain the sequence of events at the cell surface by which receptor binding leads to cell death. PMID:17554045

  13. Cadherin is involved in the action of Bacillus thuringiensis toxins Cry1Ac and Cry2Aa in the beet armyworm, Spodoptera exigua.

    Science.gov (United States)

    Qiu, Lin; Hou, Leilei; Zhang, Boyao; Liu, Lang; Li, Bo; Deng, Pan; Ma, Weihua; Wang, Xiaoping; Fabrick, Jeffrey A; Chen, Lizhen; Lei, Chaoliang

    2015-05-01

    Bacillus thuringiensis (Bt) insecticidal crystal (Cry) proteins are effective against some insect pests in sprays and transgenic crops, although the evolution of resistance could threaten the long-term efficacy of such Bt use. One strategy to delay resistance to Bt crops is to "pyramid" two or more Bt proteins that bind to distinct receptor proteins within the insect midgut. The most common Bt pyramid in cotton (Gossypium hirsutum L.) employs Cry1Ac with Cry2Ab to target several key lepidopteran pests, including the beet armyworm, Spodoptera exigua (Hübner), which is a serious migratory pest of many vegetable crops and is increasingly important in cotton in China. While cadherin and aminopeptidase-N are key receptors of Cry1 toxins in many lepidopterans including S. exigua, the receptor for Cry2A toxins remains poorly characterized. Here, we show that a heterologous expressed peptide corresponding to cadherin repeat 7 to the membrane proximal extracellular domain (CR7-MPED) in the S. exigua cadherin 1b (SeCad1b) binds Cry1Ac and Cry2Aa. Moreover, SeCad1b transcription was suppressed in S. exigua larvae by oral RNA interference and susceptibility to Cry1Ac and Cry2Aa was significantly reduced. These results indicate that SeCad1b plays important functional roles of both Cry1Ac and Cry2Aa, having major implications for resistance management for S. exigua in Bt crops. PMID:25754522

  14. Genetic mapping of Bt-toxin binding proteins in a Cry1A-toxin resistant strain of diamondback moth Plutella xylostella.

    Science.gov (United States)

    Baxter, Simon W; Zhao, Jian-Zhou; Shelton, Anthony M; Vogel, Heiko; Heckel, David G

    2008-02-01

    A major mechanism of resistance to Bacillus thuringiensis (Bt) toxins in Lepidoptera is a reduction of toxin binding to sites in the midgut membrane. Genetic studies of three different species have shown that mutations in a candidate Bt receptor, a 12-cadherin-domain protein, confer Cry1A toxin resistance. Despite a similar resistance profile in a fourth lepidopteran species, Plutella xylostella, we have previously shown that the cadherin orthologue maps to a different linkage group (LG8) than Cry1Ac resistance (LG22). Here we tested the hypothesis that mutations in other genes encoding candidate Bt-binding targets could be responsible for Bt resistance, by mapping eight aminopeptidases, an alkaline phosphatase (ALP), an intestinal mucin, and a P252 glycoprotein with respect to the 29 AFLP marked linkage groups in a P. xylostella cross segregating for Cry1Ac resistance. A homologue of the Caenorhabditis elegans Bt resistance gene bre-2 was also mapped. None of the genes analysed were on the same chromosome containing the Cry1Ac resistance locus, eliminating them as candidate resistance genes in the parental resistant strain SC1. Although this finding excludes cis-acting mutations in these genes as causing resistance in this strain, one or more of the expressed proteins may still bind Cry1Ac toxin, and post-translational modifications could affect this binding and thereby exert a trans-acting effect on resistance. PMID:18207074

  15. Insulin and glucose play a role in foam cell formation and function

    Directory of Open Access Journals (Sweden)

    Keller Susanna R

    2006-06-01

    Full Text Available Abstract Background Foam cell formation in diabetic patients often occurs in the presence of high insulin and glucose levels. To test whether hyperinsulinemic hyperglycemic conditions affect foam cell differentiation, we examined gene expression, cytokine production, and Akt phosphorylation in human monocyte-derived macrophages incubated with two types of oxidized low density lipoprotein (LDL, minimally modified LDL (mmLDL and extensively oxidized LDL (OxLDL. Methods and results Using Affymetrix GeneChip® arrays, we found that several genes directly related to insulin signaling were changed. The insulin receptor and glucose-6-phosphate dehydrogenase were upregulated by mmLDL and OxLDL, whereas insulin-induced gene 1 was significantly down-regulated. In hyperinsulinemic hyperglycemic conditions, modified LDL upregulated Akt phosphorylation and expression of the insulin-regulated aminopeptidase. The level of proinflammatory cytokines, IL-lβ, IL-12, and IL-6, and of a 5-lipoxygenase eicosanoid, 5-hydroxyeicosatetraenoic acid (5-HETE, was also increased. Conclusion These results suggest that the exposure of macrophages to modified low density lipoproteins in hyperglycemic hyperinsulinemic conditions affects insulin signaling and promotes the release of proinflammatory stimuli, such as cytokines and eicosanoids. These in turn may contribute to the development of insulin resistance.

  16. Three toxins, two receptors, one mechanism: Mode of action of Cry1A toxins from Bacillus thuringiensis in Heliothis virescens.

    Science.gov (United States)

    Bretschneider, Anne; Heckel, David G; Pauchet, Yannick

    2016-09-01

    Insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) are highly active against Lepidoptera. However, field-evolved resistance to Bt toxins is on the rise. The 12-cadherin domain protein HevCaLP and the ABC transporter HevABCC2 are both genetically linked to Cry toxin resistance in Heliothis virescens. We investigated their interaction using stably expressing non-lytic clonal Sf9 cell lines expressing either protein or both together. Untransfected Sf9 cells are innately sensitive to Cry1Ca toxin, but not to Cry1A toxins; and quantitative PCR revealed negligible expression of genes involved in Cry1A toxicity such as cadherin, ABCC2, alkaline phosphatase (ALP) and aminopeptidase N (APN). Cry1Aa, Cry1Ab or Cry1Ac caused swelling of Sf9 cells expressing HevABCC2, and caused faster swelling, lysis and up to 86% mortality in cells expressing both proteins. No such effect was observed in control Sf9 cells or in cells expressing only HevCaLP. The results of a mixing experiment demonstrated that both proteins need to be expressed within the same cell for high cytotoxicity, and suggest a novel role for HevCaLP. Binding assays showed that the toxin-receptor interaction is specific. Our findings confirm that HevABCC2 is the central target in Cry1A toxin mode of action, and that HevCaLP plays a supporting role in increasing Cry1A toxicity.

  17. 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

  18. Biochemical Characterization of Hypothetical Proteins from Helicobacter pylori.

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    Han-Pil Choi

    Full Text Available The functional characterization of Open Reading Frames (ORFs from sequenced genomes remains a bottleneck in our effort to understand microbial biology. In particular, the functional characterization of proteins with only remote sequence homology to known proteins can be challenging, as there may be few clues to guide initial experiments. Affinity enrichment of proteins from cell lysates, and a global perspective of protein function as provided by COMBREX, affords an approach to this problem. We present here the biochemical analysis of six proteins from Helicobacter pylori ATCC 26695, a focus organism in COMBREX. Initial hypotheses were based upon affinity capture of proteins from total cellular lysate using derivatized nano-particles, and subsequent identification by mass spectrometry. Candidate genes encoding these proteins were cloned and expressed in Escherichia coli, and the recombinant proteins were purified and characterized biochemically and their biochemical parameters compared with the native ones. These proteins include a guanosine triphosphate (GTP cyclohydrolase (HP0959, an ATPase (HP1079, an adenosine deaminase (HP0267, a phosphodiesterase (HP1042, an aminopeptidase (HP1037, and new substrates were characterized for a peptidoglycan deacetylase (HP0310. Generally, characterized enzymes were active at acidic to neutral pH (4.0-7.5 with temperature optima ranging from 35 to 55°C, although some exhibited outstanding characteristics.

  19. Effect of adrenalectomy and hydrocortisone on ventral prostate of rats

    Institute of Scientific and Technical Information of China (English)

    Neena Nair; R.S. Bedwal; R.S. Mathur

    2001-01-01

    Aim: To study the effects of adrenalectomy and hydrocortisone on the ventral prostate of SD rats. Methods: In adrenalectomised (ADX) and ADX + hydrocortisone (1, 2, or 4 mg) treated rats, the prostatic histology and the cholesterol, protein, zinc, and copper levels and the enzymic profile (acid phosphatase, alkaline phosphatase, aryl sulphatase, lactic dehydrogenase, and leucine aminopeptidase) in the prostatic tissue were determined; the serum hormonal profile (testosterone, FSH and LH) was also assayed. Results: Adrenalectomy caused a progressive degeneration in prostatic structure that was not reversed by hydrocortisone treatment. The serum testosterone were significantly lower in ADX than in sham operated rats and lower in ADX + hydrocortisone than in ADX-C rats ( P < 0.01) The serum FSH and LH were below the detection limit of 1 mIU/mL. The enzymatic activity was higher in ADX than in sham operated rats and higher in ADX + hydrocortisone than in ADX-C rats ( P <0.05-0.01). The prostatic zinc levels were significantly higher in sham operated than in ADX, and higher in ADX-C than in ADX + hydrocortisone rats ( P < 0.05 -0.01). The prostatic copper level was significantly lower in sham operated than in ADX, and lower in ADX-C than in the ADX + hydrocortisone rats ( P < 0.01). Conclusion: In rats, adrenalectomy leads to pathological and functional changes of the prostate. Hydrocortisone treatment at the doses employed did not reverse these changes.

  20. Prolonged treatment with ursodeoxycholic acid for primary biliary cirrhosis.

    Science.gov (United States)

    Crippa, G; Cagnoni, C; Castelli, A; Concesi, C; Girometta, S; Pancotti, D; Sverzellati, E; Tacchini, G; Pierfranceschi, M G; Carrara, G C

    1995-05-01

    Eighteen patients affected with biopsy-proved primary biliary cirrhosis (PBC) (histological stage III and IV) received ursodeoxicholic acid (UDCA) 600 mg for 1 year. Signs and symptoms and biochemical tests (glutamic and oxalcetic transaminase, glutamic and pyruvic transaminase, bilirubine, gamma-glutamyl transpeptidase, alkaline phosphatase, leucine aminopeptidase, bile acids, plasma proteins electrophoresis, immunoglubulins A, G and M) and antimitochondrial antibodies were evaluated before the treatment and every four months during the treatment. The results were compared with those obtained in 8 untreated patients affected PBC. The control group of patients were comparable (as far as age, histological stage, biochemical tests are concerned) to the group who received UDCA. Bilirubine, ALP, gamma-GT and LAP decreased during the treatment with UDCA and remained lower than baseline values until the end of the observation (12 months), while no changes occurred in the untreated patients. Both in the treated and untreated group plasma protein electrophoresis, serum immunoglubulins A, G and M remained unchanged, as well as anti-mitochondrial antibody. A moderate reduction of transaminases and bile acids was observed in the group of patients receiving UDCA but it did not reach statistical significance. In 16 out of the 18 treated patients pruritus disappeared and resulted diminished in the remaining 2 patients. No significant amelioration of pruritus was observed in the patients who did not receive UDCA. In conclusion, our data show that prolonged treatment with UDCA drastically reduces pruritus and improves cholestasis biochemical tests in patients affected with symptomatic PBC.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Multiple hydrolases in bean leaves (Phaseolus vulgaris L.) and the effect of the halo blight disease caused by Pseudomonas phaseolicola (Burkh.) Dowson.

    Science.gov (United States)

    Rudolph, K; Stahmann, M A

    1966-03-01

    Multiple forms of hydrolytic enzymes were demonstrated in extracts of healthy bean leaves (Phascolus vulgaris L.) and bean leaves infected with the halo blight organism [Pseudomonas phaseolicola (Burkh.) Dowson] by polyacrylamide disc electrophoresis. Bean leaves contained up to 4 acid phosphatase bands, 9 esterase bands active towards alpha-naphthyl acetate, and 7 esterase bands towards alpha-naphthyl butyrate. Only low or no activity was found for alkaline phosphatase, lipase, and aminopeptidase. Two artifacts are described which were observed with the lead phosphate method for acid phosphatase and the Tween method for demonstration of lipase. After infection with the halo blight organism the major acid phosphatase of the host increased during early and decreased at later infection stages. An acid phosphatase of bacterial origin with a more neutral pH optimum could be demonstrated in infected leaves. It is suggested that the bacterial acid phosphatase plays a role in uptake of metabolites by the pathogen. Several esterase bands decreased after infection. One host band with activity towards alpha-naphthyl butyrate increased. Also the pathogen showed an esterase band with high activity towards alpha-naphthyl butyrate. PMID:5906374

  2. Opiorphin causes a panicolytic-like effect in rat panic models mediated by μ-opioid receptors in the dorsal periaqueductal gray.

    Science.gov (United States)

    Maraschin, Jhonatan Christian; Rangel, Marcel Pereira; Bonfim, Antonio Joaquim; Kitayama, Mariana; Graeff, Frederico Guilherme; Zangrossi, Hélio; Audi, Elisabeth Aparecida

    2016-02-01

    Reported evidence indicates that endogenous opioid peptides regulate the expression of escape behavior in rats, a panic-related defensive response, through μ-opioid receptors (MORs) in the dorsal periaqueductal gray (dPAG). These peptides are rapidly catabolized by degrading enzymes, including neutral endopeptidase (NEP) and aminopeptidase N (APN). Opiorphin is a peptide inhibitor of both NEP and APN and potentiates the action of endogenous enkephalins. This study evaluated the effects of intravenous and intra-dPAG administration of opiorphin on escape responses in the elevated T-maze and in a dPAG electrical stimulation test in rats. We also evaluated the involvement of MORs in the effects of opiorphin using the selective MOR antagonist CTOP. A dose of 2.0 mg/kg, i.v., of opiorphin impaired escape performance in both tests. Similar effects were observed with intra-dPAG administration of 5.0 nmol of opiorphin. Local pretreatment with 1.0 nmol CTOP antagonized the anti-escape effects of intra-dPAG opiorphin in both tests, as well as the effect of systemically administered opiorphin (2.0 mg/kg, i.v.) in the electrical stimulation test. These results indicate that opiorphin has an antipanic-like effect that is mediated by MORs in the dPAG. They may open new perspectives for the development of opiorphin analogues with greater bioavailability and physicochemical characteristics in the pursuit of new medications for the treatment of panic disorder.

  3. A toxin-binding alkaline phosphatase fragment synergizes Bt toxin Cry1Ac against susceptible and resistant Helicoverpa armigera.

    Directory of Open Access Journals (Sweden)

    Wenbo Chen

    Full Text Available Evolution of resistance by insects threatens the continued success of pest control using insecticidal crystal (Cry proteins from the bacterium Bacillus thuringiensis (Bt in sprays and transgenic plants. In this study, laboratory selection with Cry1Ac yielded five strains of cotton bollworm, Helicoverpa armigera, with resistance ratios at the median lethal concentration (LC50 of activated Cry1Ac ranging from 22 to 1700. Reduced activity and reduced transcription of an alkaline phosphatase protein that binds Cry1Ac was associated with resistance to Cry1Ac in the four most resistant strains. A Cry1Ac-binding fragment of alkaline phosphatase from H. armigera (HaALP1f was not toxic by itself, but it increased mortality caused by Cry1Ac in a susceptible strain and in all five resistant strains. Although synergism of Bt toxins against susceptible insects by toxin-binding fragments of cadherin and aminopeptidase N has been reported previously, the results here provide the first evidence of synergism of a Bt toxin by a toxin-binding fragment of alkaline phosphatase. The results here also provide the first evidence of synergism of a Bt toxin by any toxin-binding peptide against resistant insects.

  4. Identification of differentially expressed microRNAs between Bacillus thuringiensis Cry1Ab-resistant and -susceptible strains of Ostrinia furnacalis.

    Science.gov (United States)

    Xu, Li-Na; Ling, Ying-Hui; Wang, Yue-Qin; Wang, Zhen-Ying; Hu, Ben-Jin; Zhou, Zi-Yan; Hu, Fei; He, Kang-Lai

    2015-01-01

    The Asian corn borer (ACB), Ostrinia furnacalis (Guenée), can develop strong resistance to Cry1Ab, the most widely commercialized Cry toxin for Bt maize worldwide. It is essential to understand the mechanism of resistance for management of this species, but information on the post-transcriptional regulation of Bt resistance in this target insect is limited. In the present study, RNA was extracted from the ACB in various larval stages (1-5 instar) from Cry1Ab-sensitive (ACB-BtS) and -resistant (ACB-AbR) strains, each of which included two biological replicates. Using Illumina sequencing, a total of 23,809,890 high-quality reads were collected from the four ACB libraries. The numbers of known microRNAs (miRNAs) were 302 and 395 for ACB-BtS and 268 and 287 for ACB-AbR. Using Mireap software, we identified 32 and 16 potential novel miRNAs for ACB-BtS and 18 and 22 for ACB-AbR. Among them, 21 known and 1 novel miRNAs had significantly different expression between ACB-BtS and ACB-AbR. Several miRNAs were observed to target potential Bt receptor genes, such as aminopeptidase N and cadherin-like protein. The glycosylphosphatidylinositol-anchor biosynthetic process and ABC transporters pathway were identified through Gene Ontology and KEGG pathway analysis of target genes of the differentially expressed miRNAs. PMID:26486179

  5. A toxin-binding alkaline phosphatase fragment synergizes Bt toxin Cry1Ac against susceptible and resistant Helicoverpa armigera.

    Science.gov (United States)

    Chen, Wenbo; Liu, Chenxi; Xiao, Yutao; Zhang, Dandan; Zhang, Yongdong; Li, Xianchun; Tabashnik, Bruce E; Wu, Kongming

    2015-01-01

    Evolution of resistance by insects threatens the continued success of pest control using insecticidal crystal (Cry) proteins from the bacterium Bacillus thuringiensis (Bt) in sprays and transgenic plants. In this study, laboratory selection with Cry1Ac yielded five strains of cotton bollworm, Helicoverpa armigera, with resistance ratios at the median lethal concentration (LC50) of activated Cry1Ac ranging from 22 to 1700. Reduced activity and reduced transcription of an alkaline phosphatase protein that binds Cry1Ac was associated with resistance to Cry1Ac in the four most resistant strains. A Cry1Ac-binding fragment of alkaline phosphatase from H. armigera (HaALP1f) was not toxic by itself, but it increased mortality caused by Cry1Ac in a susceptible strain and in all five resistant strains. Although synergism of Bt toxins against susceptible insects by toxin-binding fragments of cadherin and aminopeptidase N has been reported previously, the results here provide the first evidence of synergism of a Bt toxin by a toxin-binding fragment of alkaline phosphatase. The results here also provide the first evidence of synergism of a Bt toxin by any toxin-binding peptide against resistant insects. PMID:25885820

  6. Three toxins, two receptors, one mechanism: Mode of action of Cry1A toxins from Bacillus thuringiensis in Heliothis virescens.

    Science.gov (United States)

    Bretschneider, Anne; Heckel, David G; Pauchet, Yannick

    2016-09-01

    Insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) are highly active against Lepidoptera. However, field-evolved resistance to Bt toxins is on the rise. The 12-cadherin domain protein HevCaLP and the ABC transporter HevABCC2 are both genetically linked to Cry toxin resistance in Heliothis virescens. We investigated their interaction using stably expressing non-lytic clonal Sf9 cell lines expressing either protein or both together. Untransfected Sf9 cells are innately sensitive to Cry1Ca toxin, but not to Cry1A toxins; and quantitative PCR revealed negligible expression of genes involved in Cry1A toxicity such as cadherin, ABCC2, alkaline phosphatase (ALP) and aminopeptidase N (APN). Cry1Aa, Cry1Ab or Cry1Ac caused swelling of Sf9 cells expressing HevABCC2, and caused faster swelling, lysis and up to 86% mortality in cells expressing both proteins. No such effect was observed in control Sf9 cells or in cells expressing only HevCaLP. The results of a mixing experiment demonstrated that both proteins need to be expressed within the same cell for high cytotoxicity, and suggest a novel role for HevCaLP. Binding assays showed that the toxin-receptor interaction is specific. Our findings confirm that HevABCC2 is the central target in Cry1A toxin mode of action, and that HevCaLP plays a supporting role in increasing Cry1A toxicity. PMID:27456115

  7. PENG: a neural gas-based approach for pharmacophore elucidation. method design, validation, and virtual screening for novel ligands of LTA4H.

    Science.gov (United States)

    Moser, Daniel; Wittmann, Sandra K; Kramer, Jan; Blöcher, René; Achenbach, Janosch; Pogoryelov, Denys; Proschak, Ewgenij

    2015-02-23

    The pharmacophore concept is commonly employed in virtual screening for hit identification. A pharmacophore is generally defined as the three-dimensional arrangement of the structural and physicochemical features of a compound responsible for its affinity to a pharmacological target. Given a number of active ligands binding to a particular target in the same manner, it can reasonably be assumed that they have some shared features, a common pharmacophore. We present a growing neural gas (GNG)-based approach for the extraction of the relevant features which we called PENG (pharmacophore elucidation by neural gas). Results of retrospective validation indicate an acceptable quality of the generated models. Additionally a prospective virtual screening for leukotriene A4 hydrolase (LTA4H) inhibitors was performed. LTA4H is a bifunctional zinc metalloprotease which displays both epoxide hydrolase and aminopeptidase activity. We could show that the PENG approach is able to predict the binding mode of the ligand by X-ray crystallography. Furthermore, we identified a novel chemotype of LTA4H inhibitors. PMID:25625859

  8. Automated Solution-Phase Synthesis of Insect Glycans to Probe the Binding Affinity of Pea Enation Mosaic Virus.

    Science.gov (United States)

    Tang, Shu-Lun; Linz, Lucas B; Bonning, Bryony C; Pohl, Nicola L B

    2015-11-01

    Pea enation mosaic virus (PEMV)--a plant RNA virus transmitted exclusively by aphids--causes disease in multiple food crops. However, the aphid-virus interactions required for disease transmission are poorly understood. For virus transmission, PEMV binds to a heavily glycosylated receptor aminopeptidase N in the pea aphid gut and is transcytosed across the gut epithelium into the aphid body cavity prior to release in saliva as the aphid feeds. To investigate the role of glycans in PEMV-aphid interactions and explore the possibility of viral control through blocking a glycan interaction, we synthesized insect N-glycan terminal trimannosides by automated solution-phase synthesis. The route features a mannose building block with C-5 ester enforcing a β-linkage, which also provides a site for subsequent chain extension. The resulting insect N-glycan terminal trimannosides with fluorous tags were used in a fluorous microarray to analyze binding with fluorescein isothiocyanate-labeled PEMV; however, no specific binding between the insect glycan and PEMV was detected. To confirm these microarray results, we removed the fluorous tag from the trimannosides for isothermal titration calorimetry studies with unlabeled PEMV. The ITC studies confirmed the microarray results and suggested that this particular glycan-PEMV interaction is not involved in virus uptake and transport through the aphid.

  9. A cadherin-like protein functions as a receptor for Bacillus thuringiensis Cry1Aa and Cry1Ac toxins on midgut epithelial cells of Bombyx mori larvae.

    Science.gov (United States)

    Hara, Hirotaka; Atsumi, Shogo; Yaoi, Katsuro; Nakanishi, Kazuko; Higurashi, Satoshi; Miura, Nami; Tabunoki, Hiroko; Sato, Ryoichi

    2003-03-13

    Aminopeptidase N (APN) and cadherin-like protein (BtR175) from Bombyx mori larvae were examined for their roles in Cry1Aa- and Cry1Ac-induced lysis of B. mori midgut epithelial cells (MECs). APNs and BtR175 were present in all areas of the midgut, were particularly abundant in the posterior region, and were found only on columnar cell microvilli and not on the lateral membrane that makes cell-cell contacts. This distribution was in accordance with the distribution of Cry1A-susceptible MECs in the midgut. The lytic activity of Cry1Aa and Cry1Ac on collagenase-dissociated MECs was linearly dependent on toxin concentration. Although pre-treatment of MECs with anti-BtR175 antibody was observed to partially inhibit the lytic activity exerted by 0.1-1 nM Cry1Aa toxin or 5 nM Cry1Ac toxin, no significant inhibition was observed when MECs were pre-treated with anti-APN antibody. These results suggest that BtR175 functions as a major receptor for Cry1A toxins in the midgut of B. mori larvae. PMID:12633848

  10. Periphytic photosynthetic stimulation of extracellular enzyme activity in aquatic microbial communities associated with decaying typha litter.

    Science.gov (United States)

    Francoeur, Steven N; Schaecher, Mark; Neely, Robert K; Kuehn, Kevin A

    2006-11-01

    We examined the effect of light on extracellular enzyme activities of periphytic/endogenous microbial assemblages associated with decomposing litter of an emergent macrophyte Typha angustifolia within a small inland wetland in southeastern Michigan. Standing-dead Typha leaf litter was collected, placed into floating wire mesh litter baskets, and submerged in a wetland pool. Enzyme saturation assays were conducted on three occasions following litter submergence (days 9, 28, and 44) to generate saturation curves for the individual enzymes tested and to examine potential differences in enzyme saturation kinetics during microbial colonization and development. Experimental light manipulations were conducted on two occasions during microbial development (days 10 and 29). Short-term (30 min) light exposure significantly increased extracellular beta-glucosidase activity of litter-associated microbial communities. Activities of beta-xylosidase and leucine-aminopeptidase were not stimulated, and stimulation of phosphatase activity was variable. The exact mechanism for increased enzyme activity remains unknown, but it may have been increased pH arising from periphytic algal photosynthesis. These results suggest that extracellular enzyme activity in microbial communities colonizing natural organic substrata may be influenced by light/photosynthesis, as has previously been demonstrated for periphyton communities grown on artificial, inert substrata. Thus, light/photosynthetic mediated stimulation of extracellular enzyme activities may be a common occurrence in microbial communities associated with natural decaying plant litter in wetlands and might engender diurnal patterns in other microbial decay processes (e.g., production, organic matter decomposition, and mineralization). PMID:17082997

  11. Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C.

    Science.gov (United States)

    Poreba, Marcin; Mihelic, Marko; Krai, Priscilla; Rajkovic, Jelena; Krezel, Artur; Pawelczak, Malgorzata; Klemba, Michael; Turk, Dusan; Turk, Boris; Latajka, Rafal; Drag, Marcin

    2014-04-01

    Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.

  12. Protective mechanisms against homocysteine toxicity: the role of bleomycin hydrolase.

    Science.gov (United States)

    Zimny, Jaroslaw; Sikora, Marta; Guranowski, Andrzej; Jakubowski, Hieronim

    2006-08-11

    Homocysteine (Hcy) editing by methionyl-tRNA synthetase results in the formation of Hcy-thiolactone and initiates a pathway that has been implicated in human disease. In addition to being cleared from the circulation by urinary excretion, Hcy-thiolactone is detoxified by the serum Hcy-thiolactonase/paraoxonase carried on high density lipoprotein. Whether Hcy-thiolactone is detoxified inside cells was unknown. Here we show that Hcy-thiolactone is hydrolyzed by an intracellular enzyme, which we have purified to homogeneity from human placenta and identified by proteomic analyses as human bleomycin hydrolase (hBLH). We have also purified an Hcy-thiolactonase from the yeast Saccharomyces cerevisiae and identified it as yeast bleomycin hydrolase (yBLH). BLH belongs to a family of evolutionarily conserved cysteine aminopeptidases, and its only known biologically relevant function was deamidation of the anticancer drug bleomycin. Recombinant hBLH or yBLH, expressed in Escherichia coli, exhibits Hcy-thiolactonase activity similar to that of the native enzymes. Active site mutations, C73A for hBLH and H369A for yBLH, inactivate Hcy-thiolactonase activities. Yeast blh1 mutants are deficient in Hcy-thiolactonase activity in vitro and in vivo, produce more Hcy-thiolactone, and exhibit greater sensitivity to Hcy toxicity than wild type yeast cells. Our data suggest that BLH protects cells against Hcy toxicity by hydrolyzing intracellular Hcy-thiolactone. PMID:16769724

  13. Seasonal flexibility of organ mass and intestinal function for the Andean lizard Liolaemus nigroviridis.

    Science.gov (United States)

    Naya, Daniel E; Veloso, Claudio; Sabat, Pablo; Bozinovic, Francisco

    2009-04-01

    One of the most fundamental questions in organismal ecology is how animals work in a continuously changing environment. In order to contribute to the current understanding of this question, this study evaluated seasonal changes in digestive enzymes activities, organs size, and energy reserves in Liolaemus nigroviridis, a medium-size lizard that inhabit extreme environments in the Andes range. We found that digestive enzymes (trehalase, maltase, and aminopeptidase-N) hydrolytic activities, dry masses of digestive organs and liver, and energy reserve (dry mass of fat bodies and tail energy density) were greater during summer than during winter months. By contrast, dry mass of the kidneys, lungs, heart, and gonads were greater during winter (though significance was reach only for the last two organs). In summary, obtained results reinforce the idea that hibernation is connected with phenotypic adjustments at different organizational levels, which in turn, potentially affects rates of energy acquisition and expenditure, organisms' fitness, and, ultimately, ecological and evolutionary success of species living in highly seasonal environments. We suggest that, owing to the pressing need to explain and predict the impact of climatic change on the biota, more studies destined to determine the levels and limits of physiological flexibility are necessary. PMID:19204910

  14. Isolation and characterisation of dipeptidyl peptidase IV from Prevotella loescheii ATCC 15930.

    Science.gov (United States)

    Koreeda, Y; Hayakawa, M; Ikemi, T; Abiko, Y

    2001-08-01

    A proline-specific dipeptidyl aminopeptidase, dipeptidyl peptidase IV (EC 3.4.14.5), was purified from a cell sonicate soluble fraction of Prevotella loescheii ATCC 15930 by sequential column chromatography. The molecular mass of the native enzyme was estimated as 160 kDa by high-pressure liquid gel filtration column chromatography and unheated sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The subunit molecular mass was 80 kDa when the enzyme was heated to 100 degrees C in the presence of 2-mercaptoethanol before SDS-PAGE, suggesting that the native enzyme consists of two identical subunits and is folded in 2% SDS. The optimum pH, with glycyl-prolyl-4-methyl-coumaryl-7-amide as the substrate, was 8.0; the isoelectric point was 5.2. Purified enzyme showed a strong preference for dipeptide substrates containing proline and, less efficiently, alanine in the P1 position. The enzyme was markedly inhibited by Cd(2+), Zn(2+), Hg(2+), Co(2+), and serine proteinase inhibitor di-isopropylfluorophosphate. PMID:11389867

  15. The Anopheles-midgut APN1 structure reveals a new malaria transmission-blocking vaccine epitope.

    Science.gov (United States)

    Atkinson, Sarah C; Armistead, Jennifer S; Mathias, Derrick K; Sandeu, Maurice M; Tao, Dingyin; Borhani-Dizaji, Nahid; Tarimo, Brian B; Morlais, Isabelle; Dinglasan, Rhoel R; Borg, Natalie A

    2015-07-01

    Mosquito-based malaria transmission-blocking vaccines (mTBVs) target midgut-surface antigens of the Plasmodium parasite's obligate vector, the Anopheles mosquito. The alanyl aminopeptidase N (AnAPN1) is the leading mTBV immunogen; however, AnAPN1's role in Plasmodium infection of the mosquito and how anti-AnAPN1 antibodies functionally block parasite transmission have remained elusive. Here we present the 2.65-Å crystal structure of AnAPN1 and the immunoreactivity and transmission-blocking profiles of three monoclonal antibodies (mAbs) to AnAPN1, including mAb 4H5B7, which effectively blocks transmission of natural strains of Plasmodium falciparum. Using the AnAPN1 structure, we map the conformation-dependent 4H5B7 neoepitope to a previously uncharacterized region on domain 1 and further demonstrate that nonhuman-primate neoepitope-specific IgG also blocks parasite transmission. We discuss the prospect of a new biological function of AnAPN1 as a receptor for Plasmodium in the mosquito midgut and the implications for redesigning the AnAPN1 mTBV. PMID:26075520

  16. Assessing approaches to determine the effect of ocean acidification on bacterial processes

    Science.gov (United States)

    Burrell, Timothy J.; Maas, Elizabeth W.; Teesdale-Spittle, Paul; Law, Cliff S.

    2016-08-01

    Bacterial extracellular enzymes play a significant role in the degradation of labile organic matter and nutrient availability in the open ocean. Although bacterial production and extracellular enzymes may be affected by ocean acidification, few studies to date have considered the methodology used to measure enzyme activity and bacterial processes. This study investigated the potential artefacts in determining the response of bacterial growth and extracellular glucosidase and aminopeptidase activity to ocean acidification as well as the relative effects of three different acidification techniques. Tests confirmed that the observed effect of pH on fluorescence of artificial fluorophores, and the influence of the MCA fluorescent substrate on seawater sample pH, were both overcome by the use of Tris buffer. In experiments testing different acidification methods, bubbling with CO2 gas mixtures resulted in higher β-glucosidase activity and 15-40 % higher bacterial abundance, relative to acidification via gas-permeable silicon tubing and acid addition (HCl). Bubbling may stimulate carbohydrate degradation and bacterial growth, leading to the incorrect interpretation of the impacts of ocean acidification on organic matter cycling.

  17. Detecting groups of coevolving positions in a molecule: a clustering approach

    Directory of Open Access Journals (Sweden)

    Galtier Nicolas

    2007-11-01

    Full Text Available Abstract Background Although the patterns of co-substitutions in RNA is now well characterized, detection of coevolving positions in proteins remains a difficult task. It has been recognized that the signal is typically weak, due to the fact that (i amino-acid are characterized by various biochemical properties, so that distinct amino acids changes are not functionally equivalent, and (ii a given mutation can be compensated by more than one mutation, at more than one position. Results We present a new method based on phylogenetic substitution mapping. The two above-mentioned problems are addressed by (i the introduction of a weighted mapping, which accounts for the biochemical effects (volume, polarity, charge of amino-acid changes, (ii the use of a clustering approach to detect groups of coevolving sites of virtually any size, and (iii the distinction between biochemical compensation and other coevolutionary mechanisms. We apply this methodology to a previously studied data set of bacterial ribosomal RNA, and to three protein data sets (myoglobin of vertebrates, S-locus Receptor Kinase and Methionine Amino-Peptidase. Conclusion We succeed in detecting groups of sites which significantly depart the null hypothesis of independence. Group sizes range from pairs to groups of size ≃ 10, depending on the substitution weights used. The structural and functional relevance of these groups of sites are assessed, and the various evolutionary processes potentially generating correlated substitution patterns are discussed.

  18. Early Morphological and Physiological Events Occurring During Germination of Maize Seeds

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The early morphological and physiological events occurring during maize (Zea mays cv. Nongda 108) seed imbibition and germination were studied. Water uptake of seeds exhibited a triphasic pattern with a marked increase during the initial phase of imbibition, and then a slow increase, followed by a second substantial increase. Imbibition time for 10 and 50% of seed germination was about 26 and 46 h at 30℃, respectively. The relative conductivity of maize seeds dramatically decreased during the initial phase of imbibition, followed by a substantial increase. Respiratory rate of seeds gradually increased with imbibition. Length of root cap cells decreased during the initial phase and then increased; those of meristematic zone cells increased during the initial phase and then decreased; and those of elongation zone cells and of the whole elongation zone of the radicle gradually increased during germination. The contents of soluble sugars and starch in embryos gradually decreased as the activities of α- and β-amylase strikingly increased with imbibition. In the meantime, protein contents of embryos gradually decreased and free amino acid content increased. The activities of aminopeptidase and endopeptidase increased until 12 h of imbibition and then decreased. It is concluded that germination of maize seeds is mainly completed by extension of cells in the elongation zone of the radicle, and that mobilization of stored reserves in the embryo during the initial phase of imbibition is also an early event during seed germination.

  19. Development and validation of a simple cell-based fluorescence assay for dipeptidyl peptidase 1 (DPP1) activity.

    Science.gov (United States)

    Thong, Bob; Pilling, James; Ainscow, Edward; Beri, Raj; Unitt, John

    2011-01-01

    Dipeptidyl peptidase 1 (DPP1) (EC 3.4.14.1; also known as cathepsin C, cathepsin J, dipeptidyl aminopeptidase, and dipeptidyl aminotransferase) is a lysosomal cysteinyl protease of the papain family involved in the intracellular degradation of proteins. Isolated enzyme assays for DPP1 activity using a variety of synthetic substrates such as dipeptide or peptide linked to amino-methyl-coumarin (AMC) or other fluorophores are well established. There is, however, no report of a simple whole-cell-based assay for measuring lysosomal DPP1 activity other than the use of flow cytometry (fluorescence-activated cell sorting) or the use of invasive activity-based probes or the production of physiological products such as neutrophil elastase. The authors investigated a number of DPP1 fluorogenic substrates that have the potential to access the lysosome and enable the measurement of DPP1 enzyme activity in situ. They describe the development and evaluation of a simple noninvasive fluorescence assay for measuring DPP1 activity in fresh or cryopreserved human THP-1 cells using the substrate H-Gly-Phe-AFC (amino-fluoro-coumarin). This cell-based fluorescence assay can be performed in a 96-well plate format and is ideally suited for determining the cell potency of potential DPP1 enzyme inhibitors.

  20. Selecting protein N-terminal peptides by combined fractional diagonal chromatography.

    Science.gov (United States)

    Staes, An; Impens, Francis; Van Damme, Petra; Ruttens, Bart; Goethals, Marc; Demol, Hans; Timmerman, Evy; Vandekerckhove, Joël; Gevaert, Kris

    2011-07-14

    In recent years, procedures for selecting the N-terminal peptides of proteins with analysis by mass spectrometry have been established to characterize protease-mediated cleavage and protein α-N-acetylation on a proteomic level. As a pioneering technology, N-terminal combined fractional diagonal chromatography (COFRADIC) has been used in numerous studies in which these protein modifications were investigated. Derivatization of primary amines--which can include stable isotope labeling--occurs before trypsin digestion so that cleavage occurs after arginine residues. Strong cation exchange (SCX) chromatography results in the removal of most of the internal peptides. Diagonal, reversed-phase peptide chromatography, in which the two runs are separated by reaction with 2,4,6-trinitrobenzenesulfonic acid, results in the removal of the C-terminal peptides and remaining internal peptides and the fractionation of the sample. We describe here the fully matured N-terminal COFRADIC protocol as it is currently routinely used, including the most substantial improvements (including treatment with glutamine cyclotransferase and pyroglutamyl aminopeptidase to remove pyroglutamate before SCX, and a sample pooling scheme to reduce the overall number of liquid chromatography-tandem mass spectrometry analyses) that were made since its original publication. Completion of the N-terminal COFRADIC procedure takes ~5 d.