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. Aminopeptidases do not directly degrade tau protein

    Directory of Open Access Journals (Sweden)

    Hersh Louis B

    2010-11-01

    Full Text Available Abstract Background Tau hyperphosphorylation and aggregation to form intracellular neurofibrillar tangles is prevalent in a number of tauopathies. Thus there is current interest in the mechanisms involved in Tau clearance. It was recently reported that Tau can be degraded by an aminopeptidase known as the puromycin sensitive aminopeptidase (PSA. Until now PSA has been reported to only cleave peptides, with the largest reported substrates having 30-50 amino acids. We have studied this unique PSA cleavage reaction using a number of different PSA preparations. Results An N-terminally His tagged-PSA was expressed and purified from Sf9 insect cells. Although this PSA preparation cleaved Tau, product analysis with N and C terminal Tau antibodies coupled with mass spectrometry showed an endoproteolytic cleavage atypical for an aminopeptidase. Furthermore, the reaction was not blocked by the general aminopeptidase inhibitor bestatin or the specific PSA inhibitor puromycin. In order to test whether Tau hydrolysis might be caused by a protease contaminant the enzyme was expressed in E. coli as glutathione S-transferase and maltose binding protein fusion proteins or in Sf9 cells as a C-terminally His-tagged protein. After purification to near homogeneity none of these other recombinant forms of PSA cleaved Tau. Further, Tau-cleaving activity and aminopeptidase activities derived from the Sf9 cell expression system were separable by molecular sieve chromatography. When tested in a cellular context we again failed to see a PSA dependent cleavage of Tau. A commercial preparation of a related aminopeptidase, aminopeptidase N, also exhibited Tau cleaving activity, but this activity could also be separated from aminopeptidase activity. Conclusion It is concluded that PSA does not directly cleave Tau.

  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. 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.

  6. 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

  7. 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.

  8. Aminopeptidase A is a functional target in angiogenic blood vessels.

    NARCIS (Netherlands)

    Marchio, S.; Lahdenranta, J.; Schlingemann, R.O.; Valdembri, D.; Wesseling, P.; Arap, M.A.; Hajitou, A.; Ozawa, M.G.; Trepel, M.; Giordano, R.J.; Nanus, D.M.; Dijkman, H.B.P.M.; Oosterwijk, E.; Sidman, R.L.; Cooper, M.D.; Bussolino, F.; Pasqualini, R.; Arap, W.

    2004-01-01

    We show that a membrane-associated protease, aminopeptidase A (APA), is upregulated and enzymatically active in blood vessels of human tumors. To gain mechanistic insight, we evaluated angiogenesis in APA null mice. We found that, although these mice develop normally, they fail to mount the expected

  9. 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.

  10. "Leucine aminopeptidase" (neutral arylamidase) in sheep sera: improved resolution with gradient gel electrophoresis.

    Science.gov (United States)

    Manwell, C; Baker, C M

    1986-01-01

    Electrophoretic resolution of the heterogeneity of sheep serum "leucine aminopeptidase" is greatly improved by the use of gradients of acrylamide polymer, together with enzyme localisation involving L-alanyl beta-naphthylamide and cobaltous ion. The improved resolution contradicts an earlier claim of the existence of only two patterns of individual variation in the heterogeneity of sheep serum "leucine aminopeptidase", with one pattern completely dominant to the other. While the sheep enzyme is unusual among mammalian serum "leucine aminopeptidases" in its complex heterogeneity, it does conform to the typical mammalian pattern of codominant individual variation. The complexity of sheep serum "leucine aminopeptidase" is useful in the study of sheep evolution.

  11. Characterization of Aspergillus oryzae aspartyl aminopeptidase expressed in Escherichia coli.

    Science.gov (United States)

    Watanabe, Jun; Tanaka, Hisaki; Akagawa, Takumi; Mogi, Yoshinobu; Yamazaki, Tatsuo

    2007-10-01

    To characterize aspartyl aminopeptidase from Aspergillus oryzae, the recombinant enzyme was expressed in Escherichia coli. The enzyme cleaves N-terminal acidic amino acids. About 30% activity was retained in 20% NaCl. Digestion of defatted soybean by the enzyme resulted in an increase in the glutamic acid content, suggesting that the enzyme is potentially responsible for the release of glutamic acid in soy sauce mash.

  12. Structural basis for the inhibition of M1 family aminopeptidases by the natural product actinonin: Crystal structure in complex with E. coli aminopeptidase N.

    Science.gov (United States)

    Ganji, Roopa Jones; Reddi, Ravikumar; Gumpena, Rajesh; Marapaka, Anil Kumar; Arya, Tarun; Sankoju, Priyanka; Bhukya, Supriya; Addlagatta, Anthony

    2015-05-01

    Actinonin is a pseudotripeptide that displays a high affinity towards metalloproteases including peptide deformylases (PDFs) and M1 family aminopeptidases. PDF and M1 family aminopeptidases belong to thermolysin-metzincin superfamily. One of the major differences in terms of substrate binding pockets between these families is presence (in M1 aminopeptidases) or absence (in PDFs) of an S1 substrate pocket. The binding mode of actinonin to PDFs has been established previously; however, it is not clear how the actinonin, without a P1 residue, would bind to the M1 aminopeptidases. Here we describe the crystal structure of Escherichia coli aminopeptidase N (ePepN), a model protein of the M1 family aminopeptidases in complex with actinonin. For comparison we have also determined the structure of ePepN in complex with a well-known tetrapeptide inhibitor, amastatin. From the comparison of the actinonin and amastatin ePepN complexes, it is clear that the P1 residue is not critical as long as strong metal chelating head groups, like hydroxamic acid or α-hydroxy ketone, are present. Results from this study will be useful for the design of selective and efficient hydroxamate inhibitors against M1 family aminopeptidases.

  13. 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.

  14. 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...

  15. Tissue-specific interactions between nuclear proteins and the aminopeptidase N promoter

    DEFF Research Database (Denmark)

    Kärnström, U; Sjöström, H; Norén, O;

    1991-01-01

    Aminopeptidase N/CD13 is a metallopeptidase found in many tissues. Aminopeptidase N activity is high in the small intestinal mucosa, moderate in the liver, and low in the spleen. Using DNase I footprinting and electrophoretic mobility shift assays with nuclear extracts from these tissues, three cis...... intestinal mucosa. The UF region (-112 to -90) interacts with nuclear factors which seem to be expressed differentially in the liver and the small intestine. Transfection of promoter deletions into HepG2 cells showed that the LF-B1 region is necessary for high expression of the aminopeptidase N gene in liver...

  16. 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......The biogenesis of pig small intestinal aminopeptidase N (EC 3. 4. 11. 2) was studied by cell-free translation of intestinal mRNA and by labelling of organ cultured intestinal explants. In cell-free translation, the primary mRNA translation product of aminopeptidase N was a polypeptide of Mr 115...

  17. 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.

  18. Superoxol and aminopeptidase tests for identification of pathogenic Neisseria species and Moraxella (Branhamella) catarrhalis.

    Science.gov (United States)

    Pérez, J L; Pulido, A; Gómez, E; Sauca, G; Martín, R

    1990-06-01

    The superoxol test, and prolyl aminopeptidase and gammaglutamyl aminopeptidase tests were evaluated for the detection of pathogenic Neisseria spp. using 317 strains of Neisseria-ceae. The superoxol test was positive for all 116 gonococci and 62 Moraxella (Branhamella) catarrhalis strains, but also for three strains of Neisseria meningitidis, one strain of Neisseria lactamica and eight saprophytic neisseriae. When using strains grown on Thayer-Martin medium, the positive and negative predictive values of the superoxol test for the identification of Neisseria gonorrhoeae were 96.7% and 100% respectively. Meningococci were the only neisseriae growing on Thayer-Martin medium that showed gamma-glutamyl aminopeptidase activity. The prolyl aminopeptidase test showed low specificity.

  19. Identification of Aminopeptidase N as a Cellular Receptor for Human Coronavirus-229E

    Science.gov (United States)

    1992-05-12

    feline enteric coronav irus feline infectious peritonitis virus hUman adult intestine hUman aminopeptidase N human aminopeptidase with 39 amino...coronavirus (TCV), rat coronavirus (RCV), cat feline infectious peritonitis virus (FIPV), and the hUman coronaviruses. These include the slow, patchy...While the cat, dog and pig serve as natural hosts for the other coronavirus group 1 viruses, feline infectious peritonitis virus (FIPV), canine

  20. 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...... that microsomal fractions should be added before about 25% of the polypeptide was synthesized to ensure processing to the high-mannose glycosylated form. This suggests that the signal sequence is situated in the N-terminal part of the aminopeptidase N. The size of the cell-free translation product in the absence...

  1. 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.

  2. 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.

  3. 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.

  4. The monoclonal antibody ER-BMDM1 recognizes a macrophage and dendritic cell differentiation antigen with aminopeptidase activity

    NARCIS (Netherlands)

    P.J. Leenen (Pieter); M.L. Melis (Marleen); G. Kraal (Georg); A.T. Hoogeveen (Andre); W. van Ewijk (Willem)

    1992-01-01

    markdownabstractAbstract Here we describe the reactivity of monoclonal antibody (mAb) ER-BMDM1, directed against a 160-kDa cell membrane-associated antigen (Ag) with aminopeptidase activity. The aminopeptidase recognized by ER-BMDM1 is present on various mouse macrophage (MΦ) and dendritic cell (D

  5. 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-embe...... that postembedding immunogold labelling can be used quantitatively....

  6. Aminopeptidase N inhibition could be involved in the anti-angiogenic effect of dobesilates

    Directory of Open Access Journals (Sweden)

    Farsa Oldřich

    2015-01-01

    Full Text Available Calcium, magnesium and zinc 2,5-dihydroxybenzenesulfonates (dobesilates were synthesized by sulfonation of hydroquinone with sulfuric acid under mild conditions. To form the salts, neutralization with calcium carbonate followed by cation exchange by means of magnesium or zinc sulfates was performed. The dobesilates were characterized by standard spectral methods and by AAS for metal content and then tested for inhibitory activity against aminopeptidase N. Calcium and magnesium 2,5-dihydroxybenzene sulfonates exhibited rather weak inhibitory activity to aminopeptidase N as demonstrated by IC50 values of 978.0 and 832.1 mmol l-1 respectively while zinc 2,5-dihydroxybenzene sulfonate reached the more significant inhibitory activity characterized by IC50 77.4 mmol l-1. The inhibitory activity results suggest that the inhibition of aminopeptidase N could play a role in the anti-angiogenic activity of 2,5-dihydroxybenzenesulfonates.

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

    DEFF Research Database (Denmark)

    Danielsen, E M

    1990-01-01

    The pig intestinal brush border enzymes aminopeptidase N (EC 3.4.11.2) and lactase-phlorizin hydrolase (EC 3.2.1.23-62) are present in the microvillar membrane as homodimers. Dimethyl adipimidate was used to cross-link the two [35S]methionine-labeled brush border enzymes from cultured mucosal...... 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...

  8. 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.

  9. HNF1 alpha activates the aminopeptidase N promoter in intestinal (Caco-2) cells

    DEFF Research Database (Denmark)

    Olsen, Jørgen; Laustsen, Lotte; Troelsen, J

    1994-01-01

    The importance of HNF1 binding proteins for intestinal aminopeptidase N expression was investigated using the Caco-2 cell-line. Aminopeptidase N promoter activity in Caco-2 cells depends on the HNF1 element (positions -85 to -58) and co-transfection with an HNF1 alpha expression vector demonstrates...... a direct activation of the promoter by HNF1 alpha through this element. Electrophoretic mobility shift assays using nuclear extracts from Caco-2 cells show the presence of high amounts of HNF1 binding proteins irrespective of their state of differentiation....

  10. Enhanced lysosomal activity by overexpressed aminopeptidase Y in Saccharomyces cerevisiae.

    Science.gov (United States)

    Yoon, Jihee; Sekhon, Simranjeet Singh; Kim, Yang-Hoon; Min, Jiho

    2016-06-01

    Saccharomyces cerevisiae contains vacuoles corresponding to lysosomes in higher eukaryotes. Lysosomes are dynamic (not silent) organelles in which enzymes can be easily integrated or released when exposed to stressful conditions. Changes in lysosomal enzymes have been observed due to oxidative stress, resulting in an increased function of lysosomes. The protein profiles from H2O2- and NH4Cl-treated lysosomes showed different expression patterns, observed with two-dimensional gel electrophoresis. The aminopeptidase Y protein (APE3) that conspicuously enhanced antimicrobial activity than other proteins was selected for further studies. The S. cerevisiae APE3 gene was isolated and inserted into pYES2.0 expression vector. The GFP gene was inserted downstream to the APE3 gene for confirmation of APE3 targeting to lysosomes, and S. cerevisiae was transformed to pYES2::APE3::GFP. The APE3 did not enter in lysosomes and formed an inclusion body at 30 °C, but it inserted to lysosomes as shown by the merger of GFP with lysosomes at 28 °C. Antimicrobial activity of the cloned S. cerevisiae increased about 5 to 10 % against eight strains, compared to normal cells, and galactose induction is increased more two folds than that of normal cells. Therefore, S. cerevisiae was transformed to pYES2::APE3::GFP, accumulating a large amount of APE3, resulting in increased lysosomal activity. Increase in endogenous levels of lysosomes and their activity following genetic modification can lead to its use in applications such as antimicrobial agents and apoptosis-inducing materials for cancer cells, and consequently, it may also be possible to use the organelles for improving in vitro functions.

  11. 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.

  12. Immunomicroscopic localization of aminopeptidase N in the pig enterocyte. Implications for the route of intracellular transport

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Sjöström, H; Norén, Ove

    1987-01-01

    . Labelling was demonstrated in the Golgi apparatus and in a minor fraction of the intracellular smooth vesicles positioned between the Golgi apparatus and the microvillar membrane. These observations are compatible with the view that newly synthesized aminopeptidase N is delivered directly to the microvillar...

  13. 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

    Pig small intestinal mRNA was translated in a rabbit reticulocyte lysate system supplemented with microsomal membranes. Castanospermine, an inhibitor of glucosidase I, induced a high mannose-glycosylated form of microvillar aminopeptidase N (EC 3.4.11.2) of increased molecular mass, indicating...

  14. Haemonchus contortus: Characterization of the baculovirus expressed form of aminopeptidase H11

    NARCIS (Netherlands)

    Reszka, N.; Rijsewijk, F.A.M.; Zelnik, V.; Moskwa, B.; Bienkowska-Szewczyk, K.

    2007-01-01

    Recombinant form of Haemonchus contortus aminopeptidase H11, an intestinal membrane glycoprotein considered to be in its native form the most promising vaccine candidate, was produced in insect cells, characterised and tested in pilot vaccination-challenge trial on sheep. The sequence of the cloned

  15. 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

  16. 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.

  17. 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.

  18. 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...

  19. An enkephalin degrading aminopeptidase of human brain preserved during the vertebrate phylogeny.

    Science.gov (United States)

    de Souza, A N; Bruno, J A; Carvalho, K M

    1991-01-01

    1. A soluble human brain aminopeptidase which hydrolyses the Tyr-Gly bond of Met-enkephalin and Leu-enkephalin was identified in the brains of the following vertebrates: mammals (Callithrix jacchus and Rattus norvegicus), bird (Gallus domesticus), reptile (Tupinambis teguixin), amphibia (Bufo paracnemis), fish (Sarotherdon niloticus) and elasmobranchy (Galeocerdo cuvieri). 2. The properties of this enzyme are: molecular weight near 100,000 Da, isoelectric point near 4.9, optimum pH near 7.5, activation by dithiothreitol, strong inhibition by Cu2+, Zn2+, Ni2+, puromycin and bacitracin, hydrolysis of enkephalins and basic and neutral aminoacid-beta-naphythylamide substrates. 3. The results indicate the preservation of this human brain aminopeptidase during the course of vertebrate phylogeny.

  20. 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

    . 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...... oxygen, which is part of either a water molecule or a hydroxide ion. Within the site of hMetAP-2 the results strongly indicate that a hydroxide ion bridges the metal ions. By contrast, the nature of the oxygen bridging the metal ions within the metal binding site of eMetAP-1 cannot be determined based...... on the results here, due to the similar structural results obtained with a bridging water molecule and a bridging hydroxide ion....

  1. 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.

  2. 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...

  3. 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.

  4. Urinary aminopeptidase activities as early and predictive biomarkers of renal dysfunction in cisplatin-treated rats.

    Directory of Open Access Journals (Sweden)

    Andrés Quesada

    Full Text Available This study analyzes the fluorimetric determination of alanyl- (Ala, glutamyl- (Glu, leucyl-cystinyl- (Cys and aspartyl-aminopeptidase (AspAp urinary enzymatic activities as early and predictive biomarkers of renal dysfunction in cisplatin-treated rats. Male Wistar rats (n = 8 each group received a single subcutaneous injection of either saline or cisplatin 3.5 or 7 mg/kg, and urine samples were taken at 0, 1, 2, 3 and 14 days after treatment. In urine samples we determined Ala, Glu, Cys and AspAp activities, proteinuria, N-acetyl-β-D-glucosaminidase (NAG, albumin, and neutrophil gelatinase-associated lipocalin (NGAL. Plasma creatinine, creatinine clearance and renal morphological variables were measured at the end of the experiment. CysAp, NAG and albumin were increased 48 hours after treatment in the cisplatin 3.5 mg/kg treated group. At 24 hours, all urinary aminopeptidase activities and albuminuria were significantly increased in the cisplatin 7 mg/kg treated group. Aminopeptidase urinary activities correlated (p0.259 with plasma creatinine, creatinine clearance and/or kidney weight/body weight ratio at the end of the experiment and they could be considered as predictive biomarkers of renal injury severity. ROC-AUC analysis was made to study their sensitivity and specificity to distinguish between treated and untreated rats at day 1. All aminopeptidase activities showed an AUC>0.633. We conclude that Ala, Cys, Glu and AspAp enzymatic activities are early and predictive urinary biomarkers of the renal dysfunction induced by cisplatin. These determinations can be very useful in the prognostic and diagnostic of renal dysfunction in preclinical research and clinical practice.

  5. A novel dipeptidyl aminopeptidase in rat brain membranes. Its isolation, purification, and characterization.

    Science.gov (United States)

    Hui, K S

    1988-05-15

    A new type of dipeptidyl aminopeptidase, which releases basic aminoacyl dipeptides from the NH2-terminal end of oligopeptides, was purified about 2100-fold with 6.8% recovery from rat brain membranes by column chromatography on Cellex D, Arg-Tyr-AH-Sepharose 4B, hydroxylapatite, and Sephadex G-75, after the membranes were solubilized with Nonidet P-40. Activity was assayed by high performance liquid chromatography (HPLC) using Arg0-Met5-enkephalin (Arg0-enk)* as substrate in the presence of bestatin, thiorphan, and captopril. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme is apparently homogeneous with a mass of 64,000 daltons. This thiol enzyme is optimally active at pH 7 and is selectively activated by Mn(II). It loses 94% of its activity after EDTA treatment and can be reactivated by Mn(II), Co(II), and Zn(II). It splits Arg0-enk into equimolar amounts of Arg-Tyr and Gly-Gly-Phe-Met with a Km of 100 microM, and Vmax of 3.8 mumol/mg of protein/min. Dipeptidyl aminopeptidase does not hydrolyze model substrates for dipeptidyl aminopeptidases I, II, III, and IV, aminoacyl beta-naphthylamides, actin, desmin, tubulin, glial fibrillary acidic protein, and cytoskeletal neurofilament proteins. The enzyme is insensitive to puromycin, but is inhibited by several neuropeptides. Angiotensin III is the most potent with a Ki of 0.3 microM. Substrate specificity, pH optimum, molecular weight, activators, and catalytic site demonstrate that this enzyme is distinct from dipeptidyl aminopeptidases previously described.

  6. 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Danielsen, E.M. (Univ. of Cophenhagen (Denmark))

    1990-01-09

    The pig intestinal brush border enzymes aminopeptidase and lactase-phlorizin hydrolase are present in the microvilla membrane as homodimers. Dimethyl adipimidate was used to cross-link the two ({sup 35}S)methionine-labeled brush border enzymes from cultured mucosal 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 seems to occur in different organelles of the enterocyte.

  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. 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

  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. 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...... for the transport of aminopeptidase N to its final destination. A different type of processing was observed to take place in the presence of swainsonine, resulting in a considerable increase in apparent Mr (from 140000 to 160000). This processing could not be ascribed to N-linked glycosylation, since treatment...

  13. 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.

  14. Essential Role for an M17 Leucine Aminopeptidase in Encystation of Acanthamoeba castellanii.

    Science.gov (United States)

    Lee, Yu-Ran; Na, Byoung-Kuk; Moon, Eun-Kyung; Song, Su-Min; Joo, So-Young; Kong, Hyun-Hee; Goo, Youn-Kyoung; Chung, Dong-Il; Hong, Yeonchul

    2015-01-01

    Encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions such as starvation, low temperatures, and exposure to biocides. During encystation, a massive turnover of intracellular components occurs, and a large number of organelles and proteins are degraded by proteases. Previous studies with specific protease inhibitors have shown that cysteine and serine proteases are involved in encystation of Acanthamoeba, but little is known about the role of metalloproteases in this process. Here, we have biochemically characterized an M17 leucine aminopeptidase of Acanthamoeba castellanii (AcLAP) and analyzed its functional involvement in encystation of the parasite. Recombinant AcLAP shared biochemical properties such as optimal pH, requirement of divalent metal ions for activity, substrate specificity for Leu, and inhibition profile by aminopeptidase inhibitors and metal chelators with other characterized M17 family LAPs. AcLAP was highly expressed at a late stage of encystation and mainly localized in the cytoplasm of A. castellanii. Knockdown of AcLAP using small interfering RNA induced a decrease of LAP activity during encystation, a reduction of mature cyst formation, and the formation of abnormal cyst walls. In summary, these results indicate that AcLAP is a typical M17 family enzyme that plays an essential role during encystation of Acanthamoeba.

  15. Essential Role for an M17 Leucine Aminopeptidase in Encystation of Acanthamoeba castellanii.

    Directory of Open Access Journals (Sweden)

    Yu-Ran Lee

    Full Text Available Encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions such as starvation, low temperatures, and exposure to biocides. During encystation, a massive turnover of intracellular components occurs, and a large number of organelles and proteins are degraded by proteases. Previous studies with specific protease inhibitors have shown that cysteine and serine proteases are involved in encystation of Acanthamoeba, but little is known about the role of metalloproteases in this process. Here, we have biochemically characterized an M17 leucine aminopeptidase of Acanthamoeba castellanii (AcLAP and analyzed its functional involvement in encystation of the parasite. Recombinant AcLAP shared biochemical properties such as optimal pH, requirement of divalent metal ions for activity, substrate specificity for Leu, and inhibition profile by aminopeptidase inhibitors and metal chelators with other characterized M17 family LAPs. AcLAP was highly expressed at a late stage of encystation and mainly localized in the cytoplasm of A. castellanii. Knockdown of AcLAP using small interfering RNA induced a decrease of LAP activity during encystation, a reduction of mature cyst formation, and the formation of abnormal cyst walls. In summary, these results indicate that AcLAP is a typical M17 family enzyme that plays an essential role during encystation of Acanthamoeba.

  16. Overexpression, purification and biochemical characterization of the wound-induced leucine aminopeptidase of tomato.

    Science.gov (United States)

    Gu, Y Q; Holzer, F M; Walling, L L

    1999-08-01

    Wounding of tomato leaves results in the accumulation of an exoprotease called leucine aminopeptidase (LAP-A). While the expression of LapA genes are well characterized, the specificity of the LAP-A enzyme has not been studied. The LAP-A preprotein and mature polypeptide were overexpressed in Escherichia coli. PreLAP-A was not processed and was inactive accumulating in inclusion bodies. In contrast, 55-kDa mature LAP-A subunits assembled into an active, 357-kDa enzyme in E. coli. LAP-A from E. coli cultures was purified to apparent homogeneity and characterized relative to its animal (porcine LAP) and prokaryotic (E. coli PepA) homologues. Similar to the porcine and E. coli enzymes, the tomato LAP-A had high temperature and pH optima. Mn2+ was a strong activator for all three enzymes, while chelators, zinc ion, and the slow-binding aminopeptidase inhibitors (amastatin and bestatin) strongly inhibited activities of all three LAPs. The substrate specificities of porcine, E. coli and tomato LAPs were determined using amino-acid-p-nitroanilide and -beta-naphthylamide substrates. The tomato LAP-A preferentially hydrolyzed substrates with N-terminal Leu, Met and Arg residues. LAP-A had substantially lower levels of activity on other chromogenic substrates. Several differences in substrate specificities for the animal, plant and prokaryotic enzymes were noted.

  17. Phase I/II Clinical Study of Tosedostat, an Inhibitor of Aminopeptidases, in Patients With Acute Myeloid Leukemia and Myelodysplasia

    NARCIS (Netherlands)

    Lowenberg, Bob; Morgan, Gareth; Ossenkoppele, Gert J.; Burnett, Alan K.; Zachee, Pierre; Duehrsen, Ulrich; Dierickx, Daan; Mueller-Tidow, Carsten; Sonneveld, Pieter; Krug, Utz; Bone, Elisabeth; Flores, Nicolas; Richardson, Alison F.; Hooftman, Leon; Jenkins, Chris; Zweegman, Sonja; Davies, Faith

    2010-01-01

    Purpose To identify the maximum-tolerated dose (MTD) and to evaluate the antileukemic activity of tosedostat (formerly CHR-2797), an orally bioavailable aminopeptidase inhibitor. Patients and Methods In phase I, the MTD of once daily oral doses of tosedostat in hematologic malignancies was defined.

  18. 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.

  19. 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

  20. 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

  1. The Activity of a Hexameric M17 Metallo-Aminopeptidase Is Associated With Survival of Mycobacterium tuberculosis

    Science.gov (United States)

    Correa, Andre F.; Bastos, Izabela M. D.; Neves, David; Kipnis, Andre; Junqueira-Kipnis, Ana P.; de Santana, Jaime M.

    2017-01-01

    Mycobacterium tuberculosis is one of the most prevalent human pathogens causing millions of deaths in the last years. Moreover, tuberculosis (TB) treatment has become increasingly challenging owing to the emergence of multidrug resistant M. tuberculosis strains. Thus, there is an immediate need for the development of new anti-TB drugs. Proteases appear to be a promising approach and may lead to shortened and effective treatments for drug-resistant TB. Although the M. tuberculosis genome predicts more than 100 genes encoding proteases, only a few of them have been studied. Aminopeptidases constitute a set of proteases that selectively remove amino acids from the N-terminus of proteins and peptides and may act as virulence factors, essential for survival and maintenance of many microbial pathogens. Here, we characterized a leucine aminopeptidase of M. tuberculosis (MtLAP) as a cytosolic oligomeric metallo-aminopeptidase. Molecular and enzymatic properties lead us to classify MtLAP as a typical member of the peptidase family M17. Furthermore, the aminopeptidase inhibitor bestatin strongly inhibited MtLAP activity, in vitro M. tuberculosis growth and macrophage infection. In murine model of TB, bestatin treatment reduced bacterial burden and lesion in the lungs of infected mice. Thus, our data suggest that MtLAP participates in important metabolic pathways of M. tuberculosis necessary for its survival and virulence and consequently may be a promising target for new anti-TB drugs.

  2. 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.

  3. 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.

  4. Aminopeptidase N (APN/CD13 inhibitor, Ubenimex, enhances radiation sensitivity in human cervical cancer

    Directory of Open Access Journals (Sweden)

    Nawa Akihiro

    2008-03-01

    Full Text Available Abstract Background Radiotherapy can be used to treat all stages of cervical cancer. For improving local control via radiotherapy, it is important to use additional antitumor agents. Aminopeptidase N (APN/CD13, a 150-kDa metalloproteinase, is a multifunctional cell surface aminopeptidase with ubiquitous expression. Recent studies have suggested that APN/CD13 plays an important role in tumor progression in several human malignancies. Methods We investigated whether the suppression of APN/CD13 using Ubenimex, an inhibitor of APN/CD13 activity, may affect tumor radiosensitivity in cervical cancer cells both in vitro and in vivo. Cell surface APN/CD13 activity in HeLa cells was calculated using alanine-p-nitroanilido as a substrate. For colony formation assays, single-dose radiation and/or Ubenimex were administered to each dish of HeLa cells, and these dishes were cultured for 14 days. Molecular changes of apoptosis were determined by Western blot. Apoptosis was evaluated by Annexin-V PI staining (flow cytometry analysis and the Tunel method. Moreover, we investigated the effect of combining Ubenimex and low-dose radiation on tumor growth using nude mice. Results We demonstrated that Ubenimex enhanced the effectiveness of radiotherapy, acting as a radiosensitizer both in vitro and in vivo. In colony formation assays, a significant decline in clonogenic survival was observed in Ubenimex-treated cells. Mice treated with a combination of radiation and Ubenimex showed a significant prolongation of the tumor-doubling time compared with the control, Ubenimex, or radiation-alone groups. We also showed that ubenimex enhanced radiation-induced apoptosis in vitro and in vivo. Conclusion Although further studies are needed, this report suggests that Ubeniemx acts as a radiosensitizer in cervical cancer treatment, and that the inhibition of APN/CD13 activity may represent a new approach for improving the therapeutic efficacy of radiotherapy for uterine

  5. 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.

  6. 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.

  7. 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.

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. 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.

  16. Selective targeting of the conserved active site cysteine of Mycobacterium tuberculosis methionine aminopeptidase with electrophilic reagents.

    Science.gov (United States)

    Reddi, Ravikumar; Arya, Tarun; Kishor, Chandan; Gumpena, Rajesh; Ganji, Roopa J; Bhukya, Supriya; Addlagatta, Anthony

    2014-09-01

    Methionine aminopeptidases (MetAPs) cleave initiator methionine from ~ 70% of the newly synthesized proteins in every living cell, and specific inhibition or knockdown of this function is detrimental. MetAPs are metalloenzymes, and are broadly classified into two subtypes, type I and type II. Bacteria contain only type I MetAPs, and the active site of these enzymes contains a conserved cysteine. By contrast, in type II enzymes the analogous position is occupied by a conserved glycine. Here, we report the reactivity of the active site cysteine in a type I MetAP, MetAP1c, of Mycobacterium tuberculosis (MtMetAP1c) towards highly selective cysteine-specific reagents. The authenticity of selective modification of Cys105 of MtMetAP1c was established by using site-directed mutagenesis and crystal structure determination of covalent and noncovalent complexes. On the basis of these observations, we propose that metal ions in the active site assist in the covalent modification of Cys105 by orienting the reagents appropriately for a successful reaction. These studies establish, for the first time, that the conserved cysteine of type I MetAPs can be targeted for selective inhibition, and we believe that this chemistry can be exploited for further drug discovery efforts regarding microbial MetAPs.

  17. 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.

  18. Identification of the FTBL protein of Sumner and Dounce as a leucine aminopeptidase.

    Science.gov (United States)

    Dounce, A L; Allen, P Z

    1987-08-15

    The crystalline beef liver protein of Sumner and Dounce (A. L. Dounce, P. Z. Allen, and G. A. Mourtzikos (1978) Arch. Biochem. Biophys. 188, 251-265) termed FTBL (football) protein because of the shape of its crystals, has been identified as a crystalline leucine aminopeptidase (LAP), on the basis of its high specific LAP activity and coincidence of its N terminal amino acid sequence (30 amino acids) with that of beef eye lens LAP. Amino acid analyses of the two proteins are also in reasonable agreement when based on the exact monomer molecular weight of beef eye lens protein obtained by the van Loon group ((1982) J. Biol. Chem. 257, 7077-7081). Our previously published monomer molecular weight of the FTBL protein was 25% too high, leading to the erroneous conclusion that the beef liver FTBL-LAP protein was a tetramer rather than a hexamer, as found by the van Loon group for beef lens LAP. The present report, taken together with our first paper on the FTBL protein establishes that the FTBL-LAP protein has been isolated from beef kidney and beef spleen as well as from beef liver. We now find that the properties of FTBL-LAP protein indicate that it is the same protein as beef eye lens LAP. The cellular and intracellular distributions of the FTBL-LAP protein have been considered in our first publication on the FTBL protein.

  19. 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.

  20. Fungal Mimicry of a Mammalian Aminopeptidase Disables Innate Immunity and Promotes Pathogenicity.

    Science.gov (United States)

    Sterkel, Alana K; Lorenzini, Jenna L; Fites, J Scott; Subramanian Vignesh, Kavitha; Sullivan, Thomas D; Wuthrich, Marcel; Brandhorst, Tristan; Hernandez-Santos, Nydiaris; Deepe, George S; Klein, Bruce S

    2016-03-09

    Systemic fungal infections trigger marked immune-regulatory disturbances, but the mechanisms are poorly understood. We report that the pathogenic yeast of Blastomyces dermatitidis elaborates dipeptidyl-peptidase IVA (DppIVA), a close mimic of the mammalian ectopeptidase CD26, which modulates critical aspects of hematopoiesis. We show that, like the mammalian enzyme, fungal DppIVA cleaved C-C chemokines and GM-CSF. Yeast producing DppIVA crippled the recruitment and differentiation of monocytes and prevented phagocyte activation and ROS production. Silencing fungal DppIVA gene expression curtailed virulence and restored recruitment of CCR2(+) monocytes, generation of TipDC, and phagocyte killing of yeast. Pharmacological blockade of DppIVA restored leukocyte effector functions and stemmed infection, while addition of recombinant DppIVA to gene-silenced yeast enabled them to evade leukocyte defense. Thus, fungal DppIVA mediates immune-regulatory disturbances that underlie invasive fungal disease. These findings reveal a form of molecular piracy by a broadly conserved aminopeptidase during disease pathogenesis.

  1. Hormonal regulation of dipeptidyl-aminopeptidase I activity in cultured human fibroblasts.

    Science.gov (United States)

    Davis, M H

    1987-05-01

    Human male fibroblasts, cell line GM2987, were grown in 10% Nu-Serum or fetal bovine serum. Dipeptidyl-aminopeptidase I (DAP-I) activity was higher in cells grown with Nu-Serum and cells grown in 10% fetal bovine serum purchased from Grand Island Biological Company (GIBCO) and lower in cells grown in 10% fetal bovine serum obtained from Sterile Systems, Inc. (Hyclone). The addition of 0.3 microM cortisol to all three types of sera resulted in cells that had similar levels of DAP-I activity (maximum of 800-900 nmol of beta-naphthylamine released from glycyl-L-phenylanine-beta-naphthylamine per hour per milligram of cellular protein). The addition of cortisol to Hyclone fetal bovine serum increased the DAP-I levels by up to threefold with a half-maximal response occurring at 30 nM cortisol. Triiodothyronine also could increase DAP-I levels, but only between 1.5- and 2.0-fold. Testosterone propionate increased DAP-I levels by 1.4-fold. These changes in growth media and hormones had little effect on other lysosomal enzymes or the growth characteristics of the cells.

  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. T cell responses affected by aminopeptidase N (CD13)-mediated trimming of major histocompatibility complex class II-bound peptides

    DEFF Research Database (Denmark)

    Larsen, S L; Pedersen, L O; Buus, S;

    1996-01-01

    the exopeptidase Aminopeptidase N (APN, CD13) as one of the enzymes involved in the observed cell-surface antigen processing. The NH2-terminal end of the longer peptide could, even while bound to major histocompatibility complex (MHC) class II molecules, be digested by APN with dramatic consequences for T cell...... antigen recognition. This could be demonstrated both in cell-free systems using purified reagents and in cellular systems. Thus, MHC class II and APN may act in concert to generate the final T cell epitopes....

  4. Biosynthesis of intestinal microvillar proteins. Translational evidence in vitro that aminopeptidase N is synthesized as a Mr-115000 polypeptide

    DEFF Research Database (Denmark)

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

    1982-01-01

    was immunopurified from the translation mixture and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It was found to have an apparent Mr of 115000 regardless of whether the translation was performed in the absence or presence of proteinase inhibitors. This result contradicts the possibility......A crude RNA fraction, prepared from pig small intestine, was found to be more efficient than a fraction enriched in polyadenylated RNA in directing translation of polypeptides with Mr greater than 100000 in a rabbit reticulocyte lysate system. Aminopeptidase N (EC 3.4.11.2) synthesized in vitro...

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. 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.

  8. 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

    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......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...... in the gut lumen of pancreatic proteases. In neonatal animals, the level of mRNA for lactase-phlorizin hydrolase in both proximal and distal regions of the intestine was of the same magnitude as in the proximal jejunum of the post-weaned pigs. Our results point to two mechanisms that affect the expression...

  9. RNAi in the striped stem borer, Chilo suppressalis, establishes a functional role for aminopeptidase N in Cry1Ab intoxication.

    Science.gov (United States)

    Wang, X Y; Du, L X; Liu, C X; Gong, L; Han, L Z; Peng, Y F

    2017-02-01

    The striped stem borer, Chilo suppressalis, is a major target pest of transgenic rice expressing the Cry1Ab protein from the bacterium Bacillus thuringiensis (Bt) in China. Evolution of resistance in this pest is a major threat to the durability of Bt rice. Since Bt exerts its activity through binding to specific receptors in the midgut of target insects, identification of functional Cry1Ab receptors in the midgut of C. suppressalis larvae is crucial to evaluate potential resistance mechanisms and develop effective strategies for delaying insect resistance. In this work, we identified the putative Cry1Ab toxin-binding protein, aminopeptidase-N (APN), in the midgut of C. suppressalis by ligand blot and mass spectrometry. After cloning the full-length cDNAs encoding APN isoforms from the C. suppressalis larval midgut, we studied their spatiotemporal expression in different gut tissues and developmental stages. Furthermore, RNA interference (RNAi) against C. suppressalis aminopeptidases (CsAPNs) was employed to illustrate a functional role for CsAPNs in Cry1Ab toxicity to C. suppressalis larvae using injection and oral delivery of Stealth™ siRNA. Down-regulating the expression of CsAPNs by RNAi was closely associated with reduced susceptibility of C. suppressalis to Cry1Ab. These data provide the first direct evidence that CsAPNs participate in the mode of Cry1Ab action and may act as the functional receptor of Cry1A in C. suppressalis larvae.

  10. 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.

  11. Cloning of the pig aminopeptidase N gene. Identification of possible regulatory elements and the exon distribution in relation to the membrane-spanning region

    DEFF Research Database (Denmark)

    Sjöström, H; Norén, O; Olsen, Jørgen

    1989-01-01

    We have isolated four lambda-phages covering the complete pig aminopeptidase N/CD13 gene. The sequence of 2.85 kbp encompasses 1.18 kbp of the 5' upstream region and 1.67 kbp of the structural gene. In the promoter region we find a TATA box and potential binding sites for CTF-1/NF-1 and AP-2...

  12. 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...

  13. 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

  14. 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

  15. 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

  16. Chemical shift assignments of zinc finger domain of methionine aminopeptidase 1 (MetAP1) from Homo sapiens.

    Science.gov (United States)

    Rachineni, Kavitha; Arya, Tarun; Singarapu, Kiran Kumar; Addlagatta, Anthony; Bharatam, Jagadeesh

    2015-10-01

    Methionine aminopeptidase Type I (MetAP1) cleaves the initiator methionine from about 70 % of all newly synthesized proteins in almost every living cell. Human MetAP1 is a two domain protein with a zinc finger on the N-terminus and a catalytic domain on the C-terminus. Here, we report the chemical shift assignments of the amino terminal zinc binding domain (ZBD) (1-83 residues) of the human MetAP1 derived by using advanced NMR spectroscopic methods. We were able to assign the chemical shifts of ZBD of MetAP1 nearly complete, which reveal two helical fragments involving residues P44-L49 (α1) and Q59-K82 (α2). The protein structure unfolds upon complex formation with the addition of 2 M excess EDTA, indicated by the appearance of amide resonances in the random coil chemical shift region of (15)NHSQC spectrum.

  17. Identification of the molecular basis of inhibitor selectivity between the human and streptococcal type I methionine aminopeptidases.

    Science.gov (United States)

    Arya, Tarun; Reddi, Ravikumar; Kishor, Chandan; Ganji, Roopa Jones; Bhukya, Supriya; Gumpena, Rajesh; McGowan, Sheena; Drag, Marcin; Addlagatta, Anthony

    2015-03-12

    The methionine aminopeptidase (MetAP) family is responsible for the cleavage of the initiator methionine from newly synthesized proteins. Currently, there are no small molecule inhibitors that show selectivity toward the bacterial MetAPs compared to the human enzyme. In our current study, we have screened 20 α-aminophosphonate derivatives and identified a molecule (compound 15) that selectively inhibits the S. pneumonia MetAP in low micromolar range but not the human enzyme. Further bioinformatics, biochemical, and structural analyses suggested that phenylalanine (F309) in the human enzyme and methionine (M205) in the S. pneumonia MetAP at the analogous position render them with different susceptibilities against the identified inhibitor. X-ray crystal structures of various inhibitors in complex with wild type and F309M enzyme further established the molecular basis for the inhibitor selectivity.

  18. 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.

  19. TsPAP1 encodes a novel plant prolyl aminopeptidase whose expression is induced in response to suboptimal growth conditions

    Energy Technology Data Exchange (ETDEWEB)

    Szawlowska, Urszula; Grabowska, Agnieszka [Department of Biochemistry, Warsaw University of Life Sciences - SGGW, Nowoursynowska 159, 02-776 Warsaw (Poland); Zdunek-Zastocka, Edyta, E-mail: edyta_zdunek_zastocka@sggw.pl [Department of Biochemistry, Warsaw University of Life Sciences - SGGW, Nowoursynowska 159, 02-776 Warsaw (Poland); Bielawski, Wieslaw [Department of Biochemistry, Warsaw University of Life Sciences - SGGW, Nowoursynowska 159, 02-776 Warsaw (Poland)

    2012-03-02

    Highlights: Black-Right-Pointing-Pointer A cDNA encoding a novel plant prolyl aminopeptidase, TsPAP1, was obtained from triticale. Black-Right-Pointing-Pointer The cloned TsPAP1 cDNA is 1387 bp long and encodes a protein of 390 amino acids. Black-Right-Pointing-Pointer The deduced TsPAP1 protein revealed characteristics of the monomeric bacterial PAPs. Black-Right-Pointing-Pointer The TsPAP1 mRNA level increased under drought, salinity and in the presence of metal ions. -- Abstract: A triticale cDNA encoding a prolyl aminopeptidase (PAP) was obtained by RT-PCR and has been designated as TsPAP1. The cloned cDNA is 1387 bp long and encodes a protein of 390 amino acids with a calculated molecular mass of 43.9 kDa. The deduced TsPAP1 protein exhibits a considerable sequence identity with the biochemically characterized bacterial and fungal PAP proteins of small molecular masses ({approx}35 kDa). Moreover, the presence of conserved regions that are characteristic for bacterial monomeric PAP enzymes (the GGSWG motif, the localization of the catalytic triad residues and the segment involved in substrate binding) has also been noted. Primary structure analysis and phylogenetic analysis revealed that TsPAP1 encodes a novel plant PAP protein that is distinct from the multimeric proteins that have thus far been characterized in plants and whose counterparts have been recognized only in bacteria and fungi. A significant increase in the TsPAP1 transcript level in the shoots of triticale plants was observed under drought and saline conditions as well as in the presence of cadmium and aluminium ions in the nutrient medium. This paper is the first report describing changes in the transcript levels of any plant PAP in response to suboptimal growth conditions.

  20. 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

  1. 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.

  2. Vaccination with cathepsin L proteinases and with leucine aminopeptidase induces high levels of protection against fascioliasis in sheep.

    Science.gov (United States)

    Piacenza, L; Acosta, D; Basmadjian, I; Dalton, J P; Carmona, C

    1999-04-01

    The potential of different parasite proteinases for use as vaccine candidates against fascioliasis in sheep was studied by vaccinating animals with the cathepsin L proteinases CL1 and CL2 and with leucine aminopeptidase (LAP) purified from adult flukes. In the first trial, sheep were immunized with CL1 or CL2 and the mean protection levels obtained were 33 and 34%, respectively. Furthermore, a significant reduction in egg output was observed in sheep vaccinated either with CL1 (71%) or with CL2 (81%). The second trial was performed to determine the protective potential of the two cathepsin L proteinases assayed together, as well as in combination with LAP, and of LAP alone. The combination of CL1 and CL2 induced higher levels of protection (60%) than those produced when these enzymes were administered separately. Those sheep that received the cocktail vaccine including CL1, CL2, and LAP were significantly protected (78%) against metacercarial challenge, but vaccination with LAP alone elicited the highest level of protection (89%). All vaccine preparations induced high immunoglobulin G titers which were boosted after the challenge infection, but no correlations between antibody titers and worm burdens were found. However, the sera of those animals vaccinated with LAP contained LAP-neutralizing antibodies. Reduced liver damage, as assessed by the level of the liver enzyme gamma-glutamyl transferase, was observed in the groups vaccinated with CL1, CL2, and LAP or with LAP alone.

  3. Construction and evaluation of a chimeric protein made from Fasciola hepatica leucine aminopeptidase and cathepsin L1.

    Science.gov (United States)

    Hernández-Guzmán, K; Sahagún-Ruiz, A; Vallecillo, A J; Cruz-Mendoza, I; Quiroz-Romero, H

    2016-01-01

    Leucine aminopeptidase (LAP) and cathepsin L1 (CL1) are important enzymes for the pathogenesis and physiology of Fasciola hepatica. These enzymes were analysed in silico to design a chimeric protein containing the most antigenic sequences of LAP (GenBank; AAV59016.1; amino acids 192-281) and CL1 (GenBank CAC12806.1; amino acids 173-309). The cloned 681-bp chimeric fragment (rFhLAP-CL1) contains 270 bp from LAP and 411 bp from CL1, comprising three epitopes, DGRVVHLKY (amino acids 54-62) from LAP, VTGYYTVHSGSEVELKNLV (amino acids 119-137) and YQSQTCLPF (amino acids 161-169) from CL1. The ~25 kDa rFhLAP-CL1 chimeric protein was expressed from the pET15b plasmid in the Rosetta (DE3) Escherichia coli strain. The chimeric protein rFhLAP-CL1, which showed antigenic and immunogenic properties, was recognized in Western blot assays using F. hepatica-positive bovine sera, and induced strong, specific antibody responses following immunization in rabbits. The newly generated chimeric protein may be used as a diagnostic tool for detection of antibodies against F. hepatica in bovine sera and as an immunogen to induce protection against bovine fasciolosis.

  4. Gene cloning and biochemical characterization of eryngase, a serine aminopeptidase of Pleurotus eryngii belonging to the family S9 peptidases.

    Science.gov (United States)

    Arima, Jiro; Tokai, Shota; Chiba, Masanori; Ichiyanagi, Tsuyoshi; Yabuta, Yukinori; Mori, Nobuhiro; Aimi, Tadanori

    2014-01-01

    Pleurotus eryngii serine aminopeptidase that has peptide bond formation activity, redesignated as eryngase, was cloned and expressed. Eryngase has a family S9 peptidase unit in the C-terminal region having a catalytic triad of Ser, Asp, and His. In the phylogenetic relations among the subfamilies of family S9 peptidase (S9A, prolyl oligopeptidase; S9B, dipeptidyl peptidase; S9C, acylaminoacyl peptidase; S9D, glutamyl endopeptidase), eryngase existed alone in the neighbor of S9C subfamily. Mutation of the active site Ser524 of the eryngase with Ala eliminated its catalytic activity. In contrast, S524C mutant maintained low catalytic activity. Investigation of aminolysis activity using l-Phe-NH2 as a substrate showed that S524C mutant exhibited no hydrolysis reaction but synthesized a small amount of l-Phe-l-Phe-NH2 by the catalysis of aminolysis. In contrast, wild-type eryngase hydrolyzed the product of aminolysis l-Phe-l-Phe-NH2. Results show that the S524C mutant preferentially catalyzed aminolysis when on an l-Phe-NH2 substrate.

  5. 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.

  6. 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.

  7. 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.

  8. Early treatment with fumagillin, an inhibitor of methionine aminopeptidase-2, prevents Pulmonary Hypertension in monocrotaline-injured rats.

    Directory of Open Access Journals (Sweden)

    Daniel J Kass

    Full Text Available Pulmonary Hypertension (PH is a pathophysiologic condition characterized by hypoxemia and right ventricular strain. Proliferation of fibroblasts, smooth muscle cells, and endothelial cells is central to the pathology of PH in animal models and in humans. Methionine aminopeptidase-2 (MetAP2 regulates proliferation in a variety of cell types including endothelial cells, smooth muscle cells, and fibroblasts. MetAP2 is inhibited irreversibly by the angiogenesis inhibitor fumagillin. We have previously found that inhibition of MetAP2 with fumagillin in bleomycin-injured mice decreased pulmonary fibrosis by selectively decreasing the proliferation of lung myofibroblasts. In this study, we investigated the role of fumagillin as a potential therapy in experimental PH. In vivo, treatment of rats with fumagillin early after monocrotaline injury prevented PH and right ventricular remodeling by decreasing the thickness of the medial layer of the pulmonary arteries. Treatment with fumagillin beginning two weeks after monocrotaline injury did not prevent PH but was associated with decreased right ventricular mass and decreased cardiomyocyte hypertrophy, suggesting a direct effect of fumagillin on right ventricular remodeling. Incubation of rat pulmonary artery smooth muscle cells (RPASMC with fumagillin and MetAP2-targeting siRNA inhibited proliferation of RPASMC in vitro. Platelet-derived growth factor, a growth factor that is important in the pathogenesis of PH and stimulates proliferation of fibroblasts and smooth muscle cells, strongly increased expression of MetP2. By immunohistochemistry, we found that MetAP2 was expressed in the lesions of human pulmonary arterial hypertension. We propose that fumagillin may be an effective adjunctive therapy for treating PH in patients.

  9. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-07-01

    N0 -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 ¼ 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.

  11. Biosynthesis of intestinal microvillar proteins. The intracellular transport of aminopeptidase N and sucrase-isomaltase occurs at different rates pre-Golgi but at the same rate post-Golgi

    DEFF Research Database (Denmark)

    Danielsen, E M; Cowell, G M

    1985-01-01

    The kinetics of processing and microvillar expression of aminopeptidase N (EC 3.4.11.2) and sucrose alpha-D-glucohydrolase-oligo-1,6-glucosidase (sucrase-isomaltase, EC 3.2.1.48 and EC 3.2.1.10) were compared by labelling of pig small intestinal mucosal explants with [35S]methionine. The conversi...... from transient (high mannose glycosylated) to mature (complex glycosylated) form was 1.7-times slower for sucrase-isomaltase than for aminopeptidase N, indicating a slower rate of migration from the rough endoplasmic reticulum to the Golgi complex. Likewise, sucrase-isomaltase appeared...... in the microvillar fraction at a slower rate than aminopeptidase N. The relative pool sizes of mature and transient forms of both enzymes in intracellular membranes (Mg2+-precipitated fraction) were determined to obtain information on the relative time, spent pre- and post-Golgi, respectively, prior to microvillar...... expression. This ratio was 0.24 +/- 0.06 (mean +/- SD) for sucrase-isomaltase as compared to 0.40 +/- 0.04 (mean +/- SD) for aminopeptidase N. Considering the slower rate of pre-Golgi transport for sucrase-isomaltase, this indicates that the two microvillar enzymes have rather similar if not identical rates...

  12. 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-

  13. 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.

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

    Directory of Open Access Journals (Sweden)

    Chitvan Khajuria

    Full Text Available 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.

  15. 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.

  16. 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

    revealed 1 g-atom of Ca/143000 g of protein. Two forms of the enzyme were purified: an amphipathic form solubilized from the membrane by Triton X-100 (detergent form) and a hydrophilic form released by incubation with trypsin (proteinase form). The detergent form exhibited charge-shift in crossed...... immunoelectrophoresis when anionic or cationic detergents were present. On gel filtration, mol.wts. of 350000--400000 and 270000 were calculated for the detergent and proteinase forms. Electron microscopy after negative staining of the proteinase form revealed a dimeric structure. Electrophoresis of either form...... in the presence of sodium dodecyl sulphate revealed four polypeptides with mobilities corresponding to apparent mol.wts. of 155000, 110000, 90000 and 45000. All four bands stained positively for carbohydrate. Pig serum possesses weak aminopeptidase A activity; immunological experiments showed it to be a similar...

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. Two cytosolic puromycin-sensitive aminopeptidase isozymes in chicken brain: molecular homology to brain-specific 14-3-3 protein.

    Science.gov (United States)

    Hui, K S; Saito, M; Hui, M; Saito, M; Lajtha, A; Yamamoto, K; Osawa, T

    1993-05-01

    Two puromycin-sensitive aminopeptidase isozymes (PSA-I and PSA-II) were isolated from chicken brain cytosol by ammonium sulfate fractionation followed by column chromatography on Cellex D and AH-Sepharose 4B and separated on Bio-Gel HTP. Each was purified to homogeneity on Sephadex G-200, Arg-Tyr-AH-Sepharose, Bio-Gel HTP, and preparative gel electrophoresis. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, PSA-I appeared to be a monomer with a molecular mass of 105 kDa, and PSA-II to be composed of two subunits of 25 kDa and 100 kDa. The tryptic maps of 100 kDa and 105 kDa protein in HPLC are different in peak frequency, height, and composition. The internal peptide sequence of PSA-I has a considerable homology to PSA-II. Both isozymes have repeated copies of common peptide segments and have no significant sequence homology to other peptidases and proteinases. These thio and Co(2+)-activated isozymes have a neutral pH optimum and are inhibited by puromycin and bestatin. PSA-II is more sensitive to trypsin and heat treatment, has a lower Km to Met-enkephalin, and is more active on Arg BNA and Pro BNA. Our results suggest that PSA-I and PSA-II derive from translation of two RNAs of a new gene family related to the brain-specific 14-3-3 protein.

  5. The dipeptidyl-aminopeptidase-like protein 6 is an integral voltage sensor-interacting beta-subunit of neuronal K(V)4.2 channels.

    Science.gov (United States)

    Dougherty, Kevin; Tu, Liwei; Deutsch, Carol; Covarrubias, Manuel

    2009-01-01

    Auxiliary beta-subunits dictate the physiological properties of voltage-gated K(+) (K(V)) channels in excitable tissues. In many instances, however, the underlying mechanisms of action are poorly understood. The dipeptidyl-aminopeptidase-like protein 6 (DPP6) is a specific beta-subunit of neuronal K(V)4 channels, which may promote gating through interactions between the single transmembrane segment of DPP6 and the channel's voltage sensing domain (VSD). A combination of gating current measurements and protein biochemistry (in-vitro translation and co-immunoprecipitations) revealed preferential physical interaction between the isolated K(V)4.2-VSD and DPP6. Significantly weaker interactions were detected between DPP6 and K(V)1.3 channels or the K(V)4.2 pore domain. More efficient gating charge movement resulting from a direct interaction between DPP6 and the K(V)4.2-VSD is unique among the known actions of K(V) channel beta-subunits. This study shows that the modular VSD of a K(V) channel can be directly regulated by transmembrane protein-protein interactions involving an extrinsic beta-subunit. Understanding these interactions may shed light on the pathophysiology of recently identified human disorders associated with mutations affecting the dpp6 gene.

  6. 冠状病毒的氨基肽酶N受体的研究进展%Review of aminopeptidase N as the receptor of coronavirus

    Institute of Scientific and Technical Information of China (English)

    陈杰; 黄小波; 张雨迪; 卿盈; 李亚青; 文心田; 曹三杰; 文翼平; 伍锐

    2016-01-01

    氨基肽酶N(Aminopeptidase N,APN)是一种锌离子依赖性膜结合Ⅱ型的金属跨膜糖蛋白,其广泛存在于人和哺乳动物的多种组织细胞中.APN是多种冠状病毒的黏附性受体,参与冠状病毒的粘附与侵袭,与病毒感染密切相关.本文对多种冠状病毒(HCoV-229E、FCoV、CCoV、TGEV、PEDV)的APN受体的结构、功能、抑制剂等研究进展进行了总结,重点介绍了其作为人和猪冠状病毒功能受体的进展.本文为深入研究和应用冠状病毒APN受体提供参考.

  7. 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.

  8. Affinity between TBC1D4 (AS160 phosphotyrosine-binding domain and insulin-regulated aminopeptidase cytoplasmic domain measured by isothermal titration calorimetry

    Directory of Open Access Journals (Sweden)

    SangYoun Park*, Keon Young Kim, Sunmin Kim & Young Seok Yu

    2012-06-01

    Full Text Available Uptake of circulating glucose into the cells happens via the insulin-mediated signalling pathway, which translocates the glucosetransporter 4 (GLUT4 vesicles from the intracellular compartmentto the plasma membrane. RabㆍGTPases are involvedin this vesicle trafficking, where RabㆍGTPase-activatingproteins (RabGAP enhance the GTP to GDP hydrolysis.TBC1D4 (AS160 and TBC1D1 are functional RabGAPs in theadipocytes and the skeletonal myocytes, respectively. Theseproteins contain two phosphotyrosine-binding domains (PTBsat the amino-terminus of the catalytic RabGAP domain. Thesecond PTB has been shown to interact with the cytoplasmicregion of the insulin-regulated aminopeptidase (IRAP of theGLUT4 vesicle. In this study, we quantitatively measured the∼μM affinity (KD between TBC1D4 PTB and IRAP using isothermaltitration calorimetry, and further showed that IRAP residues1-49 are the major region mediating this interaction. Wealso demonstrated that the IRAP residues 1-15 are necessarybut not sufficient for the PTB interaction.

  9. TNF-alpha, produced by feline infectious peritonitis virus (FIPV)-infected macrophages, upregulates expression of type II FIPV receptor feline aminopeptidase N in feline macrophages.

    Science.gov (United States)

    Takano, Tomomi; Hohdatsu, Tsutomu; Toda, Ayako; Tanabe, Maki; Koyama, Hiroyuki

    2007-07-20

    The pathogenicity of feline infectious peritonitis virus (FIPV) is known to depend on macrophage tropism, and this macrophage infection is enhanced by mediation via anti-S antibody (antibody-dependent enhancement, ADE). In this study, we found that TNF-alpha production was increased with viral replication in macrophages inoculated with a mixture of FIPV and anti-S antibody, and demonstrated that this culture supernatant had feline PBMC apoptosis-inducing activity. We also demonstrated that the expression level of the FIPV virus receptor, feline aminopeptidase N (fAPN), was increased in macrophages of FIP cats. For upregulation of TNF-alpha and fAPN in macrophages, viral replication in macrophages is necessary, and their expressions were increased by ADE of FIPV infection. It was demonstrated that a heat-resistant fAPN-inducing factor was present in the culture supernatant of FIPV-infected macrophages, and this factor was TNF-alpha: fAPN expression was upregulated in recombinant feline TNF-alpha-treated macrophages, and FIPV infectivity was increased in these macrophages. These findings suggested that FIPV replication in macrophages increases TNF-alpha production in macrophages, and the produced TNF-alpha acts and upregulates fAPN expression, increasing FIPV sensitivity.

  10. In Vitro and In Vivo Antimalarial Activity Assays of Seeds from Balanites aegyptiaca: Compounds of the Extract Show Growth Inhibition and Activity against Plasmodial Aminopeptidase

    Directory of Open Access Journals (Sweden)

    Peter Kusch

    2011-01-01

    Full Text Available Balanites aegyptiaca (Balanitaceae is a widely grown desert plant with multiuse potential. In the present paper, a crude extract from B. aegyptiaca seeds equivalent to a ratio of 1 : 2000 seeds to the extract was screened for antiplasmodial activity. The determined IC50 value for the chloroquine-susceptible Plasmodium falciparum NF54 strain was 68.26 g/L±3.5. Analysis of the extract by gas chromatography-mass spectrometry detected 6-phenyl-2(H-1,2,4-triazin-5-one oxime, an inhibitor of the parasitic M18 Aspartyl Aminopeptidase as one of the compounds which is responsible for the in vitro antiplasmodial activity. The crude plant extract had a of 2.35 g/L and showed a dose-dependent response. After depletion of the compound, a significantly lower inhibition was determined with a of 4.8 g/L. Moreover, two phenolic compounds, that is, 2,6-di-tert-butyl-phenol and 2,4-di-tert-butyl-phenol, with determined IC50 values of 50.29 M±3 and 47.82 M±2.5, respectively, were detected. These compounds may contribute to the in vitro antimalarial activity due to their antioxidative properties. In an in vivo experiment, treatment of BALB/c mice with the aqueous Balanite extract did not lead to eradication of the parasites, although a reduced parasitemia at day 12 p.i. was observed.

  11. Prognostic value of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and aminopeptidase N/CD13 in breast cancer patients.

    Science.gov (United States)

    Ranogajec, Irena; Jakić-Razumović, Jasminka; Puzović, Velibor; Gabrilovac, Jelka

    2012-06-01

    The aim of this study was to analyse the expression of matrix metalloproteinase-2(MMP-2), matrix metalloproteinase-9 (MMP-9) and aminopeptidase APN/CD13 in breast carcinoma samples, and their possible prognostic value in breast cancer patients. The expression of MMP-2, MMP-9 and APN/CD13 in tumor cells was analysed in 138 breast carcinomas by immunohistochemical staining of tumor cells using the semiquantitative method for the detection of cytoplasmic and membrane reaction in tumor cells as well as stromal cells positivity. MMP-2 was positive in tumor cells of 52.9% patients and in stromal cells of 74.6% patients, while MMP-9 positive tumor and stromal cells were found in 84.8 and 63.8% patients, respectively. Tumor cell APN/CD13 expression was found in 36.2% patients. Stromal cell MMP-2 expression correlated significantly with tumor size and neoangiogenesis. A positive correlation was also observed between tumor cell MMP-9 expression and hormone receptor status. Stromal cell coexpression of MMP-2/MMP-9 correlated significantly with tumor size. APN/CD13 expression in tumor cells significantly correlated with tumor type and neoangiogenesis. Overall survival was significantly shorter in patients with MMP-2, MMP-2/MMP-9 positive tumor cells, and tended to be shorter in patients with APN/CD13 positive tumor cells. Coexpression of MMP-2/MMP-9 in tumor cells was an independent risk factor for patient survival (OD = 13.9). Our results suggest that MMP-2, MMP-9, APN/CD13 expression and MMP-2/MMP-9 coexpression in combination with other standard prognostic factors can serve as a poor prognostic factor in the evaluation of breast cancer prognosis.

  12. 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.

  13. 阿奇霉素联合脾氨肽治疗小儿哮喘38例疗效观察%Effect Observation of Azithromycin Combined with Spleen Aminopeptidase for 38 Children with Asthma

    Institute of Scientific and Technical Information of China (English)

    2013-01-01

    Objective:To observe the clinical effect of azithromycin combined with spleen aminopeptidase for the treatment of asthma in children. Method:76 patients were randomly divided into two groups:test group and control group,38 cases in each group.The two groups were given conventional treatment.The control group was given 10 mg/kgod of azithromycine tablets. The test group was given spleen aminopeptidase on the base of control group. Comparing the clinical effects of the different groups in a 2-month course.Result:The total effective rate of test group was 92.1%,it was significantly higher than that of control group(P<0.05). Disappearance time of symptoms and signs and hospital stay of the test group were significantly shorter than the that of control group(P<0.05).Conclusion:The clinical effect of azithromycin combined with spleen aminopeptidase on the treatment for asthma in children is satisfactory.The patients recovery faster and less adverse reactions,is worthy of clinical use.%  目的:观察阿奇霉素联合脾氨肽治疗小儿哮喘的临床疗效和安全性。方法:将76例支气管哮喘患儿随机分为观察组和对照组各38例,两组均给予常规治疗,对照组给予阿奇霉素口服,10 mg/(kg·d);观察组在对照组用药基础上给予脾氨肽口服冻干粉,1支/d,一月后改为隔日1支;2个月后比较两组疗效。结果:观察组有效率92.1%,明显高于对照组的81.6%(P<0.05);观察组的症状体征消失时间和住院时间也明显优于对照组(P<0.05)。结论:阿奇霉素联合脾氨肽治疗小儿哮喘疗效显著,患儿恢复快且不良反应少,值得在临床推广。

  14. Analysis of the mechanism of antibody-dependent enhancement of feline infectious peritonitis virus infection: aminopeptidase N is not important and a process of acidification of the endosome is necessary.

    Science.gov (United States)

    Takano, Tomomi; Katada, Yukari; Moritoh, Saiko; Ogasawara, Mika; Satoh, Kumi; Satoh, Ryoichi; Tanabe, Maki; Hohdatsu, Tsutomu

    2008-04-01

    Infection of the monocyte/macrophage lineage with feline infectious peritonitis virus (FIPV) is enhanced in the presence of anti-FIPV antibodies (antibody-dependent enhancement or ADE). We investigated the following unclear points concerning ADE of FIPV infection: (i) involvement of the virus receptor, feline aminopeptidase N (fAPN), in ADE activity in FIPV infection; (ii) necessity of acidification of the endosome in cellular invasion of FIPV. Virus receptor-blocking experiments using anti-fAPN antibodies at 4 or 37 degrees C and experiments using fAPN-negative U937 cells revealed that fAPN is not involved in ADE of FIPV infection. Experiments using lysosomotropic agents clarified that acidification of the endosome is necessary for cellular invasion by FIPV, regardless of the presence or absence of antibodies. These findings may be very important for understanding the mechanism of ADE of FIPV infection.

  15. 水稻干尖线虫氨基肽酶基因克隆及功能初步分析%Cloning and Functional Analysis of Aminopeptidase Gene from Aphelenchoides besseyi

    Institute of Scientific and Technical Information of China (English)

    王步勇; 王峰; 李丹蕾; 马玲; 陈俏丽; 王博文

    2015-01-01

    To study the characteristics of the aminopeptidase gene in white-tip nematode,and to provide theory support for the prevention.According to the transcriptome resequencing,an aminopeptidase gene of A.besseyi was i-solated by RACE method,and designed as Ab-Ampep.Bioinformatic and in-situ hybridization were used for the gene analysis.The coding sequence was 2 787 bp including a 2 661 bp open reading frame (ORF),which encoded an 886 amino acid protein.The putative protein molecular weight was 1 01 .74 kDa and its theoretical isoelectric point was 5.78.The deduced amino acid sequence contains two conserved zinc-binding sites and a PepN Multi-domain.It belongs to GluZincin Superfamily,which contained the characteristic conserved sequence of HExxH.The multiple sequence alignment showed that the functional region was conserved,and the highest level of similarity was 50% to a aminopeptidase(ERG83308.1 )from Ascaris suum in GenBank.The phylogenetic tree constructed on the basis of amino acid sequences clearly showed that the relationship of Ab-Ampep was most intimate with aminopeptidase (ERG83308.1 )from A.suum.Protein structure analysis indicated that the main structural element in Ab-Ampep protein was α-Helix.In situ hybridization indicated that enhanced hybridization signals were observed in genital are-a,and the expression of Ab-Ampep was similarly in genital area of A.besseyi.Though the functional analysis of aminopeptidase gene,molecular technology or enzyme inhibitor was used to inhibit the expression of aminopeptid-ase.It may affect the nematode development and provide a new way for prevention.%为防治水稻干尖线虫病提供理论支持,研究了水稻干尖线虫氨基肽酶基因特征,并对其编码蛋白结构与功能进行分析。基于水稻干尖线虫转录组测序,并通过 RACE 技术,从水稻干尖线虫中克隆得到了一氨基肽酶基因 Ab-Ampep。生物信息学进行基因分析,原位杂交实验进行

  16. 小菜蛾中肠氨肽酶N2在昆虫细胞中的表达%Expression of aminopeptidase N (APN2) from Plutella xylostella larval midgut in Sf9 cells

    Institute of Scientific and Technical Information of China (English)

    王俊华; 周小毛; 吴青君; 王少丽; 谢文; 陈得峰; 徐宝云; 张友军

    2012-01-01

    利用昆虫细胞-杆状病毒表达系统(Bac-to-Bac baculovirus expression system)表达获得小菜蛾Plutella xylostella(L.)中肠氨肽酶N2( aminopeptidase N,APN2)蛋白,成功建立了该蛋白在昆虫细胞中的表达体系.将对Cry1Ac毒素敏感和已产生抗性的小菜蛾中肠APN2基因插入表达载体pFAST Bac HTB中,并在草地夜蛾Spodoptera frugiperda细胞系(Sf9)中进行了重组表达.聚丙烯酰胺凝胶电泳( SDS-PAGE)及蛋白免疫印迹技术(Western-blotting)结果显示,在107 kDa处有1条特异性蛋白条带.研究表明,小菜蛾中肠APN2基因可在Sf9细胞中成功表达,这为继续研究其功能及抗性的关系奠定了基础.%Using the insect cells-Bac-to-Bac baculovirus expression system to express APN2 (aminopeptidase N) protein, the APN2 expression system in Sf9 cells was successfully established. APN2 gene from susceptible and resistant Plutella xylostella (L. ) larval midgut were inserted into pFAST Bac HTB vector, expressed in insect cells Spodoptera frugiperda (Sf9) cells, respectively, with Bac-to-Bac baculovirus expression system. A 107 kDa protein was examined and confirmed by SDS-PAGE and Western-blotting. The results showed that P. Xylostella larval midgut APN2 gene was successfully expressed in Sf9 cells which provided a foundation for further investigation on function of APN2 and its relationship with Bt resistance.

  17. 评价亮氨酸氨基肽酶在甲状腺功能亢进中的诊断价值%Evaluation on diagnostic value of leucine aminopeptidase in hyperthyroidism

    Institute of Scientific and Technical Information of China (English)

    王静; 李治锋; 夏寿扬

    2015-01-01

    Objective To detect serum leucine aminopeptidase (LAP) level in the patients with hyperthyroidism for investiga‐ting the changes of LAP in the patients with thyroid dysfunction and its clinical significance .Methods 117 patients with hyperthy‐roidism were taken as the test group and 109 healthy people as the control group .The differences of various laboratory indexes were comparatively analyzed and the correlation between LAP with FT 3 ,FT4 and TSH was analyzed .117 patients with hyperthyroidism were divided into the liver damage group(52 cases) and non‐liver damage group(65 cases) and performed the LAP determination . Results The serum LAP ,FT3 ,FT4 and TSH levels in the hyperthyroidism whole group ,hyperthyroidism liver damage group and hyperthyroidism non‐liver damage group had statistically significant differences compared with those in the control group (P0 .05) .Conclusion The serum LAP content is closely related with the thyroid hormones and has certain clinical reference value for the diagnosis ,disease condition evaluation and medication .Lap can serve as an independent diagnostic indicator .%目的:检测甲状腺功能亢进患者血清亮氨酸氨基肽酶(leucine aminopeptidase ,LAP)指标,探讨LAP在甲状腺功能异常患者中的变化及其临床意义。方法选取117例甲状腺功能亢进(简称甲亢)患者作为试验组,109例健康者作为对照组。对比分析不同组间各项实验室指标的差异,并进行LAP和FT3、FT4、TSH的相关性分析。将117例甲亢患者分为肝损害组52例,肝未损害组65例,并进行LAP测定。结果甲亢整体组、甲亢肝损害组、甲亢肝未损害组LAP和FT3、FT4、TSH与正常对照组对比均有统计学差异(P<0.01),血清LAP与FT3、FT4成正相关,与TSH成负相关;甲亢肝损害组LAP和FT3、FT4、TSH与甲亢肝未损伤组对比无统计学差异(P>0.05)。结论血清LAP含量与甲状腺激素关系密切,对

  18. 棉铃虫中肠Bt毒素受体蛋白(APN)的分离与纯化%Isolation and Purification of Aminopeptidase N (APNs), the Main Receptor Proteins for Bacillus thuringiensis toxin in Midgut of Helicoverpa armigera Larva

    Institute of Scientific and Technical Information of China (English)

    梁革梅; 徐广; 王桂荣; 吴孔明; 郭予元

    2005-01-01

    利用Mg/EGTA方法,以不同的速度进行离心,成功地分离了棉铃虫(Bacillus thuringiensis)中肠刷状缘膜囊泡(BBMV0,BBMV中保留了大部分氨肽酶N(aminopeptidase N,APN)活性,BBMV中APN的特异活性较粗匀浆液相比提高了3.29倍;CHAPS3-[(3-chlor-amidopropyl)dimethylammonio]-1-propane sulphonate可以增加BBMV的溶解,加入1%(CHAPS)之后APN特异活性增加至5.21倍;利用磷酸酰肌醇特异性的磷脂酶(PI-PLC)能将APN从膜上解离下来,BBMV经PI-PLC处理后,SDS-PAGE电泳结果仅有120 kD的1条带;Mono-Q和FPLC结合的方法可以纯化部分APN,SDS-PAGE电泳结果显示,经Mono-Q柱处理后的BBMV中分子量约100 kD的蛋白条带加粗;棉铃虫3个抗性品系与敏感品系相比,APN特异活性差异不显著,说明棉铃虫对Bt产生抗性与APN活性的变化无关,抗性的产生可能与Bt毒素和APN结合能力的改变有关.

  19. 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.

  20. Inhibitory Mechanisms of Apigenin on Aminopeptidase N of Lung Cancer Cells%芹菜素对肺癌细胞氨肽酶N抑制作用机制的研究

    Institute of Scientific and Technical Information of China (English)

    周云峰; 罗飞; 付欢欢; 肖江卫; 廖娟

    2015-01-01

    目的:探讨芹菜素对肺癌细胞氨肽酶N活性的影响及其对肺癌细胞A549生长抑制作用的机制。方法采用酶抑制动力学方法,观察芹菜素对氨肽酶N的抑制作用和延滞时间;进行肺癌细胞A549和肺正常细胞MRC-5生长的抑制试验;通过锌离子螯合实验和分子对接研究芹菜素与氨肽酶N的相互作用机制。结果芹菜素能够对肺癌细胞A549的生长产生抑制作用,对正常细胞MRC-5具有较低的毒性,且是一种可逆的竞争型氨肽酶N抑制剂[半数抑制浓度(IC50)为(71.22±2.43)μmol/L];锌离子螯合实验和分子模拟结果表明,芹菜素能够优先结合到氨肽酶N的活性中心锌离子附近,并与催化基团His383和Glu350形成氢键。结论芹菜素是一种竞争性的氨肽酶N抑制剂,能够特异性地对肺癌细胞A549的生长起到抑制作用。%Objective To investigate the effect of Apigenin on the Aminopeptidase N (APN) activity of lung cancer cells and inhibitory mechanisms for mouse hepatitis virus A549 (A549) growth. Methods The inhibition effect and lag time of Apigenin on APN were analyzed using enzyme inhibition and kinetic method. The inhibitory tests of the growth of lung cancer cell A549 and lung normal cell MRC-5 were performed, and the interaction mechanisms of Apigenin on APN was performed by zinc chelation experiment and molecular docking. Results Apigenin had the inhibitory effect on the growth of lung cancer cell A549 and little toxicity to normal lung cell MRC-5, and it was a reversible and competi-tive inhibitor [inhibitory concentration 50 (IC50):(71. 22 ± 2. 43)μmol/L]; zinc chelation experiment and molecular docking showed that Apigenin combined with Zn ions in APN active center preferentially, and formed hydrogen bonds with catalytic groups of His383 and Glu350. Conclusion Apigenin is a competitive APN inhibitor, and it has a special inhibitory effect on the growth of lung cancer cells A549.

  1. 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.

  2. 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

  3. 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.

  4. The influence of spleen aminopeptidase lyophilized powder on serum levels of TGF -β1,MCP -1,SDF -1 in pediatric asthma%脾氨肽口服冻干粉对小儿哮喘血清 TGF-β1、MCP-1、SDF-1水平的影响

    Institute of Scientific and Technical Information of China (English)

    严波

    2016-01-01

    目的探讨孟鲁司特联合脾氨肽口服冻干粉对小儿哮喘疗效及血清转化生长因子-β1(TGF-β1)、单核细胞趋化蛋白-1(MCP-1)、基质细胞衍生因子-1(SDF-1)水平的影响。方法选取128例小儿哮喘患儿,根据抽签法分为观察组(64例)及对照组(64例),对照组给予孟鲁司特口服治疗,观察组在对照组基础上加服脾氨肽口服冻干粉。观察两组治疗效果及肺功能改善情况。治疗前后应用 ELISA 法测定两组血清TGF-β1、MCP-1、SDF-1水平。结果观察组总有效率为96.87%高于对照组84.37%,差异有统计学意义(χ2=5.885,P =0.015)。观察组治疗后哮喘控制测试表(ACT)评分(24.25±3.98)分、第一秒呼气容积(FEV1)为(80.25±4.25)%和呼气峰值流速(PEF)(7.25±0.69)L/min,显著高于对照组(20.12±4.02)分、(75.02±3.96)%、(5.82±0.70)L/min(t =7.203,6.757,14.459,26.677,均 P <0.01)。观察组治疗后血清TGF-β1(42.23±6.02)ng/mL、MCP-1(48.56±3.96)ρg/mL、SDF-1(252.36±32.22)ng/L 水平低于对照组(42.23±6.02)ng/mL、(59.02±4.22)ρg/mL、(425.25±40.62)ng/L,差异有统计学意义(t =6.757,14.459,26.677,均 P <0.01)。结论孟鲁司特联合脾氨肽口服冻干粉能有效提高哮喘患儿治疗效果,改善患儿肺功能,其可能机制与降低哮喘患儿血清 TGF-β1、MCP-1、SDF-1水平有关。%Objective To investigate the influence of spleen aminopeptidase lyophilized powder on serum levels of transforming growth factor -β1 (TGF -β1),monocyte chemoattractant protein -1 (MCP -1),stromal cell derived factor 1 (SDF -1 )in pediatric asthma.Methods 128 patients with asthma were randomly divided into observation group(64 cases)and control group (64 cases).Two groups of children were given symptomatic

  5. Postnatal development of aminopeptidase (arylamidase) activity in rat brain.

    Science.gov (United States)

    de Gandarias, J M; Ramírez, M; Zulaica, J; Iribar, C; Casis, L

    1989-01-01

    Changes in the activities of Leu- and Arg-arylamidase in rat frontal and parietal cortices and the subcortical area (including thalamus, hypothalamus, and striatum) were examined in the 2nd, 4th, 8th, 12th, and 24th weeks of life. Average levels found in the subcortical region were greater than those in the cortical areas. The most marked changes in enzymatic activity in the course of brain development were found in the subcortical structure. Leu-arylamidase activity increased from the 2nd week up to the 8th week, returning to the 2nd week level at the 12th and 24th weeks. The maximum levels of Arg-arylamidase activity were found at the 4th and 8th weeks. These data suggest that proteolytic activity is involved in the postnatal development of rat brain.

  6. 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

  7. Effects of antiserums of cadherin, aminopeptidase N and alkaline phosphatase on the toxicities of Cry1Ac and Cry2Aa in Helicoverpa armigera (Lepidoptera: Noctuidae)%棉铃虫中肠钙粘蛋白、氨肽酶N及碱性磷酸酯酶的抗血清对Cry1Ac和Cry2Aa毒力的影响

    Institute of Scientific and Technical Information of China (English)

    王冰洁; 袁向东; 赵曼; 刘臣; 陈琳; 梁革梅

    2016-01-01

    [Aim] Cry1A and Cry2A toxins play insecticidal roles by specifically binding with receptor proteins on insect midgut,and are widely used in Bt transgenic crops now.This study aims to further understand the action mechanisms of Cry2A and the roles of the functional receptor proteins of Cry1 A in the toxicity of Cry2A.[Methods] We firstly extracted brush border membrane vesicles (BBMV) of midgut,prepared antibody and antiserums of cadherin (CAD),aminopeptidase N (APN),and alkaline phosphatase (ALP) in Helicoverpa armigera.Then,after detecting the antiserums of these three receptor proteins in BBMV by Western blot,we compared the effects of antiserums of these three receptor proteins on the toxicities of Cry1Ac and Cry2Aa in the susceptible and Cry1Ac-resistant H.armigera (BtR) by the antibody blocking technology.[Results] For the susceptible H.armigera,the antiserums of these three receptor proteins not only significantly reduced the toxicity of Cry1 Ac,but also remarkably reduced the toxicity of Cry2Aa.Among them,anti-APN antiserum had the biggest impact on the toxicity of Cry1Ac,causing the mortality of H.armigera larvae to be reduced by 84.44%,and anti-ALP antiserum had the greatest effect on the toxicity of Cry2Aa,causing the larval mortality to be reduced by 71.04% compared with the control (without treatment by antiserum).The toxicity of Cry1Ac to Cry1Ac-resistant H.armigera (BtR) was obviously reduced and the toxicity of Cry2Aa also became less to these resistant larvae.The influences of the antiserums of these three receptors on the toxicity of Cry1Ac to the Cry1Acresistant strain of H.armigera (BtR) were smaller than those to the susceptible strain.Especially,the inhibition percentage of anti-CAD and-APN antiserums to the toxicity of Cry1 Ac decreased significantly.The effects of anti-CAD and-ALP antiserums on the toxicity of Cry2Aa to the Cry1Ac-resistant strain (BtR) and the susceptible strain were not significantly different,but the anti-APN antiserum

  8. 尿N-乙酰-β-D氨基葡萄糖酶、丙氨酸氨肽酶和α1-微球蛋白检测在唑来膦酸致肾损伤早期诊断作用%Effects of detecting of N-acety-beta-D-glucosaminidase,Alanine aminopeptidase andα1-microglobulin in urine on early diag-nosis of kidney injury induced by zoledronic acid

    Institute of Scientific and Technical Information of China (English)

    刘星; 刘大为; 谢晓冬; 郭占文; 郑振东; 张丽辉; 张娇蕊; 刘永叶

    2016-01-01

    目的 探讨尿N-乙酰-β-D氨基葡萄糖酶(NAG)、丙氨酸氨肽酶(AAP)和α1-微球蛋白(α1-MG)检测在唑来膦酸致肾损伤早期诊断中的作用.方法 选取2012年2月至2014年7月沈阳军区总医院收治的100例应用唑来膦酸治疗的恶性肿瘤骨转移患者为研究对象.其中,男性48例,女性52例,年龄25~82岁,平均(60±11)岁.患者接受唑来膦酸注射液4 mg静脉滴注,1次/25~35 d,30 min滴完,连续应用3次.每次用药前和用药次日,留取患者清晨尿液及静脉血,检测并比较尿NAG、AAP、α1-MG和血肌酐(Scr)、尿素氮(BUN)水平.结果 每次用药前、后比较尿NAG水平,差异均有统计学意义(P0.05);而第2次和第3次用药前后,尿α1-MG水平比较,差异有统计学意义(P0.05).每次用药前后,Scr、BUN水平比较,差异均无统计学意义(P>0.05).3次用药后,尿NAG、α1-MG水平组间比较,差异均有统计学意义(P0.05).结论 唑来膦酸致肾损伤患者尿NAG、AAP和α1-MG水平变化早于血肌酐.尿NAG、AAP和α1-MG联合检测可作为肾损伤的早期诊断指标.%Objective To explore the effects of detecting of N-acety-beta-D-glucosaminidase( NAG) ,Alanine aminopeptidase( AAP) andα1-microglobulin(α1-MG) in urine on early diagnosis of kidney injury induced by zoledronic acid. Methods A total of 100 pa-tients comprised 48 males and 52 females with a mean age of (60 ± 11)years were enrolled into the study. The patients with malignant tumor bone metastasis and received zoledronic acid from February 2012 to July 2014 in the General Hospital of Shenyang Military Command. They received IV infusion of zoledronic acid 4 mg,once for 25 to 35 days,finished within 30 minutes. The therapy was ap-plied 3 times continuously. The patients′urine and venous blood samples were taken before and after each medication′s morning. The level of NAG,AAP and α1-MG in urine and serum creatinine,blood urea nitrogen were detected and compared. Results The differ

  9. 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.

  10. Evolutionary diversification of aminopeptidase N in Lepidoptera by conserved clade-specific amino acid residues.

    Science.gov (United States)

    Hughes, Austin L

    2014-07-01

    Members of the aminopepidase N (APN) gene family of the insect order Lepidoptera (moths and butterflies) bind the naturally insecticidal Cry toxins produced by the bacterium Bacillus thuringiensis. Phylogenetic analysis of amino acid sequences of seven lepidopteran APN classes provided strong support for the hypothesis that lepidopteran APN2 class arose by gene duplication prior to the most recent common ancestor of Lepidoptera and Diptera. The Cry toxin-binding region (BR) of lepidopteran and dipteran APNs was subject to stronger purifying selection within APN classes than was the remainder of the molecule, reflecting conservation of catalytic site and adjoining residues within the BR. Of lepidopteran APN classes, APN2, APN6, and APN8 showed the strongest evidence of functional specialization, both in expression patterns and in the occurrence of conserved derived amino acid residues. The latter three APN classes also shared a convergently evolved conserved residue close to the catalytic site. APN8 showed a particularly strong tendency towards class-specific conserved residues, including one of the catalytic site residues in the BR and ten others in close vicinity to the catalytic site residues. The occurrence of class-specific sequences along with the conservation of enzymatic function is consistent with the hypothesis that the presence of Cry toxins in the environment has been a factor shaping the evolution of this multi-gene family.

  11. 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.

  12. Aminopeptidase N/CD13 is associated with raft membrane microdomains in monocytes

    DEFF Research Database (Denmark)

    Navarrete Santos, A; Roentsch, J; Danielsen, E M;

    2000-01-01

    of monocytes were characterized by the presence of GM1 ganglioside as raft marker molecule and by the high level of tyrosine-phosphorylated proteins. Furthermore, similar to polarized cells, rafts in monocytic cells lack Na(+), K(+)-ATPase. Cholesterol depletion of monocytes by methyl-beta-cyclodextrin greatly...

  13. 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... manufacture of cheddar cheese, in accordance with § 133.113 of this chapter, and in the preparation of...

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

    DEFF Research Database (Denmark)

    Danielsen, E M

    1984-01-01

    in the crypt cells and a moderate increase of about 20% in the villus cells. Whereas the latter can possibly be ascribed to a general protective effect of dexamethasone on villus architecture, these experiments indicate that crypt cells of mucosa from adult individuals exhibit the same sensitivity...

  15. 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...

  16. 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...

  17. 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...

  18. 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.

  19. 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.

  20. 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 includin...

  1. Demethionylation of Pro-1 variants of 4-oxalocrotonate tautomerase in Escherichia coli by co-expression with an engineered methionine aminopeptidase

    NARCIS (Netherlands)

    Baas, Bert-Jan; Zandvoort, Ellen; Wasiel, Anna A; Poelarends, Gerrit J

    2014-01-01

    4-Oxalocrotonate tautomerase (4-OT) catalyzes the enol-keto tautomerization of 2-hydroxymuconate, utilizing its N-terminal proline (Pro-1) as general base catalyst. Substituting Pro-1 with bulky or charged residues will result in poor or no post-translational removal of the translation-initiating me

  2. 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....

  3. 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

    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....

  4. 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.

  5. Progress of studies on insect midgut aminopeptidase N serving as Bacillus thuringiensis insecticidal crystal protein toxin receptor%昆虫中肠Bt杀虫晶体蛋白毒素受体氨肽酶N的研究进展

    Institute of Scientific and Technical Information of China (English)

    刘凯于; 姚汉超; 杨红; 洪华珠

    2004-01-01

    鳞翅目昆虫中肠上皮细胞刷状缘膜(BBM)上的Bt杀虫晶体蛋白毒素受体氨肽酶N(APN)的结构和位点密度的改变是昆虫对Bt毒素的主要抗性机制之一,该文简要综述了APN受体的研究进展.每种昆虫中肠上皮细胞中有数种APNs,彼此间同源性较高,其中部分APNs为Cry1A家族毒素的功能性受体.不同种类昆虫的APNs受体,甚至同一种昆虫的不同类型APNs,其所结合的毒素种类可能不同.APNs决定该昆虫对Cry1A类毒素的敏感程度差异.有些抗性昆虫的APNs基因编码区发生了多个点突变.

  6. Detection of urine glycyl-proline dipeptidyl aminopeptidase for adjunct diagnosing early kidney injury in pregnancy induced hypertension%检测尿GPDA辅助诊断妊娠高血压早期肾损害

    Institute of Scientific and Technical Information of China (English)

    许会彬; 王青; 张代民; 李萍

    2006-01-01

    目的 研究尿甘氨酰脯氨酸二肽氨基肽酶(GPDA)诊断妊娠高血压早期肾损害的价值.方法 选择妊娠高血压患者51例,分为轻、中、重三组,正常孕妇20名作对照组.采用GPDA连续监测法,分别对其尿中GPDA活性进行测定并与其他反映早期肾损害的指标N-乙酰β-D氨基葡萄糖苷酶(NAG)及尿微量白蛋白(UMA)相比较.结果 妊娠高血压患者尿液GPDA显著高于对照组(P<0.01),且尿GPDA活性与妊娠高血压的严重程度有关.尿GPDA与NAG及UMA均呈显著正相关(r=0.738,r=0.652,P<0.01).结论 尿液GPDA测定可作为一项新的监测妊娠高血压早期肾损害的敏感指标.

  7. 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.

  8. 高压脉冲电场对草莓汁抗氧化活性的影响%Research of the Ultrafiltration on the Bacillus subtilis Aminopeptidase Extraction

    Institute of Scientific and Technical Information of China (English)

    陈晓婵; 赵伟; 杨瑞金; 顾艳洁; 李春阳

    2013-01-01

    研究了高压脉冲电场在不同电场强度(20~35 kV/cm)和处理时间(100~400μs)处理对草莓汁杀菌效果及其抗氧化活性的影响,还进一步比较了高压脉冲电场和传统热杀菌(95℃,30s)处理后和贮藏过程中草莓汁抗氧化活性的变化.结果表明:随着高压脉冲电场的电场强度和处理时间的增加,草莓汁抗氧化活性略有降低,但其损失程度比传统热杀菌小,且在储藏过程中保存较好.%The effects of high-intensity pulsed electric fields in different levels of electric field intensity(20~35 kV/cm) and treatment time (100-400 μs) on microbial inactivation and antioxidant activity of strawberry juice were investigated in this manuscript. The changes of antioxidant activity of strawberry juice immediately after pulsed electric fields and conventional thermal processing (95 ℃ ,30 s) treatments and during the storage were further investigated. The results showed that compared with the conventional thermal pasteurization methods, the antioxidant activity of strawberry juice was less affected initially after PEF processing and were maintained in higher quality throughout storage period.

  9. SwissProt search result: AK100736 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100736 J023118B01 (P46101) Dipeptidyl aminopeptidase-like protein 6 (Dipeptidylpeptidase VI) (Dipeptidy...lpeptidase 6) (Dipeptidyl peptidase IV-like protein) (Dipeptidyl aminopeptidase-related protein) (DPPX) DPP6_RAT 7e-43 ...

  10. SwissProt search result: AK060335 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK060335 001-008-C02 (P42658) Dipeptidyl aminopeptidase-like protein 6 (Dipeptidylpeptidase VI) (Dipeptidy...lpeptidase 6) (Dipeptidyl peptidase IV-like protein) (Dipeptidyl aminopeptidase-related protein) (DPPX) DPP6_HUMAN 6e-28 ...

  11. SwissProt search result: AK060335 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK060335 001-008-C02 (Q9Z218) Dipeptidyl aminopeptidase-like protein 6 (Dipeptidylpeptidase VI) (Dipeptidy...lpeptidase 6) (Dipeptidyl peptidase IV-like protein) (Dipeptidyl aminopeptidase-related protein) (DPPX) DPP6_MOUSE 7e-29 ...

  12. SwissProt search result: AK100736 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100736 J023118B01 (P42658) Dipeptidyl aminopeptidase-like protein 6 (Dipeptidylpeptidase VI) (Dipeptidy...lpeptidase 6) (Dipeptidyl peptidase IV-like protein) (Dipeptidyl aminopeptidase-related protein) (DPPX) DPP6_HUMAN 1e-41 ...

  13. SwissProt search result: AK110189 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110189 002-162-A04 (Q9Z218) Dipeptidyl aminopeptidase-like protein 6 (Dipeptidylpeptidase VI) (Dipeptidy...lpeptidase 6) (Dipeptidyl peptidase IV-like protein) (Dipeptidyl aminopeptidase-related protein) (DPPX) DPP6_MOUSE 1e-52 ...

  14. SwissProt search result: AK110189 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110189 002-162-A04 (P46101) Dipeptidyl aminopeptidase-like protein 6 (Dipeptidylpeptidase VI) (Dipeptidy...lpeptidase 6) (Dipeptidyl peptidase IV-like protein) (Dipeptidyl aminopeptidase-related protein) (DPPX) DPP6_RAT 6e-53 ...

  15. SwissProt search result: AK100736 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100736 J023118B01 (P42659) Dipeptidyl aminopeptidase-like protein 6 (Dipeptidylpeptidase VI) (Dipeptidy...lpeptidase 6) (Dipeptidyl peptidase IV-like protein) (Dipeptidyl aminopeptidase-related protein) (DPPX) DPP6_BOVIN 8e-42 ...

  16. SwissProt search result: AK110189 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110189 002-162-A04 (P42659) Dipeptidyl aminopeptidase-like protein 6 (Dipeptidylpeptidase VI) (Dipeptidy...lpeptidase 6) (Dipeptidyl peptidase IV-like protein) (Dipeptidyl aminopeptidase-related protein) (DPPX) DPP6_BOVIN 3e-56 ...

  17. SwissProt search result: AK060335 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK060335 001-008-C02 (P42659) Dipeptidyl aminopeptidase-like protein 6 (Dipeptidylpeptidase VI) (Dipeptidy...lpeptidase 6) (Dipeptidyl peptidase IV-like protein) (Dipeptidyl aminopeptidase-related protein) (DPPX) DPP6_BOVIN 1e-27 ...

  18. SwissProt search result: AK060335 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK060335 001-008-C02 (P46101) Dipeptidyl aminopeptidase-like protein 6 (Dipeptidylpeptidase VI) (Dipeptidy...lpeptidase 6) (Dipeptidyl peptidase IV-like protein) (Dipeptidyl aminopeptidase-related protein) (DPPX) DPP6_RAT 9e-29 ...

  19. SwissProt search result: AK100736 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100736 J023118B01 (Q9Z218) Dipeptidyl aminopeptidase-like protein 6 (Dipeptidylpeptidase VI) (Dipeptidy...lpeptidase 6) (Dipeptidyl peptidase IV-like protein) (Dipeptidyl aminopeptidase-related protein) (DPPX) DPP6_MOUSE 6e-43 ...

  20. SwissProt search result: AK110189 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110189 002-162-A04 (P42658) Dipeptidyl aminopeptidase-like protein 6 (Dipeptidylpeptidase VI) (Dipeptidy...lpeptidase 6) (Dipeptidyl peptidase IV-like protein) (Dipeptidyl aminopeptidase-related protein) (DPPX) DPP6_HUMAN 4e-54 ...

  1. NCBI nr-aa BLAST: CBRC-AGAM-02-0044 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-02-0044 ref|YP_001012824.1| Leucyl aminopeptidase, AmpS [Hyperthermus but...ylicus DSM 5456] gb|ABM80479.1| Leucyl aminopeptidase, AmpS [Hyperthermus butylicus DSM 5456] YP_001012824.1 0.37 29% ...

  2. 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 dipeptidyl...

  3. 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

    , 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...

  4. Long-term changes in microbial and biochemical parameters in the Central Indian Basin

    Digital Repository Service at National Institute of Oceanography (India)

    Raghukumar, C.; Nath, B.N.; Sharma, R.; LokaBharathi, P.A.; Dalal, S.G.

    an artificial sediment disturbance was created by a hydraulic benthic disturber. Besides the above- mentioned parameters, extracellular enzymes, alkaline phosphatase, aminopeptidase and lipase were also monitored at these 9 locations. Postdisturbance (PoD1...

  5. Immunoelectrophoretic studies on pig intestinal brush border proteins

    DEFF Research Database (Denmark)

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

    1977-01-01

    aminopeptidase (EC 3.4.11.2), aspartate aminopeptidase (EC 3.4.11.7), dipeptidyl peptidase IV (EC 3.4.14.X), lactase (EC 3.2.1.23), glucoamylase (EC 3.2.1.3), sucrase (EC 3.2.1.48), isomaltase (EC 3.2.1.10) and alkaline phosphatase (EC 3.1.3.1). In addition, at least four faint immunoprecipitates were formed...

  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 application of urine glycyl-proline dipeptidyl aminopeptidase on the study of kidney injury in lead and mercury poisoning%尿甘氨酰脯氨酸二肽氨基肽酶测定对铅、汞中毒肾损害探讨

    Institute of Scientific and Technical Information of China (English)

    郗厚富

    2008-01-01

    目的 研究尿液甘氨酰脯氨酸二肽氨基肽酶(GPDA)测定对铅、汞中毒肾损害的临床应用价值.方法 选择铅中毒45例、汞中毒38例和正常对照组40例,其中铅汞中毒分为轻、中、重3组,采用GPDA连续检测法,分别对其尿中GPDA活力进行测定,并与其他反映早期肾损害的指标N-乙酰-β-D氨基葡萄糖苷酶(NAG)及尿微量白蛋白(UMA)相比较.结果 铅中毒患者尿液GPDA[(36.1±32.7)u/g·cr]和汞中毒患者尿液GPDA[(38.2±35.1)u/g·Cr]显著高于正常对照组[(12.8±7.9)u/g·Cr]的水平(P<0.01),尿GPDA与NAG、UMA均呈显著的正相关.结论 尿液GPDA的操作简单、快速、稳定性好,干扰因素少,结果准确,是一项新的检测铅、汞中毒早期肾损害的敏感指标.

  8. Clinical Observation Spleen Aminopeptidase(Stoeckel care)Treatment of Recurrent Respiratory Tract ;Infections%脾氨肽(复可托)治疗小儿反复呼吸道感染的临床疗效观察

    Institute of Scientific and Technical Information of China (English)

    曾胜荣; 丁淑娟

    2014-01-01

    目的:分析探讨脾氨肽(复可托)治疗反复呼吸道感染的临床疗效。方法:选取本院近年来收治的60例反复呼吸道感染患者作为研究对象,按照数字抽签法将其随机分为研究组与对照组,每组30例,研究组在常规治疗的基础上行复可托治疗,对照组仅行常规治疗,比较两组治疗效果。结果:研究组治疗显效率为56.7%,总有效率为90.0%,对照组治疗显效率为23.3%,总有效率为50.0%,研究组患者治疗效果明显优于对照组(P<0.05)。与治疗前相比,研究组患者治疗后CD8指标出现明显下降,CD3、CD4、CD4/CD8指标出现明显上升,治疗前后研究组患者T细胞亚群指标比较差异有统计学意义(P<0.05)。结论:复可托治疗反复呼吸道感染可改善患者免疫失衡状态,具有显著的临床疗效。%Objective:To investigate the spleen ammonia peptide(complex)clinical curative effect for the treatment of recurrent respiratory tract infections. Method:To select 60 patients admitted in our hospital in recent years, patients with recurrent respiratory tract infections as the research object,according to the digital lottery method were randomly divided into research group and control group,30 cases in each group,the team in uplink compound could be the basis of routine therapy treatment,the control group only routine treatment,compare therapeutic effect of the two groups. Result:Team treatment efficiency was 56.7%,total effective rate was 90.0%,the control group’s treatment efficiency was 23.3%,total effective rate was 50.0%,the team with the treatment effect was better than the control group (P<0.05). Compared with before treatment,the team CD8 index after treatment in patients with decreased obviously, CD3,CD4,CD4/CD8 indicators appeared obvious rise,before and after treatment group patients with T cell subgroup index comparison difference was statistically significant(P<0.05). Conclusion:Compound can improve the patients' immune treatment of recurrent respiratory tract infections is imbalance,has significant clinical effect.

  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. Influence of bioremediation stimulators in soil on development of oat seedlings (Avena sativa and their aminopeptidase activity / Wpływ pozostałości substancji ropopochodnych w glebie na rozwój owsa i aktywność aminopeptydazową

    Directory of Open Access Journals (Sweden)

    Pawełczak Małgorzata

    2015-03-01

    Full Text Available Dobór technik bioremediacji jest bardzo ważny w oczyszczaniu gleby skażonej substancjami ropopochodnymi, która ma być wykorzystywanej rolniczo. Prezentowane wyniki wskazują, że na rozwój siewek owsa większy wpływ ma stosowana metoda bioremediacji niż poziom zanieczyszczenia węglowodorami. Z zastosowanych technik najbardziej przyjazne dla wegetacji owsa były te, w których wykorzystywano szczepionkę drożdżową

  11. 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.

  12. Effect of subacute benzene exposure on the activity of two neuropeptide-degrading enzymes in the rat brain.

    Science.gov (United States)

    de Gandarias, J M; Casis, O; Irazusta, J; Echevarría, E; Casis, L

    1992-04-01

    Benzene (Bz) is an important industrial chemical, a petroleum by-product, a component of unleaded gas, and thus a ubiquitous environmental pollutant. It is well established that this organic solvent possesses neurotoxic and behavioral effects. However, the neurochemical mechanism of the solvent action remains obscure. The aminopeptidases (AP) are proteolytic enzymes that have been proposed as a candidate regulator of the degradation of several neuropeptides. In this work, changes in Lys- and Leu-aminopeptidase activities in several rat brain regions after benzene administration are described. The AP activity was determined by measuring the rate of hydrolysis of the artificial substrates Lys- and Leu-2-naphthylamides (fluorimetrically detected in triplicate). Both enzyme activities decrease in the thalamus, hypothalamus, hippocampus, and amygdala after Bz treatment. It is suggested that these aminopeptidase activities play a part in the benzene action mechanism, possibly by regulating the activity of several neuroactive peptides.

  13. 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 results indicate that the lactase-phlorizin hydrolase is synthesized as a Mr 245 000 polypeptide, which is intracellularly cleaved into its mature Mr 160 000 form. Sucrase-isomaltase is shown to be synthesized as a single chain precursor (Mr 245 000 and 265 000) while the precursor of aminopeptidase N...

  14. 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...

  15. 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

  16. 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...

  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

    -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...

  18. A new procedure for N1-alkylation of imidazolidin-4-ones and its NMR characterization

    Science.gov (United States)

    Vale, Nuno; Figueiredo, Patrícia

    2016-12-01

    N1-unsubstituted imidazolidin-4-ones of primaquine (PQ) can be stabilized by N1-alkylation under basic conditions. Here we report the development, with our conditions, of peptidomimetic derivatives of PQ with L-amino acid and alkyl derivatives. The new derivatives represent potential new therapeutics for use against protozoan parasites, through enzymatic protection of aminopeptidases.

  19. 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

  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: 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

  2. 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

  3. 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

  4. 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

    from about 60 to 160 nm. Cholesterol depletion of synoviocytes by methyl-beta-cyclodextrin disrupted >90% of the caveolae and reduced the raft localization of aminopeptidase N/CD13 without affecting Ala-p-nitroanilide-cleaving activity of confluent cell cultures. In co-culture experiments with T...

  5. Bestatin Induces Specific Changes in Trypanosoma cruzi Dipeptide Pool

    OpenAIRE

    2015-01-01

    Proteases and peptidases in Trypanosoma cruzi are considered potential targets for antichagasic chemotherapy. We monitored changes in low-mass metabolites in T. cruzi epimastigotes treated with bestatin, a dipeptide metalloaminopeptidase inhibitor. After treatment, multiple dipeptides were shown to be increased, confirming in situ inhibition of the leucine aminopeptidase of T. cruzi (LAPTc) and probably other peptidases.

  6. 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...

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

    Lifescience Database Archive (English)

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

  8. 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

  9. Serum protease activity in chronic kidney disease patients: The GANI_MED renal cohort.

    Science.gov (United States)

    Wolke, Carmen; Teumer, Alexander; Endlich, Karlhans; Endlich, Nicole; Rettig, Rainer; Stracke, Sylvia; Fiene, Beate; Aymanns, Simone; Felix, Stephan B; Hannemann, Anke; Lendeckel, Uwe

    2017-03-01

    Serum or plasma proteases have been associated with various diseases including cancer, inflammation, or reno-cardiovascular diseases. We aimed to investigate whether the enzymatic activities of serum proteases are associated with the estimated glomerular filtration rate (eGFR) in patients with different stages of chronic kidney disease (CKD). Our study population comprised 268 participants of the "Greifswald Approach to Individualized Medicine" (GANI_MED) cohort. Enzymatic activity of aminopeptidase A, aminopeptidase B, alanyl (membrane) aminopeptidase, insulin-regulated aminopeptidase, puromycin-sensitive aminopeptidase, leucine aminopeptidase 3, prolyl-endopeptidase (PEP), dipeptidyl peptidase 4 (DPP4), angiotensin I-converting enzyme, and angiotensin I-converting enzyme 2 (ACE2) proteases was measured in serum. Linear regression of the respective protease was performed on kidney function adjusted for age and sex. Kidney function was modeled either by the continuous Modification of Diet in Renal Disease (MDRD)-based eGFR or dichotomized by eGFR serum protease activities showed no associations with age or sex. Our data indicate that ACE2 and DPP4 enzymatic activity are associated with the eGFR in patients with CKD. This finding distinguishes ACE2 and DPP4 from other serum peptidases analyzed and clearly indicates that further analyses are warranted to identify the precise role of these serum ectopeptidases in the pathogenesis of CKD and to fully elucidate underlying molecular mechanisms. Impact statement • Renal and cardiac diseases are very common and often occur concomitantly, resulting in increased morbidity and mortality. Understanding of molecular mechanisms linking both diseases is limited, available fragmentary data point to a role of the renin-angiotensin system (RAS) and, in particular, Ras-related peptidases. • Here, a comprehensive analysis of serum peptidase activities in patients with different stages of chronic kidney disease (CKD) is

  10. 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.

  11. 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...... membranes during starvation. Prolonged fasting caused a significant decrease in sucrase activity, but thyroidectomy partly prevented this effect. However, the amount of the corresponding mRNA dropped during long term starvation without incidence of thyroidectomy. Lactase activity in the brush border...... membranes was increased by starvation, and thyroidectomy caused a further elevation of the enzyme activity. Simultaneously, lactase mRNA content rose only slightly compared to the enzyme activity. Aminopeptidase activity and mRNA content decreased during starvation and thyroidectomy did not prevent...

  12. 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...... cell culture model, and human and porcine buccal epithelium were compared. The esterase activity in the intact cell culture model and in the porcine buccal mucosa was compared. Further, the TR146 cell culture model was used to study the permeability rate and metabolism of leu-enkephalin. The activity...... 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...

  13. In vitro quantitative study of the degradation of endomorphins.

    Science.gov (United States)

    Tömböly, Csaba; Péter, Antal; Tóth, Géza

    2002-09-01

    The catabolism of the endomorphins was investigated in detail. The endomorphins were degraded relatively slowly in the rat brain homogenate (t1/2(endomorphin-1)=4.94 min; t1/2(endomorphin-2)=3.81 min). The inhibition of metalloproteases and aminopeptidases stabilised the endomorphins to the greatest extent. The digestion of endomorphins tritiated specifically on Tyr(1), Pro(2) or Phe(3) established also that only the aminopeptidase pathways were essential for inactivation of the endomorphins, and that the tetrapeptides were degraded by cleavage of the Pro(2)-Trp(3) or Pro(2)-Phe(3) bond. The end-products of the catabolism were amino acids; the fragments Tyr-Pro-OH and Pro-Trp-Phe-NH2 were present as intermediates. Metabolites produced by brain carboxypeptidases were not detected.

  14. 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.

  15. Biosynthesis of intestinal microvillar proteins. Evidence for an intracellular sorting taking place in, or shortly after, exit from the Golgi complex

    DEFF Research Database (Denmark)

    Danielsen, E M; Cowell, G M

    1985-01-01

    the Mg2+-precipitated fraction were equally well protected from proteolytic cleavage (in the absence of Triton X-100). This indicates that the basolateral plasma membrane is unlikely to be involved in the post-Golgi transport of newly synthesized aminopeptidase N and suggests instead a direct delivery...... that for microvillar enzymes, the aspects of sorting studied take place in, or shortly after exit from, the Golgi complex....

  16. 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.

  17. Sosis Fermentasyonunda Proteoliz (İngilizce)

    OpenAIRE

    Candoğan, Kezban; Acton, James C.

    2015-01-01

    Proteolysis is one of the main biochemical reactions contributing to development of overall quality of fermented sausages. Proteolysis reactions are catalyzed by either endogenous enzymes inherent in the product or by enzymes from microbial origin. Meat proteins undergo hydrolysis first to polypeptides by endogenous muscle enzymes, and then further to small peptides by peptidases. Final step in proteolysis phenomena is free amino acid generation by bacterial aminopeptidases as well as aminope...

  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

    . The proteinase inhibitor leupeptin partially restored the suppressed synthesis, indicating that the majority of the wrongly processed enzymes, probably because of conformational instability, become degraded soon after synthesis rather than being transported to the microvillar membrane.......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. Drug: D10026 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available D10026 Drug Tosedostat (USAN/INN) C21H30N2O6 406.2104 406.4727 D10026.gif Antineopl...sed classification of drugs [BR:br08310] Enzymes Hydrolases aminopeptidase N [HSA:290] [KO:K11140] Tosedostat D100...26 Tosedostat (USAN/INN) CAS: 238750-77-1 PubChem: 135626746 LigandBox: D100

  20. Kinetic studies of novel inhibitors of endomorphin degrading enzymes

    OpenAIRE

    Perlikowska, Renata; Fichna, Jakub; do-Rego, Jean Claude; Gach, Katarzyna; Janecka, Anna

    2011-01-01

    Endomorphins (EMs), two endogenous μ-opioid receptor selective ligands, are attractive lead compounds for opioid-based pain management studies. However, these peptides are quickly degraded by peptidases, in particular by dipeptidylpeptidase IV (DPP IV) and aminopeptidase M (APM). Targeting enzymatic degradation is one approach to prolong endomorphin activity. In this study we characterized the action of two new inhibitors of similar to endomorphins structure, Tyr-Pro-Ala-NH2 (EMDB-2) and Tyr-...

  1. Peptidases prevent μ-opioid receptor internalization in dorsal horn neurons by endogenously released opioids

    OpenAIRE

    Song, Bingbing; Marvizón, Juan Carlos G.

    2003-01-01

    To evaluate the effect of peptidases on μ-opioid receptor (MOR) activation by endogenous opioids, we measured MOR-1 internalization in rat spinal cord slices. A mixture of inhibitors of aminopeptidases (amastatin), dipeptidyl carboxypeptidase (captopril), and neutral endopeptidase (phosphoramidon) dramatically increased the potencies of Leu-enkephalin and dynorphin A to produce MOR-1 internalization, and also enhanced the effects of Met-enkephalin and α-neoendorphin, but not endomorphins or β...

  2. Molecular Biological Studies on the Biogenesis of Human Cholinesterases in vivo and as Directed by Cloned Cholinesterase DNA Sequences

    Science.gov (United States)

    1990-10-24

    potentially be analogous to the well-known amplification of other genes that encode target proteins to toxic compounds. As such, it could provide cells the...occurring CHE inhibitors, including the steroidal glycoalkaloid solanine and its hydrolytic aglycone derivative solanidine, both of which may be...present in toxic concentrations in potatoes (63,64). 4.5 Suggestions for peptidase activity of cholinesterases Carboxy- and aminopeptidase activity from a

  3. Glutamate-containing dipeptides do not modulate ligand binding at excitatory amino acid receptors.

    Science.gov (United States)

    Baud, J; Fagg, G E

    1986-10-08

    Dipeptides of the structure X-Glu (e.g. X = Phe, Leu) have been proposed as allosteric modulators of excitatory amino acid receptors in rat brain membranes. Here we report that these dipeptides reduce the binding of L-[3H]Glu (predominantly N-methyl-D-aspartate-sensitive sites) and of [3H]kainate to postsynaptic density preparations isolated from rat brain. However, several observations indicate that the effects of these dipeptides are mediated not by allosteric modulation, but by free L-Glu liberated by the actions of a membrane-associated aminopeptidase. The absolute and relative potencies of the dipeptides are similar at all acidic amino acid binding sites examined to date, suggesting the involvement of a factor with similar activity at each site (e.g. L-Glu). N-Acetyl-Met-Glu is a weak inhibitor of L-Glu and kainate binding, and N-blocked peptides are known to be poor substrates of aminopeptidases. Bestatin, an inhibitor of aminopeptidases, decreases or abolishes the effects of substrate dipeptides on L-Glu and kainate receptor binding, while having no effect itself.

  4. 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.

  5. 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.

  6. 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.

  7. Antiproliferative effects of palladium(II) complexes of 5-nitrosopyrimidines and interactions with the proteolytic regulatory enzymes of the renin-angiotensin system in tumoral brain cells.

    Science.gov (United States)

    Illán-Cabeza, Nuria A; García-García, Antonio R; Martínez-Martos, José M; Ramírez-Expósito, María J; Moreno-Carretero, Miguel N

    2013-09-01

    Seventeen new palladium(II) complexes of general formulaes PdCl2L, PdCl(LH-1)(solvent) and PdCl2(PPh3)2L containing pyrimidine ligands derived from 6-amino-5-nitrosouracil and violuric acid have been prepared and characterized by elemental analysis, IR and NMR ((1)H and (13)C) methods and, two of them, PdCl(DANUH-1)(CH3CN)]·½H2O and [PdCl(2MeOANUH-1)(CH3CN)] by X-ray single-crystal diffraction (DANU: 6-amino-1,3-dimethyl-5-nitrosouracil; 2MeOANU: 6-amino-2-methoxy-5-nitroso-3H-pyrimidin-4-one). The coordination environment around palladium is nearly square planar in the two compounds with different supramolecular arrangements. Crystallographic and spectral data are consistent with a bidentate coordination mode through N5 and O4 atoms when the ligands act in neutral form and N5 and N6 atoms in the monodeprotonated ones. The cytotoxicity of the complexes against human neuroblastoma (NB69) and human glioma (U373-MG) cell lines has been tested showing a considerable antiproliferative activity. Also, the study of the effects of palladium(II) complexes on the renin-angiotensin system (RAS) regulating proteolytic regulatory enzymes aminopeptidase A (APA), aminopeptidase N (APN) and insulin-regulated aminopeptidase (IRAP) shows a strong dependence on the compound tested and the tumoral cell type, also affecting different catalytic routes; the compounds affect in a different way the activities of enzymes of the RAS system, changing their functional roles as initiators of cell proliferation in tumors as autocrine/paracrine mediators.

  8. Contribution of exopeptidases to formation of nonprotein nitrogen during ensiling of alfalfa.

    Science.gov (United States)

    Tao, L; Zhou, H; Guo, X S; Long, R J; Zhu, Y; Cheng, W

    2011-08-01

    The experiment was conducted to investigate the exopeptidase classes in alfalfa (Medicago sativa L.) leaves, and to determine their contribution to the formation of nonprotein nitrogen (NPN) components during ensiling. Six classes of inhibitors that included bestatin (aminopeptidase inhibitor), potato carboxypeptidase inhibitor (PCI, carboxypeptidase inhibitor), 1,10-phenanthroline (dipeptidase inhibitor), diprotin A (dipeptidyl-peptidase inhibitor), butabindide (tripeptidyl-peptidase inhibitor), and dipeptide Phe-Arg (peptidyl-dipeptidase inhibitor) were used. To determine the contribution of each exopeptidase to the formation of NPN products, aqueous extracts of fresh alfalfa were fermented to imitate the proteolytic process of ensiled alfalfa and to ensure that each class of exopeptidase inhibitor would have immediate contact with the proteases in the alfalfa extract. Five classes of exopeptidases; namely, aminopeptidase, carboxypeptidase, dipeptidase, dipeptidyl-peptidase, and tripeptidyl-peptidase, were shown to be present in alfalfa leaves, each playing a different role in alfalfa protein degradation. Aminopeptidase, carboxypeptidase, and dipeptidase were the main exopeptidases contributing to the formation of NH(3)-N. Among the 5 exopeptidases, tripeptidyl-peptidase appeared to be the principal exopeptidase in hydrolyzing forage protein into peptides, whereas carboxypeptidase and dipeptidase appeared to be more important in contributing to the formation of amino acid-N. Dipeptidyl-peptidase and tripeptidyl-peptidase did not play a role in the formation of NH(3)-N or amino acid-N. Dipeptidase, carboxypeptidase, and tripeptidyl-peptidase were the principal exopeptidases for hydrolyzing forage protein into NPN during ensilage, and treatment with a mixture of the 5 inhibitors reduced the total NPN concentration in the fermented alfalfa extract to about 45% of that in the control after 21 d of fermentation.

  9. The analgesic activity of Bestatin as a potent APN inhibitor

    Directory of Open Access Journals (Sweden)

    Mei-Rong Jia

    2010-06-01

    Full Text Available Bestatin, a small molecular weight dipeptide, is a potent inhibitor of various aminopeptidases as well as LTA4 hydrolase. Various physiological functions of Bestatin have been identified, viz.: (1 an immunomodifier for enhancing the proliferation of normal human bone marrow granulocyte–macrophage progenitor cells to form CFU-GM colonies; Bestatin exerts a direct stimulating effect on lymphocytes via its fixation on the cell surface and an indirect effect on monocytes via aminopeptidase B inhibition of tuftsin catabolism; (2 an immunorestorator and curative or preventive agent for spontaneous tumor; Bestatin alone or its combination with chemicals can prolongate the disease-free interval and survival period in adult acute or chronic leukemia, therefore, it was primarily marketed in 1987 in Japan as an anticancer drug and servers as the only marketed inhibitor of Aminopeptidase N (APN/CD13 to cure leukemia to date; (3 a pan-hematopoietic stimulator and restorator; Bestatin promotes granulocytopoiesis and thrombocytopoiesis in vitro and restores them in myelo-hypoplastic men; (4 an inhibitor of several natural opioid peptides. Based on the knowledge that APN can cleave several bioactive neuropeptides such as Met-enkaphalins, Leu-enkaphalins, β-Endorphin, and so on, the antiaminopeptidase action of Bestatin also allows it to protect endopeptides against their catabolism, exhibiting analgesic activity. Although many scientific studies and great accomplishments have been achieved in this field, a large amount of problems are unsolved. This article reviews the promising results obtained for future development of the analgesic activity of Bestatin that can be of vital interest in a number of severe and chronic pain syndromes.

  10. Protease activities of rumen protozoa.

    Science.gov (United States)

    Forsberg, C W; Lovelock, L K; Krumholz, L; Buchanan-Smith, J G

    1984-01-01

    Intact, metabolically active rumen protozoa prepared by gravity sedimentation and washing in a mineral solution at 10 to 15 degrees C had comparatively low proteolytic activity on azocasein and low endogenous proteolytic activity. Protozoa washed in 0.1 M potassium phosphate buffer (pH 6.8) at 4 degrees C and stored on ice autolysed when they were warmed to 39 degrees C. They also exhibited low proteolytic activity on azocasein, but they had a high endogenous proteolytic activity with a pH optimum of 5.8. The endogenous proteolytic activity was inhibited by cysteine proteinase inhibitors, for example, iodoacetate (63.1%) and the aspartic proteinase inhibitor, pepstatin (43.9%). Inhibitors specific for serine proteinases and metalloproteinases were without effect. The serine and cysteine proteinase inhibitors of microbial origin, including antipain, chymostatin, and leupeptin, caused up to 67% inhibition of endogenous proteolysis. Hydrolysis of casein by protozoa autolysates was also inhibited by cysteine proteinase inhibitors. Some of the inhibitors decreased endogenous deamination, in particular, phosphoramidon, which had little inhibitory effect on proteolysis. Protozoal and bacterial preparations exhibited low hydrolytic activities on synthetic proteinase and carboxypeptidase substrates, although the protozoa had 10 to 78 times greater hydrolytic activity (per milligram of protein) than bacteria on the synthetic aminopeptidase substrates L-leucine-p-nitroanilide, L-leucine-beta-naphthylamide, and L-leucinamide. The aminopeptidase activity was partially inhibited by bestatin. It was concluded that cysteine proteinases and, to a lesser extent, aspartic proteinases are primarily responsible for proteolysis in autolysates of rumen protozoa. The protozoal autolysates had high aminopeptidase activity; low deaminase activity was observed on endogenous amino acids. PMID:6364968

  11. Liquid chromatographic study of the enzymatic degradation of endomorphins, with identification by electrospray ionization mass spectrometry.

    Science.gov (United States)

    Péter, A; Tóth, G; Tömböly, C; Laus, G; Tourwè, D

    1999-06-18

    The recently discovered native endomorphins play an important role in opioid analgesia, but their metabolic fate in the organism remains relatively little known. This paper describes the application of high-performance liquid chromatography combined with electrospray ionization mass spectrometry to identify the degradation products resulting from the incubation of endomorphins with proteolytic enzymes. The native endomorphin-1, H-Tyr-Pro-Trp-Phe-NH2 (1), and endomorphin-2, H-Tyr-Pro-Phe-Phe-NH2 (2), and an analog of endomorphin-2, H-Tyr-Pro-Phe-Phe-OH (3), were synthetized, and the levels of their resistance against carboxypeptidase A, carboxypeptidase Y, aminopeptidase M and proteinase A were determined. The patterns of peptide metabolites identified by this method indicated that carboxypeptidase Y first hydrolyzes the C-terminal amide group to a carboxy group, and then splits the peptides at the Trp3-Phe4 or Phe3-Phe4 bond. The remaining fragment peptides are stable against the enzymes investigated. Carboxypeptidase A degrades only analog 3 at the Phe3-Phe4 bond. Aminopeptidase M cleaves the peptides at the Pro2-Trp3 or Pro2-Phe3 bond. The C-terminal fragments hydrolyze further, giving amino acids and Phe-NH2-s while the N-terminal part displays a resistance to further aminopeptidase M digestion. Proteinase A exhibits a similar effect to carboxypeptidase Y: the C-terminal amide group is first converted to a carboxy group, and one amino acid is then split off from the C-terminal side.

  12. 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...... membrane trafficking in enterocytes. Cultured mucosal explants of pig small intestine were treated for 2 h with the cholesterol sequestering agent methyl-beta-cyclodextrin and lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase. The treatment reduced the cholesterol content >50...

  13. 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.

  14. 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...

  15. Dipeptidylpeptidase IV and trypsin-like enzymatic degradation of human growth hormone-releasing hormone in plasma.

    OpenAIRE

    Frohman, L A; Downs, T. R.; Heimer, E P; Felix, A M

    1989-01-01

    The plasma enzyme responsible for primary proteolytic cleavage of growth hormone-releasing hormone (GRH) at the 2-3 amino acid bond was characterized. Native GRH[GRH(1-44)-NH2 and GRH(1-40)-OH], and COOH-terminally shortened fragments [GRH(1-32)-NH2 and GRH(1-29)-NH2] were rapidly cleaved, while GRH(2-32)-NH2 was not degraded at this site. Moreover, degradation to GRH(3-44)-NH2 was unaffected by an aminopeptidase inhibitor, indicating that this metabolite was generated from a single step clea...

  16. The research of neurospecific proteins and lysosomal peptidehydrolases in frontal neocortex during forming conditioned reaction of active avoiding of rats

    Directory of Open Access Journals (Sweden)

    Vyatkin O. K.

    2009-04-01

    Full Text Available Dynamics of the activity of neuronal cell adhesion molecule (NCAM and lysosomal cysteine cathepsins B, L, H was researched in frontal neocortex of rat brain during forming a conditioned reaction of active avoiding. The quantitative estimation of NCAM content in the neocortex membrane fraction was carried on by ELISA in 3, 7, 14 and 21 days after starting animals’ training. The dynamics correlation between the NCAM content increasing and cysteine cathepsins activity was obtained, especially for aminopeptidase cathepsin H during the process of memory engram forming in frontal neocortex of rat brain.

  17. The fine structure of the excretory system in adult Nippostrongylus brasiliensis (Nematoda) and a suggested function for the 'excretory glands'.

    Science.gov (United States)

    Lee, D L

    1970-01-01

    The ultrastructure of the excretory system, including the subventral glands, of the nematode Nippostrongylus brasiliensis has been described. The walls of the lateral excretory canals contain canaliculi which open into the lumen of the canal. It is suggested that these canals play a role in osmoregulation and excretion. The sub-ventral glands contain two types of secretory granule and contain non-specific esterase, cholinesterase and aminopeptidase. It is suggested that these glands are not excretory but play an important role in feeding.

  18. AcEST: DK960899 [AcEST

    Lifescience Database Archive (English)

    Full Text Available Definition sp|P70298|CUX2_MOUSE Homeobox protein cut-like 2 OS=Mus musculus Align length 34 Score (bit) 29.6...................................................done Score E Sequences producing significant alignments: (bi...lus GN=Cux2 PE=2 SV=1 Length = 1426 Score = 29.6 bits (65), Expect = 4.9 Identities = 11/34 (32%), Positives...phosphate isomerase OS=Silicibacter sp. (strain TM1040) GN=pgi PE=3 SV=1 Length = 536 Score = 28.9 bits (63)...V Probable cytosol aminopeptidase OS=Chlamydophila caviae GN=pepA PE=3 SV=1 Length = 499 Score = 28.9 bits (

  19. 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....

  20. 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....

  1. Silencing of CYP6 and APN Genes Affects the Growth and Development of Rice Yellow Stem Borer, Scirpophaga incertulas

    OpenAIRE

    Vijaya Sudhakara Rao eKola; P eRenuka; Ayyagari Phani Padmakumari; Satendra Kumar Mangrauthia; Balachandran eS M; MAGANTI SHESHU MADHAV

    2016-01-01

    RNAi is a powerful tool to target the insect genes involved in host-pest interactions. Key insect genes are the choice for silencing to achieve pest derived resistance where resistance genes are not available in gene pool of host plant. In this study, an attempt was made to determine the effect of dsRNA designed from two genes Cytochrome P450 derivative (CYP6) and Aminopeptidase N (APN) of rice yellow stem borer (YSB) on growth and development of insect. The bioassays involved injection of ch...

  2. 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.

  3. Purification and characterization of a T-antigen specific lectin from the coelomic fluid of a marine invertebrate, sea cucumber (Holothuria scabra)

    Digital Repository Service at National Institute of Oceanography (India)

    Gowda, N.M.; Goswami, U.; Khan, M.I.

    Purification and characterization of a T-antigen specific lectin from the coelomic fluid of a marine invertebrate, sea cucumber (Holothuria scabra) Nagaraj M. Gowda 1,2 , Usha Goswami 1 and M. Islam Khan 2* 1 Gene lab, National Institute of Oceanography...–D-galactose, D-maltose, α-lactose, α-D- melibiose, raffinose, stachyose, phenyl-sepharose CL-4B, pronase-E, carboxypeptidase, aminopeptidase, fetuin (bovine), fibrinogen (human), thyroglobulin (bovine), were obtained from Sigma chemical Co. St. Louis, U.S.A...

  4. Enzymatic profile of Haemophilus ducreyi

    Energy Technology Data Exchange (ETDEWEB)

    Casin, I.M.; Sanson-Le Pors, M.J.; Gorce, M.F.; Ortenberg, M.; Perol, Y. (Universite Paris - 7, Hopital Saint-Louis, 75 - Paris (France))

    The enzymatic activities of two reference strains of Haemophilus ducreyi and thirty clinical isolates were investigated by conventional biochemical tests and the API-ZYM test kit system which included 97 synthetic substrates. No strains converted ..delta..-aminolevulinic acid to porphyrins, but they all reduced nitrates to nitrites. All strains possessed aminopeptidase activity against ..beta..-naphthylamide derivatives of L-alanine, L-arginine, L-glutamine, glycine, L-leucine, L-lysine and L-serine. No trypsin or chymotrypsin-like activities were detected. All strains had phosphatase activity with broad pH range, and phosphoamidase activity. No glycosidase was detected by the substrates tested.

  5. Is rumen development in newborn calves affected by different liquid feeds and small intestine development?

    Science.gov (United States)

    Górka, P; Kowalski, Z M; Pietrzak, P; Kotunia, A; Jagusiak, W; Zabielski, R

    2011-06-01

    The objective of the study was to determine the effect of different liquid feeds on calf small intestine and rumen development. Twenty-one bull calves (5 ± 1 d old) were randomly allocated to 3 groups and fed whole milk (WM), milk replacer (MR; 22% CP and 17.5% fat), or MR supplemented with sodium butyrate (MR+SB; 0.3% as fed). Liquid feed dry matter intake was equal between treatments and amounted to 1% of BW at the beginning of the trial. Starter diet was offered ad libitum. Animals were slaughtered at 26 (± 1) d of age. Calves fed WM had higher average daily gain in the whole trial and higher starter diet dry matter intake between d 15 to 21 of the trial as compared with calves fed MR and MR+SB. Calves fed MR lost on average 1.4 kg of BW within first 14 d of the trial and their BW tended to be lower at d 7, 14, and 21 of the study as compared with calves fed MR+SB. The empty jejunum and ileum weight, crypt depth, mitotic index in the middle jejunum were higher, and apoptotic index tended to be lower in calves fed WM as compared with calves fed MR and MR+SB. Calves fed WM also had higher aminopeptidase N activity in the middle jejunum and tended to have higher maltase activity in the distal jejunum as compared with calves fed MR and MR+SB. The mitotic index was higher and apoptotic index was lower in the middle jejunum, and aminopeptidase A activity tended to be higher in the distal jejunum of calves fed MR+SB as compared with those fed MR. Calves fed WM had greater papillae length and width, and tended to have greater muscle layer thickness as compared with calves fed MR and MR+SB. Reticulorumen weight, reticulorumen weight expressed as percent of whole stomach weight, and papillae length and width were higher in calves fed MR+SB as compared with those fed MR. Additionally, calves fed WM had higher plasma glucose and urea in the whole trial period as compared with calves fed MR and MR+SB, and plasma glucose was higher in calves fed MR+SB as compared with those

  6. Isolation of L-3-phenyllactyl-Leu-Arg-Asn-NH2 (Antho-RNamide), a sea anemone neuropeptide containing an unusual amino-terminal blocking group

    DEFF Research Database (Denmark)

    Grimmelikhuijzen, C J; Rinehart, K L; Jacob, E

    1990-01-01

    -phenyllactyl-Leu-Arg-Asn-NH2. By using reversed-phase HPLC and a chiral mobile phase, it was shown that the 3-phenyllactyl group had the L configuration. Immunocytochemical staining with antiserum against Arg-Asn-NH2 showed that L-3-phenyllactyl-Leu-Arg-Asn-NH2 (Antho-RNamide) was localized in neurons of sea...... anemones. The L-3-phenyllactyl group has not been found earlier in neuropeptides of vertebrates or higher invertebrates. We propose that this residue renders Antho-RNamide resistant to nonspecific aminopeptidases, thereby increasing the stability of the peptide after neuronal release....

  7. 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....

  8. Antibody Targeting of Caveolae in Breast Tumors

    Science.gov (United States)

    2004-08-01

    TIE2, RAGE, Glut4 , membrane. Yet, despite rigorous quality testing of each membrane ZO- 1, ICAM- 1, thrombomodulin, CD39 and aminopeptidase P were...identified 21 known Glut4 ). See Supplementary Notes online for an expanded description of blood proteins. Given that there are >500 blood plasma proteins...palmitate Giantin 516826 063714 4 - 501 364297 other 1 Glut4 4193489 Q9Z1X1 4 6 547 121159 transport 1 Wigh affinity ne-v growth factor receptor (TRK-A

  9. 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……

  10. Toxin-binding proteins isolated from yellow mealworm Tenebrio molitor and wax moth Galleria mellonella.

    Science.gov (United States)

    Bulushova, N V; Zhuzhikov, D P; Lyutikova, L I; Kirillova, N E; Zalunin, I A; Chestukhina, G G

    2011-02-01

    A 67-kDa protein that can specifically bind the activated Cry9A endotoxin under ligand-blotting conditions was purified from midgut epithelium apical membranes of wax moth Galleria mellonella by affinity chromatography. N-Terminal amino acid sequencing enabled identification of this protein as aminopeptidase N. In similar experiments, 66- and 58-kDa proteins specific to endotoxin Cry3A were isolated from the midgut epithelium apical membranes of Tenebrio molitor larvae. Mass spectrometry showed close similarity of the 58-kDa protein to the Tenebrio molitor α-amylase.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 27-Hydroxycholesterol impairs neuronal glucose uptake through an IRAP/GLUT4 system dysregulation.

    Science.gov (United States)

    Ismail, Muhammad-Al-Mustafa; Mateos, Laura; Maioli, Silvia; Merino-Serrais, Paula; Ali, Zeina; Lodeiro, Maria; Westman, Eric; Leitersdorf, Eran; Gulyás, Balázs; Olof-Wahlund, Lars; Winblad, Bengt; Savitcheva, Irina; Björkhem, Ingemar; Cedazo-Mínguez, Angel

    2017-02-17

    Hypercholesterolemia is associated with cognitively deteriorated states. Here, we show that excess 27-hydroxycholesterol (27-OH), a cholesterol metabolite passing from the circulation into the brain, reduced in vivo brain glucose uptake, GLUT4 expression, and spatial memory. Furthermore, patients exhibiting higher 27-OH levels had reduced (18)F-fluorodeoxyglucose uptake. This interplay between 27-OH and glucose uptake revealed the engagement of the insulin-regulated aminopeptidase (IRAP). 27-OH increased the levels and activity of IRAP, countered the IRAP antagonist angiotensin IV (AngIV)-mediated glucose uptake, and enhanced the levels of the AngIV-degrading enzyme aminopeptidase N (AP-N). These effects were mediated by liver X receptors. Our results reveal a molecular link between cholesterol, brain glucose, and the brain renin-angiotensin system, all of which are affected in some neurodegenerative diseases. Thus, reducing 27-OH levels or inhibiting AP-N maybe a useful strategy in the prevention of the altered glucose metabolism and memory decline in these disorders.

  16. 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.

  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. 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.

  19. 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.

  20. 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.

  1. The pro-inflammatory cytokine 14-3-3ε is a ligand of CD13 in cartilage

    Science.gov (United States)

    Nefla, Meriam; Sudre, Laure; Denat, Guillaume; Priam, Sabrina; Andre-Leroux, Gwenaëlle; Berenbaum, Francis; Jacques, Claire

    2015-01-01

    ABSTRACT Osteoarthritis is a whole-joint disease characterized by the progressive destruction of articular cartilage involving abnormal communication between subchondral bone and cartilage. Our team previously identified 14-3-3ε protein as a subchondral bone soluble mediator altering cartilage homeostasis. The aim of this study was to investigate the involvement of CD13 (also known as aminopeptidase N, APN) in the chondrocyte response to 14-3-3ε. After identifying CD13 in chondrocytes, we knocked down CD13 with small interfering RNA (siRNA) and blocking antibodies in articular chondrocytes. 14-3-3ε-induced MMP-3 and MMP-13 was significantly reduced with CD13 knockdown, which suggests that it has a crucial role in 14-3-3ε signal transduction. Aminopeptidase N activity was identified in chondrocytes, but the activity was unchanged after stimulation with 14-3-3ε. Direct interaction between CD13 and 14-3-3ε was then demonstrated by surface plasmon resonance. Using labeled 14-3-3ε, we also found that 14-3-3ε binds to the surface of chondrocytes in a manner that is dependent on CD13. Taken together, these results suggest that 14-3-3ε might directly bind to CD13, which transmits its signal in chondrocytes to induce a catabolic phenotype similar to that observed in osteoarthritis. The 14-3-3ε–CD13 interaction could be a new therapeutic target in osteoarthritis. PMID:26208633

  2. 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.

  3. 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.

  4. 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.

  5. Silencing of CYP6 and APN genes affects the growth and development of rice yellow stem borer, Scirpophaga incertulas

    Directory of Open Access Journals (Sweden)

    Vijaya Sudhakara Rao eKola

    2016-02-01

    Full Text Available RNAi is a powerful tool to target the insect genes involved in host-pest interactions. Key insect genes are the choice for silencing to achieve pest derived resistance where resistance genes are not available in gene pool of host plant. In this study, an attempt was made to determine the effect of dsRNA designed from two genes Cytochrome P450 derivative (CYP6 and Aminopeptidase N (APN of rice yellow stem borer (YSB on growth and development of insect. The bioassays involved injection of chemically synthesized 5ꞌ FAM labeled 21-nt dsRNA into rice cut stems and allowing the larvae to feed on these stems which resulted in increased mortality and observed growth and development changes in larval length and weight compared with its untreated control at 12-15 days after treatment. These results were further supported by observing the reduction in transcripts expression of these genes in treated larvae. Fluorescence detection in treated larvae also proved that dsRNA was readily taken by larvae when fed on dsRNA treated stems. These results from the present study clearly show that YSB larvae fed on dsRNA designed from Cytochrome P450 and Aminopeptidase N has detrimental effect on larval growth and development. These genes can be deployed to develop YSB resistance in rice using RNAi approach.

  6. Silencing of CYP6 and APN Genes Affects the Growth and Development of Rice Yellow Stem Borer, Scirpophaga incertulas.

    Science.gov (United States)

    Kola, Vijaya Sudhakara Rao; Renuka, P; Padmakumari, Ayyagari Phani; Mangrauthia, Satendra K; Balachandran, Sena M; Ravindra Babu, V; Madhav, Maganti S

    2016-01-01

    RNAi is a powerful tool to target the insect genes involved in host-pest interactions. Key insect genes are the choice for silencing to achieve pest derived resistance where resistance genes are not available in gene pool of host plant. In this study, an attempt was made to determine the effect of dsRNA designed from two genes Cytochrome P450 derivative (CYP6) and Aminopeptidase N (APN) of rice yellow stem borer (YSB) on growth and development of insect. The bioassays involved injection of chemically synthesized 5' FAM labeled 21-nt dsRNA into rice cut stems and allowing the larvae to feed on these stems which resulted in increased mortality and observed growth and development changes in larval length and weight compared with its untreated control at 12-15 days after treatment. These results were further supported by observing the reduction in transcripts expression of these genes in treated larvae. Fluorescence detection in treated larvae also proved that dsRNA was readily taken by larvae when fed on dsRNA treated stems. These results from the present study clearly show that YSB larvae fed on dsRNA designed from Cytochrome P450 and Aminopeptidase N has detrimental effect on larval growth and development. These genes can be deployed to develop YSB resistance in rice using RNAi approach.

  7. 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.

  8. Induced and constitutive responses of digestive enzymes to plant toxins in an herbivorous mammal.

    Science.gov (United States)

    Kohl, Kevin D; Dearing, M Denise

    2011-12-15

    Many plants produce plant secondary compounds (PSCs) that bind and inhibit the digestive enzymes of herbivores, thus limiting digestibility for the herbivore. Herbivorous insects employ several physiological responses to overcome the anti-nutritive effects of PSCs. However, studies in vertebrates have not shown such responses, perhaps stemming from the fact that previously studied vertebrates were not herbivorous. The responses of the digestive system to dietary PSCs in populations of Bryant's woodrat (Neotoma bryanti) that vary in their ecological and evolutionary experience with the PSCs in creosote bush (Larrea tridentata) were compared. Individuals from naïve and experienced populations were fed diets with and without added creosote resin. Animals fed diets with creosote resin had higher activities of pancreatic amylase, as well as luminal amylase and chymotrypsin, regardless of prior experience with creosote. The experienced population showed constitutively higher activities of intestinal maltase and sucrase. Additionally, the naïve population produced an aminopeptidase-N enzyme that was less inhibited by creosote resin when feeding on the creosote resin diet, whereas the experienced population constitutively expressed this form of aminopeptidase-N. Thus, the digestive system of an herbivorous vertebrate responds significantly to dietary PSCs, which may be important for allowing herbivorous vertebrates to feed on PSC-rich diets.

  9. 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.

  10. Identification of midgut microvillar proteins from Tenebrio molitor and Spodoptera frugiperda by cDNA library screenings with antibodies.

    Science.gov (United States)

    Ferreira, A H P; Cristofoletti, P T; Lorenzini, D M; Guerra, L O; Paiva, P B; Briones, M R S; Terra, W R; Ferreira, C

    2007-11-01

    The objective of this study was to identify midgut microvillar proteins in insects appearing earlier (Coleoptera) and later (Lepidoptera) in evolution. For this, cytoskeleton-free midgut microvillar membrane from Spodoptera frugiperda (Lepidoptera) and Tenebrio molitor (Coleoptera) were used to raise antibodies. These were used for screening midgut cDNA expression libraries. Positive clones were sequenced, assembled and searched for similarities with gene/protein databases. The predicted midgut microvillar proteins from T. molitor were: cockroach allergens (unknown function), peritrophins (peritrophic membrane proteins), digestive enzymes (aminopeptidase, alpha-mannosidase) and unknown proteins. Predicted S. frugiperda midgut proteins may be grouped into six classes: (a) proteins involved in protection of midgut (thioredoxin peroxidase, aldehyde dehydrogenase, serpin and juvenile hormone epoxide hydrolase); (b) digestive enzymes (astacin, transporter-like amylase, aminopeptidase, and carboxypeptidase); (c) peritrophins; (d) proteins associated with microapocrine secretion (gelsolin, annexin); (e) membrane-tightly bound-cytoskeleton proteins (fimbrin, calmodulin) and (f) unidentified proteins. The novel approach is compared with others and microvillar function is discussed in the light of the predicted proteins.

  11. 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

  12. 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

  13. 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

  14. 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.

  15. Effect of method of delivery of sodium butyrate on maturation of the small intestine in newborn calves.

    Science.gov (United States)

    Górka, P; Pietrzak, P; Kotunia, A; Zabielski, R; Kowalski, Z M

    2014-02-01

    The effect of sodium butyrate (SB) supplementation in milk replacer (MR), starter mixture (SM), or both on small intestine maturation in newborn calves was investigated. Twenty-eight male calves with a mean age of 5 (± 1) d were randomly allocated into 1 of 4 groups (7 animals per group) and fed (1) MR and SM, without SB (MR(-) and SM(-), respectively; MR(-)/SM(-)); (2) MR(-) and SM supplemented with SB encapsulated within triglyceride matrix (SM(+), 0.6% as fed; MR(-)/SM(+)); (3) MR supplemented with crystalline SB (MR(+), 0.3% as fed) and SM(-) (MR(+)/SM(-)); or (4) MR(+) and SM(+) (MR(+)/SM(+)). The MR was offered in amounts equal to 10% of initial body weight of the calf. The SM was blended with whole corn grain (50/50; wt/wt) and offered ad libitum as a starter diet. Calves were slaughtered at 26 d (± 1) of age and small intestine development was investigated. Treatment with MR(+) decreased villus height in the proximal jejunum and decreased villus height, crypt depth, and tunica mucosa thickness in the middle jejunum, whereas treatment with SM(+) tended to increase small intestine weight and crypt depth in the proximal jejunum, and increased villus height in the distal jejunum. In the duodenum, crypt depth and tunica mucosa thickness were greater for the MR(-)/SM(+) group compared with MR(-)/SM(-), MR(+)/SM(-), and MR(+)/SM(+) groups. In the ileum, crypt depth was less for MR(-)/SM(+) compared with MR(-)/SM(-). Supplementation with SB in both MR and SM enhanced cell proliferation and decreased apoptosis in the middle jejunum mucosa. Regarding brush border enzyme activities, addition of SB to MR increased lactase activity in the middle jejunum and maltase activity in the distal jejunum, and tended to increase lactase activity in the distal jejunum, aminopeptidase A activity in the middle jejunum and ileum, and aminopeptidase N activity in the ileum. In contrast, SM(+) increased dipeptidylpeptidase IV activity in the distal jejunum and tended to increase

  16. 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.

  17. Proteomic Analysis of Responsive Proteins Induced in Japanese Birch Plantlet Treated with Salicylic Acid

    Science.gov (United States)

    Suzuki, Hiromu; Takashima, Yuya; Ishiguri, Futoshi; Yoshizawa, Nobuo; Yokota, Shinso

    2014-01-01

    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.

  18. Ectopic cervical thymoma mimicking as papillary thyroid carcinoma: A diagnostic dilemma

    Directory of Open Access Journals (Sweden)

    Thakur Abhijit

    2010-04-01

    Full Text Available Ectopic cervical thymomas are often confused with thyroid or parathyroid swellings due to their anatomical positioning. Predominant epithelial thymoma can be misdiagnosed as papillary thyroid carcinoma on fine needle aspiration and lymph node metastasis of epithelial tumor on frozen section. Predominantly lymphocytic thymomas have often been misinterpreted as Hashimoto′s thyroiditis or malignant lymphoma, either by fine needle aspiration or on frozen section analysis. If cytology is doubtful and is not correlating with clinical, anatomical and surgical findings; immunohistochemistry is a very important tool in such cases to give final answer. Thyroid cell specific proteins such as thyroglobulin, thyroid transcription factor-1, thyroperoxidase and dipeptidyl aminopeptidase-4, neuroendocrine markers chromogranin, calcitonin and parathyroid hormone could be used to rule out thyroid or parathyroid origin. We present such rare case of ectopic cervical thymoma mimicking as papillary thyroid carcinoma.

  19. [Morphological changes in the digestive organs during prolonged space flight on the Kosmos-782 biosatellite].

    Science.gov (United States)

    Loginov, A S; Aruin, L I; Brodskiĭ, R A; Morozov, I A; Permiakov, N K

    1978-01-01

    A reduction in the content of neutral mucopolysaccharides in mucous cells of the neck, a slight decrease in the activity of succinate dehydrogenase and NAD-diaphorase in parietal cells, a decrease in the DNA synthesis rate, and an increase in the area of mitochondria and cristae were detected in the gastric mucosa of rats which were in a long-term space flight. In the small intestine, an increase in the activity of glucose-6-phosphate dehydrogenase and leucine aminopeptidase were found. Morphological changes in the liver consisted in infiltrative adiposity. A similar morphological picture was demonstrated in a synchronous experiment on the earth. These changes, however, were nonspecific and reversible (25 days after rehabilitation the picture did not differ from the animal house control).

  20. 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...

  1. 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.

  2. 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.

  3. 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...... that a formula containing maltodextrin vs. a formula containing lactose as the principal source of carbohydrate would predispose preterm pigs to a higher NEC incidence. Cesarean-derived preterm pigs were given total parenteral nutrition for 48 h followed by total enteral nutrition with a lactose-based (n = 11...... acids were observed in the maltodextrin group. In a second study, we quantified net portal absorption of aldohexoses (glucose and galactose) during acute jejunal infusion of a maltodextrin-or a lactose-based formula (n = 8) into preterm pigs. We found lower net portal aldohexose absorption (4% vs. 42...

  4. Arginyltransferase ATE1 catalyzes midchain arginylation of proteins at side chain carboxylates in vivo.

    Science.gov (United States)

    Wang, Junling; Han, Xuemei; Wong, Catherine C L; Cheng, Hong; Aslanian, Aaron; Xu, Tao; Leavis, Paul; Roder, Heinrich; Hedstrom, Lizbeth; Yates, John R; Kashina, Anna

    2014-03-20

    Arginylation is an emerging posttranslational modification mediated by Arg-tRNA-protein-transferase (ATE1). It is believed that ATE1 links Arg solely to the N terminus of proteins, requiring prior proteolysis or action by Met-aminopeptidases to expose the arginylated site. Here, we tested the possibility of Arg linkage to midchain sites within intact protein targets and found that many proteins in vivo are modified on the side chains of Asp and Glu by unconventional chemistry that targets the carboxy rather than the amino groups at the target sites. Such arginylation appears to be functionally regulated, and it can be directly mediated by ATE1, in addition to the more conventional ATE1-mediated linkage of Arg to the N-terminal alpha amino group. This midchain arginylation implies an unconventional mechanism of ATE1 action that likely facilitates its major biological role.

  5. Arginyltransferase ATE1 catalyzes mid-chain arginylation of proteins at side chain carboxylates in vivo

    Science.gov (United States)

    Wang, Junling; Han, Xuemei; Wong, Catherine C.L.; Cheng, Hong; Aslanian, Aaron; Xu, Tao; Leavis, Paul; Roder, Heinrich; Hedstrom, Lizbeth; Yates, John R.; Kashina, Anna

    2014-01-01

    Summary Arginylation is an emerging posttranslational modification mediated by Arg-tRNA-protein-transferase (ATE1). It is believed that ATE1 links Arg solely to the N-terminus of proteins, requiring prior proteolysis or action by Met-aminopeptidases to expose the arginylated site. Here, we tested the possibility of Arg linkage to mid-chain sites within intact protein targets and found that many proteins in vivo are modified on the side chains of Asp and Glu by a novel chemistry that targets the carboxy rather than the amino groups at the target sites. Such arginylation appears to be functionally regulated, and it can be directly mediated by ATE1, in addition to the more conventional Ate1-mediated linkage of Arg to the N-terminal alpha amino group. This new type of arginylation implies an unconventional mechanism of ATE1 action that likely facilitates its major biological role. PMID:24529990

  6. Rapid screening of peptide probes through in situ single-bead sequencing microarray.

    Science.gov (United States)

    Wang, Weizhi; Wei, Zewen; Zhang, Di; Ma, Huailei; Wang, Zihua; Bu, Xiangli; Li, Menglin; Geng, Lingling; Lausted, Christopher; Hood, Leroy; Fang, Qiaojun; Wang, Hao; Hu, Zhiyuan

    2014-12-02

    Peptide ligands as targeting probes for in vivo imaging and drug delivery have attracted great interest in the biomedical community. However, high affinity and specificity screening of large peptide libraries remains a tedious process. Here, we report a continuous-flow microfluidic method for one-bead-one-compound (OBOC) combinatorial peptide library screening. We screened a library with 2 × 10(5) peptide beads within 4 h and discovered 140 noncanonical peptide hits targeting the tumor marker, aminopeptidase N (APN). Using the Clustal algorithm, we identified the conserved sequence Tyr-XX-Tyr in the N terminal. We demonstrated that the novel sequence YVEYHLC peptides have both nanomolar affinity and high specificity for APN in ex vivo and in vivo models. We envision that the successful demonstration of this integrated novel nanotechnology for peptide screening and identification open a new avenue for rapid discovery of new peptide-based reagents for disease diagnostics and therapeutics.

  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. Role in Cheese Flavour Formation of Heterofermentative Lactic Acid Bacteria from Mesophilic Starter Cultures

    DEFF Research Database (Denmark)

    Pedersen, Thomas Bæk

    Undefined mesophilic cheese starters are complex ecosystems that contain both homofermentative and heterofermentative lactic acid bacteria, with the Lactococcus genera representing the former and Lceuonostoc and sometimes Lactobacillus the latter. These starters originate from old butter starters...... aminopeptidase activity compared to Lactobacillus danicus and especially Le. mesenteroides subsp. cremoris had a low and narrow activity. Aminotransferase activity was high on aromatic amino acids for Lb. danicus, and the Leuconostoc species had an activity similar to Lb. danicus only after growth in CBM...... with plant isolates, the ability to ferment citrate and lacked several genes involved in the fermentation of complex carbohydrates. The presented research in this thesis has gained insight in to the role of heterofermentative lactic acid bacteria in cheese flavour formation. The traditional DL...

  9. Virtual Screening for Transition State Analogue Inhibitors of IRAP Based on Quantum Mechanically Derived Reaction Coordinates.

    Science.gov (United States)

    Svensson, Fredrik; Engen, Karin; Lundbäck, Thomas; Larhed, Mats; Sköld, Christian

    2015-09-28

    Transition state and high energy intermediate mimetics have the potential to be very potent enzyme inhibitors. In this study, a model of peptide hydrolysis in the active site of insulin-regulated aminopeptidase (IRAP) was developed using density functional theory calculations and the cluster approach. The 3D structure models of the reaction coordinates were used for virtual screening to obtain new chemical starting points for IRAP inhibitors. This mechanism-based virtual screening process managed to identify several known peptidase inhibitors from a library of over 5 million compounds, and biological testing identified one compound not previously reported as an IRAP inhibitor. This novel methodology for virtual screening is a promising approach to identify new inhibitors mimicking key transition states or intermediates of an enzymatic reaction.

  10. 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....

  11. 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.

  12. 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...... nonmalignant and PC tissue samples, and in cell lines.Results:The APN expression was significantly downregulated in PC compared with nonmalignant prostate tissue samples. Aberrant promoter hypermethylation was frequently observed in PC tissue samples, and 5-aza-2'-deoxycytidine induced ANPEP expression...... in three hypermethylated prostate cell lines, suggesting epigenetic silencing. Negative APN immunoreactivity was significantly associated with short RFS and short CSS in the RP and CT cohort, respectively, independently of routine clinicopathological predictors. Combining APN with a known angiogenesis...

  13. 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

  14. 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

    Recombinant protein G (RPG) was conjugated to colloidal gold particles and used for immunocytochemistry. In this report, the preparation of RPG-gold conjugates (RPGG) and the application of these conjugates in spot blot tests and in double immunolabelling are described. The immunolabelling...... 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...... intensity was dependent on the size of the marker as well as on the kind of protein used for complex formation. The distributions for RPGG and PAG were respectively: for the 12 nm particles, 10.3 and 6.2 particles/micron of length of microvillar membrane, 3.5 and 1.0 particles/micron2 of Golgi profile and 5...

  15. 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

  16. Polimorfismo enzimático em populações de Melipona quadrifasciata anthidioides Lepeletier (Hymenoptera, Apidae, Meliponinae Enzymatic polymorphism in Melipona quadrifasciata anthidioides Lepeletier populations (Hymenoptera, Apidae, Meliponinae

    Directory of Open Access Journals (Sweden)

    Davi S. Aidar

    2001-06-01

    Full Text Available Them aim scope of this study is to characterize the enzymatic polymorphism found in the Melipona quadrifasciata Lepeletier, 1936 populations from Ribeirão Preto, São Paulo and Espírito Santo, Brazil and its hybrids. Samples from each colony (about 52 were prepared for starch gel electrophoresis in order to investigate the genetic variation of the following enzimes: esterase (EST, isocitrate dehydrogenase (IDH, malic enzyme (ME, phosphoglucomutase (PGM, superoxide desmutase (SOD, α-glycerophosphate dehydrogenase (αPGD, malate dehydrogenase (MDH, leucine aminopeptidase (LAP, hexokinase (HK and phosphoglucose isomerase (PGI. The analysis showed that LAP and HK did not show enzymatic activity and EST showed two alleles(est-sand and est-f while all the others were shown to be monomorphic. The allele EST-S showed a frequency of 82,6%.

  17. 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.

  18. [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.

  19. 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.

  20. 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.

  1. 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.

  2. 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...

  3. 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.

  4. 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...

  5. 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.

  6. Staurosporine as an agonist for induction of GLUT4 translocation, identified by a pH-sensitive fluorescent IRAP-mOrange2 probe.

    Science.gov (United States)

    Li, Yufeng; Zheng, Li; Wang, Dan; Zhang, Xiang; Li, Jia; Ali, Sher; Lu, Jingze; Zong, Hao; Xu, Xiaolan

    2016-11-25

    Insulin-stimulated GLUT4 translocation from GLUT4 storage vesicles (GSVs) to the plasma membrane (PM) constitutes a key process for blood glucose control. Therefore, compounds that could promote GLUT4 translocation into the PM represent potential drugs for the treatment of diabetes. In this research, we screened for agonists that induce GLUT4 translocation by using a novel pH-sensitive fluorescent probe, insulin-regulated aminopeptidase (IRAP)-mOrange2. We identified as well as validated one agonist, staurosporine, from a 64,000 compound library. Staurosporine promotes GSVs translocation into the PM and increases glucose uptake through the AMP-activated protein kinase (AMPK) pathway, serving as an effective insulin additive analogue in L6 cells. Our work highlights the convenience and efficiency of this novel pH-sensitive fluorescent probe and reveals the new biological activity of staurosporine as an agonist for GLUT4 translocation and as an effective insulin additive analogue.

  7. A Tenebrio molitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles.

    Science.gov (United States)

    Zúñiga-Navarrete, Fernando; Gómez, Isabel; Peña, Guadalupe; Bravo, Alejandra; Soberón, Mario

    2013-03-01

    Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl-phosphatidyl-inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68 kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68 kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders.

  8. Generation, characterization and therapeutic potential of anti-feline TNF-alpha MAbs for feline infectious peritonitis.

    Science.gov (United States)

    Doki, Tomoyoshi; Takano, Tomomi; Nishiyama, Yuri; Nakamura, Michiyo; Hohdatsu, Tsutomu

    2013-12-01

    Feline infectious peritonitis (FIP) is a lethal infectious disease affecting domestic and wild cats. Several reports suggested that TNF-alpha is related to the progression of FIP. Thus, the administration of a feline TNF-alpha-neutralizing antibody to cats with FIP may reduce the disease progression. In this study, we have prepared nine monoclonal antibodies (MAbs) that recognize feline TNF-alpha. All MAbs neutralized recombinant TNF-alpha. The 50% inhibitory concentrations (IC50) of the MAbs for the cytotoxicity of recombinant TNF-alpha were 5-684 ng/ml. MAb 2-4 exhibited high neutralizing activity against natural TNF-alpha derived from FIPV-infected macrophages, and was confirmed to inhibit the following feline TNF-alpha-induced conditions in vitro: (i) an increase in the survival rate of neutrophils from cats with FIP, (ii) aminopeptidase N (APN) mRNA expression in macrophages, and (iii) apoptosis of a feline T-lymphocyte cell line.

  9. 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

  10. 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.

  11. 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.

  12. 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

    2015-01-01

    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

  13. The Synthesis of L-Alanyl and β-Alanyl Derivatives of 2-Aminoacridone and Their Application in the Detection of Clinically-Important Microorganisms.

    Directory of Open Access Journals (Sweden)

    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.

  14. 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.

  15. Leukotriene A4 hydrolase: Selective abrogation of leukotriene B4 formation by mutation of aspartic acid 375

    Science.gov (United States)

    Rudberg, Peter C.; Tholander, Fredrik; Thunnissen, Marjolein M. G. M.; Samuelsson, Bengt; Haeggström, Jesper Z.

    2002-01-01

    Leukotriene A4 (LTA4, 5S-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid) hydrolase (LTA4H)/aminopeptidase is a bifunctional zinc metalloenzyme that catalyzes the final and rate-limiting step in the biosynthesis of leukotriene B4 (LTB4, 5S,12R-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid), a classical chemoattractant and immune modulating lipid mediator. Two chemical features are key to the bioactivity of LTB4, namely, the chirality of the 12R-hydroxyl group and the cis-trans-trans geometry of the conjugated triene structure. From the crystal structure of LTA4H, a hydrophilic patch composed of Gln-134, Tyr-267, and Asp-375 was identified in a narrow and otherwise hydrophobic pocket, believed to bind LTA4. In addition, Asp-375 belongs to peptide K21, a previously characterized 21-residue active site-peptide to which LTA4 binds during suicide inactivation. In the present report we used site-directed mutagenesis and x-ray crystallography to show that Asp-375, but none of the other candidate residues, is specifically required for the epoxide hydrolase activity of LTA4H. Thus, mutation of Asp-375 leads to a selective loss of the enzyme's ability to generate LTB4 whereas the aminopeptidase activity is preserved. We propose that Asp-375, possibly assisted by Gln-134, acts as a critical determinant for the stereoselective introduction of the 12R-hydroxyl group and thus the biological activity of LTB4. PMID:11917124

  16. 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.

  17. 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

  18. Performance, organ zinc concentration, jejunal brush border membrane enzyme activities and mRNA expression in piglets fed with different levels of dietary zinc.

    Science.gov (United States)

    Martin, Lena; Pieper, Robert; Schunter, Nadine; Vahjen, Wilfried; Zentek, Jürgen

    2013-06-01

    This study aimed at investigating the effect of dietary zinc on performance, jejunal brush border membrane enzyme activities and mRNA levels of enzymes and two zinc transporters in piglets. A total of 126 piglets were weaned at 26 ±1 days of age and randomly allocated into three groups fed with diets 50, 150 and 2500 mg zinc/kg. Performance was recorded and at weekly intervals, eight piglets per group were killed. The activities of isolated brush border membrane enzymes including lactase, maltase, sucrase, aminopeptidase-N and intestinal alkaline phosphatase (IAP), and the relative transcript abundance of aminopeptidase-N (APN), sucrase-isomaltase (SUC), IAP and the two zinc transporters SLC39A4 (ZIP4) and SLC30A1 (ZnT1) were investigated in the jejunum. Feeding pharmacological zinc levels increased weight gain (p < 0.001) during the first week, but performance was lower (p < 0.05) in the third week. Organ zinc concentrations were increased by high dietary zinc level. The activity of IAP was higher (p < 0.05) with the highest dietary zinc level, no effects were determined for other enzymes. Dietary zinc level had no effect on transcript abundance of digestive enzymes. The mRNA levels decreased (p < 0.001) for ZIP4, and increased for ZnT1 (p < 0.05) with pharmacological zinc levels. In conclusion, pharmacological zinc levels improved performance in the short-term. Intestinal mRNA level of zinc transporters changed with high zinc supply, but this did not prevent zinc accumulation in tissues, suggesting hampered homoeostatic regulation. This might cause impaired performance during longer supply.

  19. 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.

  20. Determinação do modo de reprodução em capim-gordura (Melinis minutiflora Beauv. por padrões enzimáticos Detection of the mode of reproduction in molasses grass (Melinis minutiflora Beauv. by enzyme patterns

    Directory of Open Access Journals (Sweden)

    Lia Rejane Machado Silveira

    1996-04-01

    Full Text Available Com o objetivo de determinar o modo de reprodução do capim-gordura (Melinis minutiflora Beauv., 24 ecótipos da coleção da Universidade Federal de Viçosa foram utilizados em cruzamentos, após emasculação massal por imersão da inflorescência em água quente, testando-se quatro faixas de temperatura. Analisaram-se, comparativamente, os padrões de isozimas dos progenitores e da descendência proveniente de panículas de polinização aberta e de panículas submetidas aos tratamentos de proteção; polinização e proteção; emasculação e proteção; e emasculação, polinização e proteção. A hipótese da apomixia ser o mecanismo reprodutivo operante no capim-gordura foi bastante favorecida pela extensiva uniformidade da progênie e semelhança isozimática com o progenitor feminino. Contribuiu para isso o fato de toda a população amostrada apresentar um padrão de bandas característico de heterozigotos para os sistemas enzimáticos leucina aminopeptidase e malato desidrogenase, uma vez que a apomixia, ao contrário da autofecundação, facilita a manutenção da heterozigosidade. Segregação isozimática entre plantas dos ecótipos CG4 e CG5, relativamente ao sistema fosfatase ácida, sugere, no entanto, a ocorrência de alguma porcentagem de fecundação cruzada, que foi confirmada pela detecção de hibridação entre os ecótipos CG2 e CG13 nas isoesterases. Esses resultados conduzem à conclusão de que um mecanismo facultativo (parcialmente sexual de apomixia atua na reprodução do capim-gordura.Panicles of 24 molasses grass ecotypes were used in an experiment involving: bagging before anthesis; cross-pollination and bagging; emasculation and bagging; and emasculation, cross-pollination and bagging. Emasculation was accomplished by immersing the inflorescences into warm water, four temperature levels, for 12 minutes. The breeding system was accessed by comparing the isozyme patterns of the estereases, acid phosphatases

  1. 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.

  2. Enzimas digestivas do bicho-mineiro do cafeeiro Leucoptera coffeella (Guérin-Mèneville & Perrottet, 1842 (Lepidoptera: Lyonetiidae Coffee leaf miner's digestive enzymes Leucoptera coffeella (Guérin-Mèneville & Perrottet, 1842 (Lepidoptera: Lyonetiidae

    Directory of Open Access Journals (Sweden)

    Guilherme Duarte Rossi

    2009-01-01

    Full Text Available Os insetos possuem diferentes enzimas digestivas que catalisam as reações de hidrólise do alimento consumido e essas se diferenciam entre os insetos de acordo com suas dietas e estado fisiológico. Foram avaliadas as atividades de algumas enzimas digestivas da praga do cafeeiro - Leucoptera coffeella (Guérin-Mèneville & Perrottet, 1842 (Lepidoptera: Lyonetiidae - popularmente conhecida como bicho-mineiro do cafeeiro, para o entendimento de seu processo digestivo. Lagartas do bicho-mineiro do cafeeiro foram coletadas em campo e em casa-de-vegetação. O extrato enzimático utilizado foi obtido pela maceração das lagartas em água (4ºC. Determinaram-se os pH's ótimos e as atividades das enzimas α e β-glicosidases, α-amilase, aminopeptidase, fosfatase alcalina, sacarase, trealase e tripsina, incubando o extrato enzimático do bicho-mineiro do cafeeiro com substratos específicos. A análise dos resultados sugere que o processo digestivo e o ambiente intestinal do bicho-mineiro do cafeeiro sejam similares com o dos demais lepidópteros encontrados na literatura.Insects are fitted with different digestive enzymes that catalyses the food hydrolysis. Those enzymes differ from one insect to another according to their diets and physiological status. In this work, one intended to verify the activities of some digestive enzymes of the coffee leaf miner - Leucoptera coffeella (Guérin-Mèneville & Perrottet, 1842 (Lepidoptera: Lyonetiidae - as a pre-requisite to understand its digest process, since this insect is a major plague in coffee production systems. Coffee leaf miner caterpillars were collected in fields and in greenhouse. The enzyme extract utilized in determining the enzyme activities was obtained through grinding the caterpillars in cold water. The optimum pH and the activities of the enzymes α and β-glucosidases, α-amylase, aminopeptidase, alkaline phosphatase, saccharase, trehalase and trypsin were measured by incubating the

  3. Stimulated bacterial growth under elevated p CO₂: results from an off-shore mesocosm study.

    Directory of Open Access Journals (Sweden)

    Sonja Endres

    Full Text Available Marine bacteria are the main consumers of freshly produced organic matter. Many enzymatic processes involved in the bacterial digestion of organic compounds were shown to be pH sensitive in previous studies. Due to the continuous rise in atmospheric CO2 concentration, seawater pH is presently decreasing at a rate unprecedented during the last 300 million years but the consequences for microbial physiology, organic matter cycling and marine biogeochemistry are still unresolved. We studied the effects of elevated seawater pCO2 on a natural plankton community during a large-scale mesocosm study in a Norwegian fjord. Nine Kiel Off-Shore Mesocosms for Future Ocean Simulations (KOSMOS were adjusted to different pCO2 levels ranging initially from ca. 280 to 3000 µatm and sampled every second day for 34 days. The first phytoplankton bloom developed around day 5. On day 14, inorganic nutrients were added to the enclosed, nutrient-poor waters to stimulate a second phytoplankton bloom, which occurred around day 20. Our results indicate that marine bacteria benefit directly and indirectly from decreasing seawater pH. During the first phytoplankton bloom, 5-10% more transparent exopolymer particles were formed in the high pCO2 mesocosms. Simultaneously, the efficiency of the protein-degrading enzyme leucine aminopeptidase increased with decreasing pH resulting in up to three times higher values in the highest pCO2/lowest pH mesocosm compared to the controls. In general, total and cell-specific aminopeptidase activities were elevated under low pH conditions. The combination of enhanced enzymatic hydrolysis of organic matter and increased availability of gel particles as substrate supported up to 28% higher bacterial abundance in the high pCO2 treatments. We conclude that ocean acidification has the potential to stimulate the bacterial community and facilitate the microbial recycling of freshly produced organic matter, thus strengthening the role of the

  4. 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

  5. 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

  6. Variabilidade isoenzimática entre linhagens de amendoim resistentes à seca Isoenzimatic variability between peanut lines resistant to drought

    Directory of Open Access Journals (Sweden)

    Roseane Cavalcanti dos Santos

    2000-04-01

    Full Text Available O uso da técnica de eletroforese para separar múltiplas formas moleculares de enzimas tem sido bastante explorada na área biológica, cujas diferenças detectadas nos tecidos podem ser eficientemente usadas para diferenciação de cultivares em qualquer fase de seu desenvolvimento fenológico. Nesse trabalho, procedeu-se ao estudo da variabilidade isoenzimática em seis linhagens de amendoim resistentes à seca, com o objetivo de se verificar as possíveis relações da variação encontrada na base desses descritores com essa aptidão no amendoim. Estudaram-se folíolos da parte apical com 5 dias após a germinação, utilizando-se a técnica de eletroforese em gel de poliacrilamida (7% sistema horizontal e contínuo de tampão. Os sistemas estudados foram fosfatase ácida (ACP, malato desidrogenase (MDH, leucina aminopeptidase (LAP, peroxidase (PO, e esterase (EST. A caracterização fenotípica dos genótipos permitiu a separação de quatro grupos para ACP, três para LAP, dois para MDH e seis para PO e EST. A partir da análise dos componentes principais dos grupos obtidos, observou-se que a cultivar IAC Tupã (sensível à seca foi separada das demais, especialmente da cultivar resistente Senegal 55437.The use of electrophoretic techniques to separate multiple molecular forms of enzymes has been used in the biological science, where differences in isozymes among tissues can be used efficiently on cultivar differentiation during any life cycle phase. In this paper, the variability of six drought resistant peanut lines was studied by isozymes analysis aiming to verify the possible relations between enzymatic descriptors and drought resistance character. Leaflets were analyzed by horizontal poliacrylamide gel electrophoresis technique and buffer continuos systems for the following systems: acid phosphatase (ACP, malate dehydrogenase (MDH, leucine aminopeptidase (LAP, peroxidase (POX and esterase (EST. The phenotypic characterization of the

  7. [Effects of ischemia and revascularization on the epithelium of the small intestine: study on swine].

    Science.gov (United States)

    Barthod, F

    1994-05-01

    Ischaemia of the small intestine leads to the destruction of the intestinal mucosa. The capacity of the epithelium to regenerate is proportional to the duration of revascularization. The aim of this work was to analyze the kinetic aspects of intestinal epithelial regeneration after destruction due to prolonged ischaemia. This study was conducted in 44 animals (swine) after development of an ischaemia-revascularization protocol of a jejunal loop and bipolar secondary cutaneous exteriorization. After a first series with ischaemia times of 1, 2, 3 and 4 hours, the 4 hour period of ischaemia was chosen for further analysis of the regeneration kinetics over a period of 21 days since it leads to regular and total destruction of the epithelium compatible with regeneration. This analysis included (1) a histological examination (semi-thin slices), (2) immunofluorescent detection of intestinal brush border proteins on frozen slices (villin, saccharase-isomaltase, aminopeptidase N, dipeptidylpeptidase-IV) and mucines, (3) measurement of specific intestinal hydrolase activities (saccharase, aminopeptidase N, dipeptidylpeptidase-IV and alkaline phosphatase) in enriched brush border fractions, and (4) an analysis of variations in intestinal flora. After the 4 hour ischaemia, total destruction of the epithelium with disappearance of the villin and intestinal hydrolases and disorganization of the mucosa invaded by mucosal lacks was observed. Epithelial regeneration was rapid and two days later the histological aspect of the mucosa showed apical expression (still discontinuous), villin and intestinal hydrolase activity. Luminal apical expression of the markers became continuous on day 4, demonstrating the total recovery of the intestinal barrier as confirmed by stable microbial flora. Mucine expression also returned to normal. This regeneration was however incomplete since the mucosa was seen to be flat, without villosities. Immunofluorescence showed the weak intensity of brush

  8. 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

  9. 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

  10. Diagnosis of. Hepatocellular Carcinoma in Patients with Liver Cir­rhosis Using Liver Function Assays

    Directory of Open Access Journals (Sweden)

    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.

  11. 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.

  12. Allelic-specific expression in relation to Bombyx mori resistance to Bt toxin.

    Science.gov (United States)

    Chen, Yazhou; Li, Muwang; Islam, Iftakher; You, Lang; Wang, Yueqiang; Li, Zhiqian; Ling, Lin; Zeng, Baosheng; Xu, Jun; Huang, Yongping; Tan, Anjiang

    2014-11-01

    Understanding the mechanism of Bt resistance is one of the key elements of the effective application of Bt in pest control. The lepidopteran model insect, the silkworm, demonstrates qualities that make it an ideal species to use in achieving this understanding. We screened 45 strains of silkworm (Bombyx mori) using a Cry1Ab toxin variant. The sensitivity levels of the strains varied over a wide range. A resistant strain (P50) and a phylogenetically related susceptible strain (Dazao) were selected to profile the expressions of 12 Bt resistance-related genes. The SNPs in these genes were detected based on EST analysis and were validated by allelic-specific PCR. A comparison of allelic-specific expression between P50 and Dazao showed that the transcript levels of heterozygous genes containing two alleles rather than an imbalanced allelic expression contribute more to the resistance of P50 against Bt. The responses of the allelic-specific expression to Bt in hybrid larvae were then investigated. The results showed that the gene expression pattern of an ATP-binding cassette transporter C2 (ABCC2) and an aminopeptidase N (APN3), changed in an allelic-specific manner, with the increase of the resistant allele expression correlated with larval survival. The results suggest that a trans-regulatory mechanism in ABCC2 and APN3 allelic-specific expression is involved in the insect's response to the Bt toxin. The potential role of allelic-specific gene regulation in insect resistance to Bt toxins is discussed.

  13. Getting to the heart of the matter in cancer: Novel approaches to targeting cancer stem cells.

    Science.gov (United States)

    Colvin, Hugh; Mori, Masaki

    2017-01-01

    Cancer is one of the leading causes of deaths worldwide. While cancers may initially show good response to chemotherapy or radiotherapy, it is not uncommon for them to recur at a later date. This phenomenon may be explained by the existence of a small population of cancer stem cells, which are inherently resistant to anti-cancer treatment as well as being capable of self-renewal. Therefore, while most of the tumour bulk consisting of cells that are not cancer stem cells respond to treatment, the cancer stem cells remain, leading to disease recurrence. Following this logic, the effective targeting of cancer stem cells holds promise for providing long-term cure in individuals with cancer. Cancer stem cells, like normal stem cells are endowed with mechanisms to protect themselves against a wide range of insults including anti-cancer treatments, such as the enhancement of the DNA damage response and the ability to extrude drugs. It is therefore important to develop new strategies if cancer stem cells are to be eradicated. In this review, we describe the strategies that we have developed to target cancer stem cells. These strategies include the targeting of the histone demethylase jumonji, AT rich interactive domain 1B (JARID1B), which we found to be functionally significant in the maintenance of cancer stem cells. Other strategies being pursued include reprogramming of cancer stem cells and the targeting of a functional cell surface marker of liver cancer stem cells, the aminopeptidase CD13.

  14. Recent Developments on 1,2,4-Triazole Nucleus in Anticancer Compounds: A Review.

    Science.gov (United States)

    Kaur, Ramandeep; Dwivedi, Ashish Ranjan; Kumar, Bhupinder; Kumar, Vinod

    2016-01-01

    1,2,4-triazole is an important nucleus present in a large number of compounds. More than thirty-five compounds containing this nucleus are introduced into the market. 1,2,4-triazole nucleus is stable to metabolism and acts as an important pharmacophore by interacting at the active site of a receptor as hydrogen bond acceptor and as a donor. Due to its polar nature, the triazole nucleus can increase the solubility of the ligand and it can significantly improve the pharmacological profile of the drug. A large number of 1,2,4-triazole derivatives are reported to possess a wide range of bioactivities including anti-cancer activity. This review article describes the role of 1,2,4-triazole nucleus in different types of anti-cancer agents such as nucleoside based anti-cancer agents, kinase inhibitors, tubulin modulators, aromatase and steroid sulfatase inhibitors, methionine aminopeptidase inhibitors, tankyrase inhibitors and metal complex based anti-cancer agents. It is expected that the current review article will provide insight into various ligand-receptor interactions and help in the rational design and development of novel 1,2,4-triazole based anti-cancer drugs with improved selectivity for cancer cells.

  15. Preliminary Evaluation of Probiotic Properties of Lactobacillus Strains Isolated from Sardinian Dairy Products

    Directory of Open Access Journals (Sweden)

    Maria Barbara Pisano

    2014-01-01

    Full Text Available Twenty-three Lactobacillus strains of dairy origin were evaluated for some functional properties relevant to their use as probiotics. A preliminary subtractive screening based on the abilities to inhibit the growth of microbial pathogens and hydrolyze conjugated bile salts was applied, and six strains were selected for further characterization including survival under gastrointestinal environmental conditions, adhesion to gut epithelial tissue, enzymatic activity, and some safety properties. All selected strains maintained elevated cell numbers under conditions simulating passage through the human gastrointestinal tract, well comparable to the values obtained for the probiotic strain Lactobacillus rhamnosus GG, and were able to adhere to Caco-2 cells to various extents (from 3 to 20%. All strains exhibited high aminopeptidase, and absent or very low proteolytic and strong β-galactosidase activities; none was found to be haemolytic or to produce biogenic amines and all were susceptible to tetracycline, chloramphenicol, erythromycin, ampicillin, and amoxicillin/clavulanic acid. Our results indicate that the Lactobacillus strains analyzed could be considered appropriate probiotic candidates, due to resistance to GIT simulated conditions, antimicrobial activity, adhesion to Caco-2 cell-line, and absence of undesirable properties. They could be used as adjunct cultures for contributing to the quality and health related functional properties of dairy products.

  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. 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.

  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. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Particle-associated extracellular enzyme activity and bacterial community composition across the Canadian Arctic Ocean.

    Science.gov (United States)

    Kellogg, Colleen T E; Deming, Jody W

    2014-08-01

    Microbial enzymatic hydrolysis of marine-derived particulate organic carbon (POC) can be a dominant mechanism for attenuating carbon flux in cold Arctic waters during spring and summer. Whether this mechanism depends on composition of associated microbial communities and extends into other seasons is not known. Bacterial community composition (BCC) and extracellular enzyme activity (EEA, for leucine aminopeptidases, glucosidases and chitobiases) were measured on small suspended particles and potentially sinking aggregates collected during fall from waters of the biologically productive North Water and river-impacted Beaufort Sea. Although other environmental variables appeared influential, both BCC and EEA varied along a marine productivity gradient in the two regions. Aggregates harbored the most distinctive bacterial communities, with a small number of taxa driving differences between particle-size classes (1.0-60 and > 60 μm) and free-living bacteria (0.2-1.0 μm). Significant relationships between patterns in particle-associated BCC and EEA suggest strong links between these two variables. Calculations indicated that up to 80% of POC in the euphotic zone of the North Water, and 20% in the Beaufort Sea, may be hydrolyzed enzymatically, underscoring the importance of this mechanism in attenuating carbon fluxes in Arctic waters even as winter approaches.

  6. Targeted γ-Secretase Inhibition To Control the Notch Pathway in Renal Diseases.

    Science.gov (United States)

    Juillerat-Jeanneret, Lucienne; Flohr, Alexander; Schneider, Manfred; Walter, Isabelle; Wyss, Jean-Christophe; Kumar, Rajesh; Golshayan, Dela; Aebi, Johannes D

    2015-10-22

    Notch is a membrane inserted protein activated by the membrane-inserted γ-secretase proteolytic complex. The Notch pathway is a potential therapeutic target for the treatment of renal diseases but also controls the function of other cells, requiring cell-targeting of Notch antagonists. Toward selective targeting, we have developed the γ-secretase inhibitor-based prodrugs 13a and 15a as substrates for γ-glutamyltranspeptidase (γ-GT) and/or γ-glutamylcyclotransferase (γ-GCT) as well as aminopeptidase A (APA), which are overexpressed in renal diseases, and have evaluated them in experimental in vitro and in vivo models. In nondiseased mice, the cleavage product from Ac-γ-Glu-γ-secretase inhibitor prodrug 13a (γ-GT-targeting and γ-GCT-targeting) but not from Ac-α-Glu-γ-secretase inhibitor prodrug 15a (APA-targeting) accumulated in kidneys when compared to blood and liver. Potential nephroprotective effects of the γ-secretase inhibitor targeted prodrugs were investigated in vivo in a mouse model of acute kidney injury, demonstrating that the expression of Notch1 and cleaved Notch1 could be selectively down-regulated upon treatment with the Ac-γ-Glu-γ-secretase-inhibitor 13a.

  7. Gene cloning and nucleotide sequencing and properties of a cocaine esterase from Rhodococcus sp. strain MB1.

    Science.gov (United States)

    Bresler, M M; Rosser, S J; Basran, A; Bruce, N C

    2000-03-01

    A strain of Rhodococcus designated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca. A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized by Rhodococcus sp. strain MB1. The structural gene encoding a cocaine esterase, designated cocE, was cloned from Rhodococcus sp. strain MB1 genomic libraries by screening recombinant strains of Rhodococcus erythropolis CW25 for growth on cocaine. The nucleotide sequence of cocE corresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. The cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with an M(r) of approximately 65,000. The apparent K(m) of the enzyme (mean +/- standard deviation) for cocaine was measured as 1.33 +/- 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine.

  8. Isolation and Identification of Concanavalin A Binding Glycoproteins from Human Seminal Plasma: A Step Towards Identification of Male Infertility Marker Proteins

    Science.gov (United States)

    Tomar, Anil Kumar; Sooch, Balwinder Singh; Raj, Isha; Singh, Sarman; Singh, Tej P.; Yadav, Savita

    2011-01-01

    Human seminal plasma contains a large array of proteins of clinical importance which are essentially needed to maintain the reproductive physiology of spermatozoa and for successful fertilization. Thus, isolation and identification of seminal plasma proteins is of paramount significance for their biophysical characterization and functional analysis in reproductive physiological processes. In this study, we have isolated Concanavalin-A binding glycoproteins from human seminal plasma and subsequently identified them by MALDI-TOF/MS analysis. The major proteins, as identified in this study, are Aminopeptidase N, lactoferrin, prostatic acid phosphatase, zinc-alpha-2-glycoprotein, prostate specific antigen, progestagen-associated endometrial protein, Izumo sperm-egg fusion protein and prolactin inducible protein. This paper also reports preliminary studies to identify altered expression of these proteins in oligospermia and azoospermia in comparison to normospermia. In oligospermia, five proteins were found to be downregulated while in azoospermia, four proteins were downregulated and two proteins were upregulated. Thus, this study is of immense biomedical interest towards identification of potential male infertility marker proteins in seminal plasma. PMID:22182811

  9. A single major QTL controls expression of larval Cry1F resistance trait in Ostrinia nubilalis (Lepidoptera: Crambidae) and is independent of midgut receptor genes.

    Science.gov (United States)

    Coates, Brad S; Sumerford, Douglas V; Lopez, Miriam D; Wang, Haichuan; Fraser, Lisa M; Kroemer, Jeremy A; Spencer, Terrence; Kim, Kyung S; Abel, Craig A; Hellmich, Richard L; Siegfried, Blair D

    2011-08-01

    The European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae), is an introduced crop pest in North America that causes major damage to corn and reduces yield of food, feed, and biofuel materials. The Cry1F toxin from Bacillus thuringiensis (Bt) expressed in transgenic hybrid corn is highly toxic to O. nubilalis larvae and effective in minimizing feeding damage. A laboratory colony of O. nubilalis was selected for high levels of Cry1F resistance (>12,000-fold compared to susceptible larvae) and is capable of survival on transgenic hybrid corn. Genetic linkage maps with segregating AFLP markers show that the Cry1F resistance trait is controlled by a single quantitative trait locus (QTL) on linkage group 12. The map position of single nucleotide polymorphism (SNP) markers indicated that midgut Bt toxin-receptor genes, alkaline phosphatase, aminopeptidase N, and cadherin, are not linked with the Cry1F QTL. Evidence suggests that genes within this genome interval may give rise to a novel Bt toxin resistance trait for Lepidoptera that appears independent of known receptor-based mechanisms of resistance.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. INSULIN AND INSULIN RESISTANCE: NEW MOLECULE MARKERS AND TARGET MOLECULE FOR THE DIAGNOSIS AND THERAPY OF DISEASES OF THE CENTRAL NERVOUS SYSTEM

    Directory of Open Access Journals (Sweden)

    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.

  15. Comparative Study on The Preventive Effect of Saffron Carotenoids, Crocin and Crocetin, in NMU-Induced Breast Cancer in Rats

    Directory of Open Access Journals (Sweden)

    S.Zahra Bathaie

    2017-01-01

    Full Text Available Objective Crocin (Cro and crocetin (Crt are two widely known saffron carotenoids, which exert anticancer effects by different mechanisms. Here, we investigated and compared the preventive effect of Cro and Crt at the initiation and promotion stages of breast cancer induction in an animal model. Materials and Methods In this experimental study, female Wistar albino rats were injected with three doses of N-methyl-N-nitrosourea (NMU. The preventive intervention was done at different times for the initiation and promotion stages. Thus, Cro/Crt was administered by gavage 20 days before, or one week after, the first NMU injection, for the prevention at the initiation or promotion stages respectively. The treatment was repeated every three days, and continued up to the end of experiment. Tumor appearance was checked by palpation and some parameters were determined after sacrifice. Results Tumor volume, latency period, and tumor number were significantly decreased in the rat groups treated with both saffron carotenoids for prevention at both the initiation and promotion stages. Tumor incidence was 77% due to NMU injection, which was decreased to 45 and 33% (on average after Cro and Crt administration, respectively. In addition, enkephaline degrading aminopeptidase (EDA was decreased significantly in the ovaries of the animals, however, changes in the brain were not significant. Conclusion Crt/Cro showed a significant protective effect against the NMU-induced breast cancer in rats. However, Crt was more effective than Cro and prevention at the initiation stage was more effective than at the promotion stage.

  16. 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.

  17. Effect of temperature on the stability of various peptidases during peptide-enriched soy sauce fermentation.

    Science.gov (United States)

    Nakahara, Takeharu; Yamaguchi, Hitomi; Uchida, Riichiro

    2012-03-01

    We previously developed a peptide-enriched soy sauce-like seasoning called Fermented Soybean Seasoning (FSS) with high-temperature fermentation, and we have reported the antihypertensive effects of FSS. Seryl-tyrosine (Ser-Tyr) and glycyl-tyrosine (Gly-Tyr) were identified from FSS as active constituents in the antihypertensive effects. They were found to be particularly enriched in FSS; more so than in regular soy sauce. In the present study, we clarified one of the mechanisms underlying the accumulation of these bioactive peptides during high temperature soy sauce brewing. Crude enzyme extracts were prepared from model soy sauce mash (moromi) fermented at various temperatures. Leucine aminopeptidase-I, II, and seryl-tyrosine hydrolytic activity were found to decrease in the moromi incubated at the fermentation temperature of FSS whereas almost no decrease was observed in that of regular soy sauce. The concentrations of ACE inhibitory peptides, Ser-Tyr and Gly-Tyr, in the moromi incubated at high temperature were revealed to be higher than those at low temperature through quantitative LC-MS/MS analysis. These results suggested that the peptidases responsible for degrading low molecular weight bioactive peptides were inactivated during the high temperature fermentation, thus, these peptides would be likely to remain in the high temperature fermentation.

  18. 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.

  19. Silver nanoparticle effects on stream periphyton during short-term exposures.

    Science.gov (United States)

    Gil-Allué, Carmen; Schirmer, Kristin; Tlili, Ahmed; Gessner, Mark O; Behra, Renata

    2015-01-20

    Silver nanoparticles (AgNP) are increasingly used as antimicrobials in consumer products. Subsequently released into aquatic environments, they are likely to come in contact with microbial communities like periphyton, which plays a key role as a primary producer in stream ecosystems. At present, however, very little is known about the effects of nanoparticles on processes mediated by periphyton communities. We assessed the effects of citrate-coated silver nanoparticles and silver ions (dosed as AgNO3) on five functional end points reflecting community and ecosystem-level processes in periphyton: photosynthetic yield, respiration potential, and the activity of three extracellular enzymes. After 2 h of exposure in experimental microcosms, AgNP and AgNO3 inhibited respiration and photosynthesis of periphyton and the activities of two of the three extracellular enzymes. Addition of a chelating ligand that complexes free silver ions indicated that, in most cases, toxicity of AgNP suspensions was caused by Ag(I) dissolved from the particles. However, these suspensions inhibited one of the extracellular enzymes (leucine aminopeptidase), pointing to a specific nanoparticle effect independent of the dissolved Ag(I). Thus, our results show that both silver nanoparticles and silver ions have potential to disrupt basic metabolic functions and enzymatic resource acquisition of stream periphyton.

  20. 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.

  1. In-silico determination of insecticidal potential of Vip3Aa-Cry1Ac fusion protein against Lepidopteran targets using molecular docking

    Directory of Open Access Journals (Sweden)

    Aftab eAhmad

    2015-12-01

    Full Text Available Study and research of Bt (Bacillus thuringiensis transgenic plants have opened new ways to combat insect pests. Over the decades, however, insect pests, especially the Lepidopteran, have developed tolerance against Bt delta-endotoxins. Such issues can be addressed through the development of novel toxins with greater toxicity and affinity against a broad range of insect receptors. In this computational study, functional domains of Bacillus thuringiensis crystal delta-endotoxin (Cry1Ac insecticidal protein and vegetative insecticidal protein (Vip3Aa have been fused to develop a broad-range Vip3Aa-Cry1Ac fusion protein. Cry1Ac and Vip3Aa are non-homologous insecticidal proteins possessing receptors against different targets within the midgut of insects. The insecticidal proteins were fused to broaden the insecticidal activity. Molecular docking analysis of the fusion protein against aminopeptidase-N (APN and cadherin receptors of five Lepidopteran insects (Agrotis ipsilon, Helicoverpa armigera, Pectinophora gossypiella, Spodoptera exigua and Spodoptera litura revealed that the Ser290, Ser293, Leu337, Thr340 and Arg437 residues of the fusion protein are involved in the interaction with insect receptors. The Helicoverpa armigera cadherin receptor, however, showed no interaction, which might be due to either loss or burial of interactive residues inside the fusion protein. These findings revealed that the Vip3Aa-Cry1Ac fusion protein has a strong affinity against Lepidopteran insect receptors and hence has a potential to be an efficient broad-range insecticidal protein.

  2. 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.

  3. 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.

  4. Poly(aspartic acid) (PAA) hydrolases and PAA biodegradation: current knowledge and impact on applications.

    Science.gov (United States)

    Hiraishi, Tomohiro

    2016-02-01

    Thermally synthesized poly(aspartic acid) (tPAA) is a bio-based, biocompatible, biodegradable, and water-soluble polymer that has a high proportion of β-Asp units and equivalent moles of D- and L-Asp units. Poly(aspartic acid) (PAA) hydrolase-1 and hydrolase-2 are tPAA biodegradation enzymes purified from Gram-negative bacteria. PAA hydrolase-1 selectively cleaves amide bonds between β-Asp units via an endo-type process, whereas PAA hydrolase-2 catalyzes the exo-type hydrolysis of the products of tPAA hydrolysis by PAA hydrolase-1. The novel reactivity of PAA hydrolase-1 makes it a good candidate for a biocatalyst in β-peptide synthesis. This mini-review gives an overview of PAA hydrolases with emphasis on their biochemical and functional properties, in particular, PAA hydrolase-1. Functionally related enzymes, such as poly(R-3-hydroxybutyrate) depolymerases and β-aminopeptidases, are compared to PAA hydrolases. This mini-review also provides findings that offer an insight into the catalytic mechanisms of PAA hydrolase-1 from Pedobacter sp. KP-2.

  5. Inflammation, Adenoma and Cancer: Objective Classification of Colon Biopsy Specimens with Gene Expression Signature

    Directory of Open Access Journals (Sweden)

    Orsolya Galamb

    2008-01-01

    Full Text Available Gene expression analysis of colon biopsies using high-density oligonucleotide microarrays can contribute to the understanding of local pathophysiological alterations and to functional classification of adenoma (15 samples, colorectal carcinomas (CRC (15 and inflammatory bowel diseases (IBD (14. Total RNA was extracted, amplified and biotinylated from frozen colonic biopsies. Genome-wide gene expression profile was evaluated by HGU133plus2 microarrays and verified by RT-PCR. We applied two independent methods for data normalization and used PAM for feature selection. Leave one-out stepwise discriminant analysis was performed. Top validated genes included collagenIVα1, lipocalin-2, calumenin, aquaporin-8 genes in CRC; CD44, met proto-oncogene, chemokine ligand-12, ADAM-like decysin-1 and ATP-binding casette-A8 genes in adenoma; and lipocalin-2, ubiquitin D and IFITM2 genes in IBD. Best differentiating markers between Ulcerative colitis and Crohn's disease were cyclin-G2; tripartite motif-containing-31; TNFR shedding aminopeptidase regulator-1 and AMICA. The discriminant analysis was able to classify the samples in overall 96.2% using 7 discriminatory genes (indoleamine-pyrrole-2,3-dioxygenase, ectodermal-neural cortex, TIMP3, fucosyltransferase-8, collectin sub-family member 12, carboxypeptidase D, and transglutaminase-2. Using routine biopsy samples we successfully performed whole genomic microarray analysis to identify discriminative signatures. Our results provide further insight into the pathophysiological background of colonic diseases. The results set up data warehouse which can be mined further.

  6. Soil degradation effect on biological activity in Mediterranean calcareous soils

    Science.gov (United States)

    Roca-Pérez, L.; Alcover-Sáez, S.; Mormeneo, S.; Boluda, R.

    2009-04-01

    Soil degradation processes include erosion, organic matter decline, compaction, salinization, landslides, contamination, sealing and biodiversity decline. In the Mediterranean region the climatological and lithological conditions, together with relief on the landscape and anthropological activity are responsible for increasing desertification process. It is therefore considered to be extreme importance to be able to measure soil degradation quantitatively. We studied soil characteristics, microbiological and biochemical parameters in different calcareous soil sequences from Valencia Community (Easter Spain), in an attempt to assess the suitability of the parameters measured to reflect the state of soil degradation and the possibility of using the parameters to assess microbiological decline and soil quality. For this purpose, forest, scrubland and agricultural soil in three soil sequences were sampled in different areas. Several sensors of the soil biochemistry and microbiology related with total organic carbon, microbial biomass carbon, soil respiration, microorganism number and enzyme activities were determined. The results show that, except microorganism number, these parameters are good indicators of a soil biological activity and soil quality. The best enzymatic activities to use like indicators were phosphatases, esterases, amino-peptidases. Thus, the enzymes test can be used as indicators of soil degradation when this degradation is related with organic matter losses. There was a statistically significant difference in cumulative O2 uptake and extracellular enzymes among the soils with different degree of degradation. We would like to thank Spanish government-MICINN for funding and support (MICINN, project CGL2006-09776).

  7. 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.

  8. 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.

  9. Effects of an extracted lectin from Citrullus colocynthis L. (Cucurbitaceae on survival, digestion and energy reserves of Ectomyelois ceratoniae Zeller (Lepidoptera: Pyralidae

    Directory of Open Access Journals (Sweden)

    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.

  10. A study of the relationship among sludge retention time, bacterial communities, and hydrolytic enzyme activities in inclined plate membrane bioreactors for the treatment of municipal wastewater.

    Science.gov (United States)

    Ittisupornrat, Suda; Tobino, Tomohiro; Yamamoto, Kazuo

    2014-11-01

    Inclined plate membrane bioreactors (ip-MBRs) have been proposed as a highly effective method in wastewater treatment. With the help of settling enhancer inclined plates, dense excess sludge can be kept in the mainstream of the process, and consequently, suitable sludge mass can be maintained in the membrane tank. In this study, the relationship among sludge retention time (SRT), bacterial communities, and hydrolytic enzyme activities was investigated. Two identical bench-scale ip-MBRs were operated 1 year in real municipal wastewater treatment. Multidimensional scaling (MDS) plots of terminal restriction fragment length polymorphism (T-RFLP) fingerprints showed similar changes in the bacterial communities in terms of bacterial members and abundance over time in both the reactors, which was primarily caused by the changes of wastewater composition. However, the impact of SRT revealed significant differences in the dominant bacterial communities when both the reactors were operated with a largely different SRT (infinite SRT and SRT of 20 days). The sequences of bacterial 16S rRNA gene were classified into six libraries of A-F. The largest group of sequences belonged to the phylum Proteobacteria. The phylum Bacteroidetes was dominant in the seed sludge retrieved from the conventional activated sludge (CAS) as Flavobacterium-like bacterium was dominantly observed. Under the MBR operation (libraries B-F), bacterial communities belonging to the phyla Proteobacteria and Chloroflexi were dominant. Most of them may be responsible for protein degradation because aminopeptidase activity increased in proportion with the abundance of these bacteria.

  11. 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.

  12. 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.

  13. Caracterização e diversidade genética do capim-elefante e seus híbridos com milheto mediante padrões isoenzimáticos

    Directory of Open Access Journals (Sweden)

    FREITAS NARA SUZY AGUIAR DE

    2000-01-01

    Full Text Available Foram avaliadas isoenzimaticamente sete cultivares de capim-elefante (Pennisetum purpureum e seus híbridos com milheto (P. americanum, selecionados pela Empresa Pernambucana de Pesquisa Agropecuária (IPA, visando à identificação de acessos. Foram estudados, em gel de poliacrilamida, os sistemas peroxidase (POX, esterase (EST, glutamato oxalacetato transaminase (GOT, leucina aminopeptidase (LAP, álcool-desidrogenase (ADH e fosfatase ácida (ACP, em folhas jovens, aos 28 dias após o corte de uniformização. Não foi observada atividade isoenzimática da ADH e observou-se baixa resolução do sistema LAP, os quais não são indicados para caracterização dos germoplasmas. Os padrões de ACP, GOT, POX e EST permitiram conhecer os fenótipos dos 14 acessos estudados. Foram revelados 9, 3, 13 e 19 diferentes padrões de bandas, respectivamente, sendo possível a identificação da coleção de forma rápida e segura utilizando apenas os padrões de esterase.

  14. Caracterização de germoplasma de soja e de feijão através de eletroforese de isoenzimas da semente

    Directory of Open Access Journals (Sweden)

    ANTI ANA BEATRIZ

    2000-01-01

    Full Text Available Foram estudadas, através de eletroforese em gel de poliacrilamida, isoenzimas de sementes de plantas de dois cultivares de soja, IAC 6 e IAC 9, e de duas linhagens de feijão da variedade Goiano Precoce, uma com folha lisa e outra com folha rugosa, tendo em vista sua caracterização. Apesar de possuírem um parental em comum, os dois cultivares de soja diferiram entre si com relação aos perfis eletroforéticos de urease, fosfatase ácida, malato desidrogenase e leucina aminopeptidase evidenciando que essa técnica pode ser usada para discriminar outros cultivares de soja; entretanto as duas linhagens de feijão não diferiram entre si nos zimogramas estudados, indicando um grande parentesco e reforçando a hipótese de que a linhagem de folha rugosa poderia ter-se originado da linhagem de folha lisa por mutação de ponto.

  15. Screening of genes related to sulfide metabolism in Urechis unicinctus (Echiura, Urechidae) using suppression subtractive hybridization and cDNA microarray analysis.

    Science.gov (United States)

    Shi, Xiaoli; Shao, Mingyu; Zhang, Litao; Ma, Yubin; Zhang, Zhifeng

    2012-09-01

    Exogenous sulfide can generally induce metabolic injuries in most organisms and even cause death. However, organisms inhabiting intertidal zones, hydrothermal vents, and cold seeps, can tolerate, metabolize, and utilize sulfide. In this study, both suppression subtractive hybridization and cDNA microarray analysis were employed to screen sulfide metabolism-related genes from the body wall in echiuran worm Urechis unicinctus, a marine sediment species. A total of 3456 monoclones were isolated and 82 were identified as differentially expressed genes in worms exposed to 50 μM sulfide for 24 h, compared to controls. The identified genes encoded proteins with multiple processes, including metabolism, cellular process, biological regulation, response to stimulus, multicellular organismal process, localization, development, and cellular component organization. Eight genes, serase, vacuolar protein, src tyrosine kinase, sulfide oxidase-like oxidoreductase, aprataxin, SN-RNP, aminopeptidase, and predicted protein, were selected to verify expression in the worm using qRT-PCR. The agreement of gene expression evaluation was 62.5% between the results of microarray analysis and qRT-PCR. These new data will provide clues for further probing of the molecular mechanism of sulfide metabolism.

  16. Characterization of the PH1704 protease from Pyrococcus horikoshii OT3 and the critical functions of Tyr120.

    Science.gov (United States)

    Zhan, Dongling; Bai, Aixi; Yu, Lei; Han, Weiwei; Feng, Yan

    2014-01-01

    The PH1704 protease from hyperthermophilic archaean Pyrococcus horikoshii OT3 is a member of DJ-1/ThiJ/PfpI superfamily with diverse functional subclasses. The recombinant PH1704 was efficiently purified and was systematically characterized by a combination of substrate specificity analysis, steady-state kinetics study and molecular docking research. The homogeneous protease was obtained as a presumed dodecamer with molecular weight of ∼240 kDa. Iodoacetamide strongly inhibited the peptidase activity, confirming that Cys100 is a nucleophilic residue. The recombinant protein was identified as both an aminopeptidase and an endopeptidase. Experimental data showed that L-R-amc was the best substrate of PH1704. Structural interaction fingerprint analysis (SIFt) indicated the binding pose of PH1704 and showed that Tyr120 is important in substrate binding. Kinetic parameters Kcat and Kcat/Km of the Y120P mutant with L-R-amc was about 7 and 7.8 times higher than that of the wild type (WT). For the endopeptidase Y120P with AAFR-amc, Kcat and Kcat/Km is 10- and 21-fold higher than that of WT. Experimental data indicate the important functions of Tyr120: involvement in enzyme activity to form a hydrogen bond with Cys100 and as an entrance gate of the substrate with Lys43. The results of this study can be used to investigate the DJ-1/ThiJ/PfpI superfamily.

  17. Identification of a mutant locus that bypasses the BsgA protease requirement for social development in Myxococcus xanthus.

    Science.gov (United States)

    Cusick, John K; Hager, Elizabeth; Gill, Ronald E

    2015-01-01

    The BsgA protease is required for the earliest morphological changes observed in Myxococcus xanthus development. We hypothesize that the BsgA protease is required to cleave an inhibitor of the developmental program, and isolation of genetic bypass suppressors of a bsgA mutant was used to identify signaling components controlling development downstream of the BsgA protease. Strain M955 was created by transposon mutagenesis of a bsgA mutant followed by screening for strains that could develop despite the absence of the BsgA protease. Strain M955 was able to aggregate, form fruiting bodies, and partially restored the production of viable spores in comparison to the parental bsgA mutant. The bsgA Tn5Ω955 strain partially restored developmental expression to a subset of genes normally induced during development, and expressed one developmentally induced fusion at higher amounts during vegetative growth in comparison to wild-type cells. The transposon in strain M955 was localized to a Ribonuclease D homolog that appears to exist in an operon with a downstream aminopeptidase-encoding gene. The identification of a third distinct bypass suppressor of the BsgA protease suggests that the BsgA protease may regulate a potentially complex pathway during the initiation of the M. xanthus developmental program.

  18. 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.

  19. Tingenone, a pentacyclic triterpene, induces peripheral antinociception due to opioidergic activation.

    Science.gov (United States)

    Veloso, Clarice de Carvalho; Rodrigues, Vanessa Gregório; Ferreira, Renata Cristina Mendes; Duarte, Lucienir Pains; Klein, Andre; Duarte, Igor Dimitri; Romero, Thiago Roberto Lima; Perez, Andrea de Castro

    2014-11-01

    Plants belonging to the genus Maytenus are routinely used in folk medicine for the treatment of pain diseases. Our previous phytochemical study of the roots of Maytenus imbricata resulted in the isolation and characterization of tingenone, a pentacyclic triterpene. Natural triterpenoids are of growing interest because they have several biological activities, including analgesic properties. The present study assessed the involvement of the opiodergic pathway in the tingenone-induced antinociceptive effect against hyperalgesia induced by prostaglandin E2 (2 µg) in the peripheral pathway. We evaluated the effect of several antagonists to opioid receptors using the mouse paw pressure test. Tingenone administered into the right hind paw induced a local antinociceptive effect that was antagonized by naloxone, a nonselective antagonist to opioid receptors. Clocinnamox, naltrindole, and nor-binaltorphimine are selective antagonists to µ, δ, and κ receptors, respectively, which reverted the peripheral antinociception induced by tingenone. Bestatine acts as an inhibitor of aminopeptidase, an enzyme that degrades endogenous opioid peptides, and was shown to intensify the antinociceptive effect of tingenone. The results suggest that the opioidergic system participates in the peripheral antinociception induced by tingenone.

  20. In vitro and in vivo anti-leukemic activity of the peptidase-potentiated alkylator melflufen in acute myeloid leukemia

    Science.gov (United States)

    Strese, Sara; Bashir Saadia, Hassan; Velander, Ebba; Haglund, Caroline; Höglund, Martin; Larsson, Rolf; Gullbo, Joachim

    2017-01-01

    The novel aminopeptidase potentiated alkylating agent melflufen, was evaluated for activity in acute myeloid leukemia in a range of in vitro models, as well as in a patient derived xenograft study. All tested AML cell lines were highly sensitive to melflufen while melphalan was considerably less potent. In the HL-60 cell line model, synergy was observed for the combination of melflufen and cytarabine, an interaction that appeared sequence dependent with increased synergy when melflufen was added before cytarabine. Also, in primary cultures of AML cells from patients melflufen was highly active, while normal PBMC cultures appeared less sensitive, indicating a 7-fold in vitro therapeutic index. Melphalan, on the other hand, was only 2-fold more potent in the AML patient samples compared with PBMCs. Melflufen was equally active against non-malignant, immature CD34+ progenitor cells and a more differentiated CD34+ derived cell population (GM14), whereas the stem cell like cells were less sensitive to melphalan. Finally, melflufen treatment showed significant anti-leukemia activity and increased survival in a patient derived xenograft of AML in mice. In conclusion, melflufen demonstrates high and significant preclinical activity in AML and further clinical evaluation seem warranted in this disease. PMID:27974676

  1. 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.

  2. Determination of the proteins encoded by BmBDV VD1-ORF4 and their interacting proteins in BmBDV-infected midguts.

    Science.gov (United States)

    Li, Guohui; Zhou, Qian; Hu, Zhaoyang; Wang, Peng; Tang, Qi; Chen, Keping; Yao, Qin

    2015-04-01

    Bombyx mori bidensovirus (BmBDV) VD1-ORF4 consists of 3,318 nucleotides, which codes for a predicted protein with molecular weight of about 127 kDa. However, the authentic proteins encoded by VD1-ORF4 in silkworm midguts infected with BmBDV and their interacting proteins are still unclear. In this study, Western blot analysis revealed that a 127-kDa protein was confirmed to be translated from the VD1-ORF4 transcript using polyclonal antibodies and monoclonal antibodies against VD1-ORF4 deduced amino acid. Moreover, four smaller proteins with molecular weight of about 70, 60, 53, and 42 kDa were also examined in the infected midguts. Transient expression assay indicated that the expression amount of VD1-ORF4 fused with egfp was at least 30-fold lower than that of egfp gene, and immunofluorescence staining result indicated that these proteins encoded by VD1-ORF4 were located in both the cytoplasm and nucleus. Co-immunoprecipitation result showed that Aminopeptidase and Heat shock protein 90 can be captured by these proteins encoded by VD1-ORF4. In conclusion, multiple proteins were produced from the transcripts of VD1-ORF4 gene by an uncertain expression strategy, which may play important roles in viral replication and assembly.

  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).

  4. Phylogeography of Littorina sitkana in the northwestern Pacific Ocean: evidence of eastward trans-Pacific colonization after the Last Glacial Maximum.

    Science.gov (United States)

    Azuma, Noriko; Zaslavskaya, Nadezhda I; Yamazaki, Tomoyasu; Nobetsu, Takahiro; Chiba, Susumu

    2017-04-01

    We investigated genetic diversity and population structure of the Sitka periwinkle Littorina sitkana along the coastlines of the northwestern Pacific (NWP) to evaluate the possibility of trans-Pacific colonization of this species from the NWP to the northeastern Pacific (NEP) after the Last Glacial Maximum. We sampled L. sitkana from 32 populations in the NWP, and sequenced a region of the mitochondrial cytochrome b oxidase gene for population genetic analyses. The results were compared with those of previous reports from the NEP. The genetic diversity of L. sitkana was much higher in the NWP than in the NEP. Genetic connectivity between the NWP and NEP populations was indicated by an extremely abundant haplotype in the NEP that was also present in eastern Hokkaido and the Kuril Islands. To confirm these results, we compared sequences of the longest intron of the aminopeptidase N gene (APN54) in the nuclear genome in four populations of L. sitkana in the NWP with previous results from the NEP. Again, much higher genetic diversity was found in the NWP than in the NEP and genetic connectivity was supported between the Kuril Islands and the NEP. These results imply postglacial colonization of this species from the NWP to the NEP, probably along the Kuril and Aleutian Island chains. This study is the first report of possible trans-Pacific postglacial colonization of a direct-developing gastropod, inferred from genetic data.

  5. 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)

  6. 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.

  7. 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.

  8. Characterization of Lactobacillus from Algerian goat's milk based on phenotypic, 16S rDNA sequencing and their technological properties

    Directory of Open Access Journals (Sweden)

    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.

  9. 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.

  10. Semen variables and sperm membrane protein profile of Saanen bucks ( Capra hircus) in dry and rainy seasons of the northeastern Brazil (3°S)

    Science.gov (United States)

    van Tilburg, M. F.; Salles, M. G. F.; Silva, M. M.; Moreira, R. A.; Moreno, F. B.; Monteiro-Moreira, A. C. O.; Martins, J. A. M.; Cândido, M. J. D.; Araújo, A. A.; Moura, A. A. A.

    2015-05-01

    The Saanen is a highly productive breed, and for this reason, it has been raised in Brazil, but mostly under climate conditions completely different from where the breed originated. The objective of this study was to investigate variations in semen parameters and sperm membrane proteins from Saanen bucks ( n = 7) raised in Northeastern Brazil, during dry season (September, October, and November) and rainy season (March, April, and May). We showed that during the dry season, sperm motility, concentration, and the percentage of normal sperm decreased as compared to the rainy season. Rectal temperatures of bucks had no significant ( p > 0.05) variations during the dry and rainy seasons. However, temperatures of left and right skin testis were higher ( p < 0.05) during the dry as compared to the rainy season. Expression of three proteins (lysine-specific demethylase 5D, adenosine triphosphate (ATP) synthase subunit d, and radial spoke head protein 9 homolog) in sperm membrane were more intense in rainy season and only one protein (cytosol aminopeptidase) had greater expression in the dry season of the year. Our results show that mechanisms of testicular thermoregulation of Saanen bucks did not prevent a decrease in seminal parameters during the dry season. This deterioration may be related to reduced expression of proteins associated with important functions in sperm membrane.

  11. 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.

  12. 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 meta-analysis showed that rs27037 and rs30187 were strongly associated with AS (P < 0.00001). Significant association was also observed for rs27434 (P = 0.001). No association was shown for rs27044 (P = 0.70). We concluded that ERAP1 variants are associated with AS in East Asian population, indicating a common pathogenic mechanism for AS in East Asians and Caucasians.

  13. Differential efficiency of simvastatin and lipoic acid treatments on Bothrops jararaca envenomation-induced acute kidney injury in mice.

    Science.gov (United States)

    Barone, Juliana Marton; Alponti, Rafaela Fadoni; Frezzatti, Rodrigo; Zambotti-Villela, Leonardo; Silveira, Paulo Flávio

    2011-01-01

    Snake bite accidents by Bothrops genus is an important public health issue in Brazil and one of its most serious complications is the acute kidney injury (AKI). Here we evaluated the effects of Bothrops jararaca venom (vBj) and the treatments with lipoic acid (LA) and simvastatin (SA) on renal function, aminopeptidase (AP) activities and renal redox status. Primordial events for establishment of AKI by vBj were hyperuricemia, hypercreatinemia, urinary hyperosmolality, renal oxidative stress and reduction of hematocrit and protein content in the membrane of renal cortex and medulla and in the plasma. In the renal cortex and medulla the changes caused by vBj in soluble and membrane-bound AP activities had a similar pattern. The beneficial effects of LA and SA on envenomed mice were similar on the hyperuricemia, renal oxidative stress and reduction of hematocrit. LA mitigated the hypercreatinemia, but exacerbated the urinary urea and creatinine, whereas SA mitigated the decrease of plasma urea, urinary hyperosmolality and hypercreatinuria induced by vBj. The beneficial effects of LA and especially of SA on renal effects of vBj open a new perspective for clinical investigations of these drugs as coadjuvant agents in the serotherapy of Bothrops envenomation.

  14. Effects of N-acetyl-L-cysteine on redox status and markers of renal function in mice inoculated with Bothrops jararaca and Crotalus durissus terrificus venoms.

    Science.gov (United States)

    Barone, Juliana Marton; Frezzatti, Rodrigo; Silveira, Paulo Flavio

    2014-03-01

    Renal dysfunction is an important aggravating factor in accidents caused by Crotalus durissus terrificus (Cdt) and Bothrops jararaca (Bj) bites. N-acetyl-l-cysteine (NAC) is well known as a nephroprotective antioxidant with low toxicity. The present study investigated the effects of NAC on redox status and markers of renal function in mice that received vehicle (controls) or venoms (v) of Cdt and Bj. In controls NAC promoted hypercreatinemia, hypouremia, hyperosmolality with decreased urea in urine, hyperproteinuria, decreased protein and increased dipeptidyl peptidase IV (DPPIV) in membrane-bound fraction (MF) from renal cortex (RC) and medulla (RM). NAC ameliorated or normalized altered creatinuria, proteinemia and aminopeptidase (AP) acid in MF, AP basic (APB) in soluble fraction (SF), and neutral AP in SF and MF from RC and RM in vBj envenomation. NAC ameliorated or normalized altered neutral AP in SF from RC and RM, and DPPIV and protein in MF from RC in vCdt envenomation. NAC ameliorated or restored renal redox status respectively in vCdt and vBj, and normalized uricemia in both envenomations. These data are promising perspectives that recommend the clinical evaluation of NAC as potential coadjuvant in the anti venom serotherapy for accidents with these snake's genera.

  15. 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.

  16. Isolation and Identification of Concanavalin A Binding Glycoproteins from Human Seminal Plasma: A Step Towards Identification of Male Infertility Marker Proteins

    Directory of Open Access Journals (Sweden)

    Anil Kumar Tomar

    2011-01-01

    Full Text Available Human seminal plasma contains a large array of proteins of clinical importance which are essentially needed to maintain the reproductive physiology of spermatozoa and for successful fertilization. Thus, isolation and identification of seminal plasma proteins is of paramount significance for their biophysical characterization and functional analysis in reproductive physiological processes. In this study, we have isolated Concanavalin-A binding glycoproteins from human seminal plasma and subsequently identified them by MALDI-TOF/MS analysis. The major proteins, as identified in this study, are Aminopeptidase N, lactoferrin, prostatic acid phosphatase, zinc-alpha-2-glycoprotein, prostate specific antigen, progestagen-associated endometrial protein, Izumo sperm-egg fusion protein and prolactin inducible protein. This paper also reports preliminary studies to identify altered expression of these proteins in oligospermia and azoospermia in comparison to normospermia. In oligospermia, five proteins were found to be downregulated while in azoospermia, four proteins were downregulated and two proteins were upregulated. Thus, this study is of immense biomedical interest towards identification of potential male infertility marker proteins in seminal plasma.

  17. Genome-wide study of the defective sucrose fermenter strain of Vibrio cholerae from the Latin American cholera epidemic.

    Directory of Open Access Journals (Sweden)

    Daniel Rios Garza

    Full Text Available The 7th cholera pandemic reached Latin America in 1991, spreading from Peru to virtually all Latin American countries. During the late epidemic period, a strain that failed to ferment sucrose dominated cholera outbreaks in the Northern Brazilian Amazon region. In order to understand the genomic characteristics and the determinants of this altered sucrose fermenting phenotype, the genome of the strain IEC224 was sequenced. This paper reports a broad genomic study of this strain, showing its correlation with the major epidemic lineage. The potentially mobile genomic regions are shown to possess GC content deviation, and harbor the main V. cholera virulence genes. A novel bioinformatic approach was applied in order to identify the putative functions of hypothetical proteins, and was compared with the automatic annotation by RAST. The genome of a large bacteriophage was found to be integrated to the IEC224's alanine aminopeptidase gene. The presence of this phage is shown to be a common characteristic of the El Tor strains from the Latin American epidemic, as well as its putative ancestor from Angola. The defective sucrose fermenting phenotype is shown to be due to a single nucleotide insertion in the V. cholerae sucrose-specific transportation gene. This frame-shift mutation truncated a membrane protein, altering its structural pore-like conformation. Further, the identification of a common bacteriophage reinforces both the monophyletic and African-Origin hypotheses for the main causative agent of the 1991 Latin America cholera epidemics.

  18. A Critical Evaluation of Liver Pathology in Humans with Danon Disease and Experimental Correlates in a Rat Model of LAMP-2 Deficiency.

    Science.gov (United States)

    Wang, Lu; Wang, Jingbo; Cai, Weile; Shi, Yongquan; Zhou, Xinmin; Guo, Guanya; Guo, Changcun; Huang, Xiaofeng; Han, Zheyi; Zhang, Shuai; Ma, Shuoyi; Zhou, Xia; Fan, Daiming; Eric Gershwin, M; Han, Ying

    2017-01-26

    Danon disease is a genetic deficiency in lysosome-associated membrane protein 2 (LAMP-2), a highly glycosylated constituent of the lysosomal membrane and characterized by a cardiomyopathy, skeletal muscle myopathy, and cognitive impairment. Patients, however, often manifest hepatic abnormalities, but liver function has not been well evaluated and the syndrome is relatively uncommon. Hence, we have taken advantage of a rat that has been deleted of LAMP-2 to study the relative role of LAMP-2 on liver function. Interestingly, rats deficient in LAMP-2 develop a striking increase in serum alkaline phosphatase (ALP) and a decrease in bile flow compared with wild-type littermates. Importantly and by ultrastructural analysis, deficient rats manifest dilated canaliculi that lack microvilli with evidence of bile-containing bodies. Moreover, following bile duct ligation, LAMP-2-deficient rats develop rapid and severe evidence of advanced cholestasis, with an increase in serum bilirubin, as early as 6 h later. In wild-type control rats, multidrug resistance-associated protein 2 (Mrp2) normally concentrates at the bile canalicular membranes to secrete conjugated bilirubin into bile. However, in LAMP-2(y/-) rats, Mrp2 was detected in hepatocytes compared with other canalicular proteins including P-glycoproteins, dipeptidyl peptidase IV (CD26), and aminopeptidase (CD13). Our data further suggest that LAMP-2 interacts with the membrane cytoskeletal proteins radixin and F-actin in determining the localization of integral membrane proteins.

  19. 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.

  20. Urinary proteins of tubular origin: basic immunochemical and clinical aspects.

    Science.gov (United States)

    Scherberich, J E

    1990-01-01

    A variety of tubular marker proteins, as compared to healthy controls, are excreted at an increased rate in the urine of patients with renal damage. Beside cytoplasmic glutathione-S-transferase and lysosomal beta-N-acetyl-glucosaminidase (beta-NAG) the majority of kidney-related urine proteins derives from membrane surface components of the most vulnerable proximal tubule epithelia, among them ala-(leu-gly)-aminopeptidase, gamma-glutamyl transpeptidase (GGT), the tubular portion of angiotensinase A, the major brush border glycoprotein 'SGP-240' and adenosine-deaminase-binding protein. Urinary tissue proteins, e.g. brush border (BB) microvilli, are immunologically identical with those antigens prepared from cell membranes of the human kidney itself. BB antigens are shed into the urine of patients with glomerulonephritis, interstitial nephritis, systemic diseases, e.g. systemic lupus erythematosus (SLE), diabetes mellitus and multiple myeloma, arterial hypertension, infectious diseases (malaria, AIDS) and after operations, renal grafting and administration of X-ray contrast media, aminoglycosides or certain cytostatics (cis-platinum). Tissue proteinuria of tubular proteins is determined by enzyme-kinetic or quantitative immunological assays applying either poly- or monoclonal antikidney antibodies. Clinical, ultrastructural and histochemical studies support the idea that both 'soluble' and high-molecular-weight membrane particles (vacuolar blebs, greater than 10(6) dalton) as well as microfilamental components of the epithelial cytoskeleton contribute to tubular 'histuria' which appears as a sensitive parameter in monitoring tubular damage under clinical conditions at a very early phase.

  1. Organisation and functional role of the brain angiotensin system

    Directory of Open Access Journals (Sweden)

    Catherine Llorens-Cortes

    2002-03-01

    Full Text Available The discovery that all components of the renin-angiotensin system (RAS are present in the brain led investigators to postulate the existence of a local brain RAS. Supporting this, angiotensin immunoreactive neurones have been visualised in the brain. Two major pathways were described: a forebrain pathway which connects circumventricular organs to the median preoptic nucleus, paraventricular and supraoptic nuclei, and a second pathway connecting the hypothalamus to the medulla oblongata. Blood-brain-barrier deficient circumventricular organs are rich in angiotensin II (Ang II receptors. By activating these receptors, circulating Ang II may act on central cardiovascular centres via angiotensinergic neurones, providing a link between peripheral and central Ang II systems. Among the effector peptides of the brain RAS, Ang II and angiotensin III (Ang III have the same affinity for type 1 and type 2 Ang II receptors. When injected into the brain, both peptides increase blood pressure (BP, water intake and pituitary hormone release and may modify learning and memory. Since Ang II is converted in vivo to Ang III, the nature of the true effector is unknown. This review summarises new insights into the predominant role of brain Ang III in the control of BP and underlines the fact that brain aminopeptidase A, the enzyme forming central Ang III, could constitute a putative central therapeutic target for the treatment of hypertension.

  2. Comparative Study on The Preventive Effect of Saffron Carotenoids, Crocin and Crocetin, in NMU-Induced Breast Cancer in Rats

    Science.gov (United States)

    Sajjadi, Meysam; Bathaie, Zahra

    2017-01-01

    Objective Crocin (Cro) and crocetin (Crt) are two widely known saffron carotenoids, which exert anticancer effects by different mechanisms. Here, we investigated and compared the preventive effect of Cro and Crt at the initiation and promotion stages of breast cancer induction in an animal model. Materials and Methods In this experimental study, female Wistar albino rats were injected with three doses of N-methyl-N-nitrosourea (NMU). The preventive intervention was done at different times for the initiation and promotion stages. Thus, Cro/Crt was administered by gavage 20 days before, or one week after, the first NMU injection, for the prevention at the initiation or promotion stages respectively. The treatment was repeated every three days, and continued up to the end of experiment. Tumor appearance was checked by palpation and some parameters were determined after sacrifice. Results Tumor volume, latency period, and tumor number were significantly decreased in the rat groups treated with both saffron carotenoids for prevention at both the initiation and promotion stages. Tumor incidence was 77% due to NMU injection, which was decreased to 45 and 33% (on average) after Cro and Crt administration, respectively. In addition, enkephaline degrading aminopeptidase (EDA) was decreased significantly in the ovaries of the animals, however, changes in the brain were not significant. Conclusion Crt/Cro showed a significant protective effect against the NMU-induced breast cancer in rats. However, Crt was more effective than Cro and prevention at the initiation stage was more effective than at the promotion stage.

  3. Digestive enzymes activity in larvae of Cameraria ohridella (Lepidoptera: Gracillariidae).

    Science.gov (United States)

    Stygar, Dominika; Dolezych, Bogdan; Nakonieczny, Mirosław; Migula, Pawel; Michalczyk, Katarzyna; Zaak, Maria

    2010-10-01

    This article presents the activity of carbohydratases and proteases in the midgut of Cameraria ohridella larvae--an oligophagous pest whose preferred feeding is horse chestnuts leaves. Optimal media pH of the assayed enzymes were similar to those of other Lepidopterans. Relatively high amylase activity, as well as maltase and sucrase activities, indicates that starch and sucrose are the main digested saccharides. Trehalase activity was similar to that described in other Lepidopterans. Activities of glycosidases were significantly lower than those of disaccharidases what suggests that neither cellulose nor glycosides are important for C. ohridella. Trypsin is the main endoprotease of this pest. Like in other leaf-eaters carboxypeptidase activity was higher than that of aminopeptidase. The activity of the majority of examined enzymes increased in the following successive pest generations, which could be explained by the decreased nutritional value of older leaves. Probably this phenomenon in hydrolases activity in Cameraria is a nonspecific mechanism present at this stage of co-evolution of the horse chestnut and its pest.

  4. Expression of the maize proteinase inhibitor (mpi) gene in rice plants enhances resistance against the striped stem borer (Chilo suppressalis): effects on larval growth and insect gut proteinases.

    Science.gov (United States)

    Vila, Laura; Quilis, Jordi; Meynard, Donaldo; Breitler, Jean Christophe; Marfà, Victoria; Murillo, Isabel; Vassal, Jean Michel; Messeguer, Joaquima; Guiderdoni, Emmanuel; San Segundo, Blanca

    2005-03-01

    The maize proteinase inhibitor (mpi) gene was introduced into two elite japonica rice varieties. Both constitutive expression of the mpi gene driven by the maize ubiquitin 1 promoter and wound-inducible expression of the mpi gene driven by its own promoter resulted in the accumulation of MPI protein in the transgenic plants. No effect on plant phenotype was observed in mpi-expressing lines. The stability of transgene expression through successive generations of mpi rice lines (up to the T(4) generation) and the production of functional MPI protein were confirmed. Expression of the mpi gene in rice enhanced resistance to the striped stem borer (Chilo suppressalis), one of the most important pests of rice. In addition, transgenic mpi plants were evaluated in terms of their effects on the growth of C. suppressalis larvae and the insect digestive proteolytic system. An important dose-dependent reduction of larval weight of C. suppressalis larvae fed on mpi rice, compared with larvae fed on untransformed rice plants, was observed. Analysis of the digestive proteolytic activity from the gut of C. suppressalis demonstrated that larvae adapted to mpi transgene expression by increasing the complement of digestive proteolytic activity: the serine and cysteine endoproteinases as well as the exopeptidases leucine aminopeptidase and carboxypeptidases A and B. However, the induction of such proteolytic activity did not prevent the deleterious effects of MPI on larval growth. The introduction of the mpi gene into rice plants can thus be considered as a promising strategy to protect rice plants against striped stem borer.

  5. Preterm as compared with full-term neonatal calves are characterized by morphological and functional immaturity of the small intestine.

    Science.gov (United States)

    Bittrich, S; Philipona, C; Hammon, H M; Romé, V; Guilloteau, P; Blum, J W

    2004-06-01

    Intestinal diseases in neonatal calves may be due to morphological and functional immaturity. We have studied histomorphology, crypt cell proliferation rates (based on incorporation of 5-bromo-2'-deoxyuridine into DNA), presence of apoptotic cells (based on terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling), and brush border enzyme activities in preterm calves (277 d of gestation), euthanized on d 1 (P0) or 8 (P8), and in full-term calves (290 d of gestation), euthanized on d 1 (F0) or 8 (F8). Vacuolated epithelial cells were present in ileum of P0 and F0 but not in P8 and F8. During the first 8 d, villus sizes, crypt depths, and proliferation rates of crypt cells in the small intestine of preterm calves did not significantly change. In contrast, in full-term calves during the first 8 d, villus sizes in jejunum decreased, crypt depths increased in small intestine and colon, and crypt cell proliferation increased in duodenum and jejunum. Submucosal thickness in jejunum was highest in P0, but in ileum it increased with gestational age and feeding. Gestational age x feeding interactions indicated increased activities of aminopeptidase N and reduced lactase activities only in F8 and reduced dipeptidylpeptidase IV activities only in P8. In conclusion, in preterm calves the small intestinal epithelium was immature and brush border enzyme activities differed in part from those in full-term calves.

  6. Evaluation of Genetic Variations in Maize Seedlings Exposed to Electric Field Based on Protein and DNA Markers

    Directory of Open Access Journals (Sweden)

    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.

  7. Pseudomonas aeruginosa vesicles associate with and are internalized by human lung epithelial cells

    Directory of Open Access Journals (Sweden)

    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.

  8. Exploring the midgut transcriptome and brush border membrane vesicle proteome of the rice stem borer, Chilo suppressalis (Walker.

    Directory of Open Access Journals (Sweden)

    Weihua Ma

    Full Text Available The rice stem borer, Chilo suppressalis (Walker (Lepidoptera: Pyralidae, is one of the most detrimental pests affecting rice crops. The use of Bacillus thuringiensis (Bt toxins has been explored as a means to control this pest, but the potential for C. suppressalis to develop resistance to Bt toxins makes this approach problematic. Few C. suppressalis gene sequences are known, which makes in-depth study of gene function difficult. Herein, we sequenced the midgut transcriptome of the rice stem borer. In total, 37,040 contigs were obtained, with a mean size of 497 bp. As expected, the transcripts of C. suppressalis shared high similarity with arthropod genes. Gene ontology and KEGG analysis were used to classify the gene functions in C. suppressalis. Using the midgut transcriptome data, we conducted a proteome analysis to identify proteins expressed abundantly in the brush border membrane vesicles (BBMV. Of the 100 top abundant proteins that were excised and subjected to mass spectrometry analysis, 74 share high similarity with known proteins. Among these proteins, Western blot analysis showed that Aminopeptidase N and EH domain-containing protein have the binding activities with Bt-toxin Cry1Ac. These data provide invaluable information about the gene sequences of C. suppressalis and the proteins that bind with Cry1Ac.

  9. Exploring the midgut transcriptome and brush border membrane vesicle proteome of the rice stem borer, Chilo suppressalis (Walker).

    Science.gov (United States)

    Ma, Weihua; Zhang, Zan; Peng, Chuanhua; Wang, Xiaoping; Li, Fei; Lin, Yongjun

    2012-01-01

    The rice stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), is one of the most detrimental pests affecting rice crops. The use of Bacillus thuringiensis (Bt) toxins has been explored as a means to control this pest, but the potential for C. suppressalis to develop resistance to Bt toxins makes this approach problematic. Few C. suppressalis gene sequences are known, which makes in-depth study of gene function difficult. Herein, we sequenced the midgut transcriptome of the rice stem borer. In total, 37,040 contigs were obtained, with a mean size of 497 bp. As expected, the transcripts of C. suppressalis shared high similarity with arthropod genes. Gene ontology and KEGG analysis were used to classify the gene functions in C. suppressalis. Using the midgut transcriptome data, we conducted a proteome analysis to identify proteins expressed abundantly in the brush border membrane vesicles (BBMV). Of the 100 top abundant proteins that were excised and subjected to mass spectrometry analysis, 74 share high similarity with known proteins. Among these proteins, Western blot analysis showed that Aminopeptidase N and EH domain-containing protein have the binding activities with Bt-toxin Cry1Ac. These data provide invaluable information about the gene sequences of C. suppressalis and the proteins that bind with Cry1Ac.

  10. 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.

  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. Debaryomyces hansenii and Rhodotorula mucilaginosa comprised the yeast core gut microbiota of wild and reared carnivorous salmonids, croaker and yellowtail.

    Science.gov (United States)

    Raggi, Patricia; Lopez, Paulina; Diaz, Angélica; Carrasco, Diana; Silva, Alfonso; Velez, Antonio; Opazo, Rafael; Magne, Fabien; Navarrete, Paola A

    2014-09-01

    This is the first study using molecular and culture-based methods aimed at investigating the composition of the intestinal yeast microbiota of wild and reared carnivorous salmonids, croaker and yellowtail, to characterize their cores and to evaluate the enzymatic activities of the cultivated yeast. Among 103 samples from salmonids, croaker and yellowtail, yeast were detected in 85.4%, with 43 species identified. The core of reared fish was composed of eight species, in contrast to the wild fish core, which consisted of two species: Debaryomyces hansenii and Rhodotorula mucilaginosa. Despite the smaller diversity of the wild fish core, similar enzymatic profiles were detected for the species from the wild and reared cores. For principal component analysis, samples grouped together independently of host species, domestication status and location. A high proportion of yeast produced aminopeptidases and lipases, which may be explained by the high proportion of protein and lipids in the carnivorous diet. This study reveals the presence of a yeast community in the fish gut that appears to be strongly shaped by a carnivorous diet. Yeast in the gut increases the repertoire of microorganisms interacting with the host intestine, which could influence health and disease.

  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. Heterotrophic bacterial production and extracellular enzymatic activity in sinking particulate matter in the western North Pacific Ocean

    Directory of Open Access Journals (Sweden)

    Namiha eYamada

    2012-10-01

    Full Text Available Heterotrophic activities on sinking particulate matter (SPM play an important role in SPM fluxes in the ocean. To demonstrate regional differences in heterotrophic activities on SPM, we measured heterotrophic bacterial production (HBP in seawater and SPM as well as potential extracellular enzyme activity (EEA in SPM on a transect along 155°E in the western North Pacific Ocean in the subarctic (44°N, the Kuroshio Extension area (35°N, and the subtropical gyre (20°N. Depth-integrated HBP in seawater from the surface to 500 m was comparable between the locations, whereas HBP in SPM at 44°N was substantially lower than at the other sites. We found the highest particulate organic carbon (POC export flux and export efficiency to bathypelagic depths, and the lowest water temperatures, at 44°N. We found significant correlations between leucine aminopeptidase (LAPase activity, ß-glucosidase (BGase activity, POC flux and particulate organic nitrogen flux. LAPase activity was two orders of magnitude higher than BGase activity, with a BGase:LAPase activity ratio of 0.027. There were no significant correlations between HBP and EEA in SPM except for lipase, and lipase activity was significantly correlated with temperature. We propose that hydrographic conditions are an important factor controlling heterotrophic bacterial activity and export efficiency of organic carbon to the deep ocean, as are the sources and abundance of SPM produced in the euphotic zone via primary production.

  15. Identification and clinical significance of Helcococcus species, with description of Helcococcus seattlensis sp. nov. from a patient with urosepsis.

    Science.gov (United States)

    Chow, Siu-Kei; Clarridge, Jill E

    2014-03-01

    Helcococcus spp. are Gram-positive, catalase-negative, facultatively anaerobic cocci that are associated with wound and prosthetic joint infections as well bacteremia and empyema. Five Helcococcus spp. strains were isolated from our patient population, including 2 strains of Helcococcus kunzii from trauma-associated wounds, 2 Helcococcus sueciensis strains from blood and abscess, and a novel Helcococcus spp. strain from blood associated with urosepsis. Based on the phenotypic and phylogenetic evidence, we propose that the unknown bacterium be classified as Helcococcus seattlensis sp. nov. We found that all 5 tested Helcococcus strains grew as satellite colonies around Staphylococcus aureus and, interestingly, both H. kunzii strains were isolated together with S. aureus. In addition to 16S rRNA gene sequencing, conventional methods for leucine aminopeptidase (LAP) and pyrrolidonyl arylamidase (PYR) testing can be cost-effective and efficient for differentiation of Helcococcus spp. from Abiotrophia and Granulicatella species. Using nonstandard methods, we found that all tested Helcococcus spp. had high MICs of >4/76 μg/ml for trimethoprim-sulfamethoxazole, an antibiotic commonly used to treat urinary tract infections. High MICs for erythromycin, azithromycin, and clindamycin, and intermediate to high MICs for moxifloxacin, levofloxacin, and gentamicin were also observed among the Helcococcus strains.

  16. Endosymbiotic and host proteases in the digestive tract of the invasive snail Pomacea canaliculata: diversity, origin and characterization.

    Directory of Open Access Journals (Sweden)

    Martín S Godoy

    Full Text Available Digestive proteases of the digestive tract of the apple snail Pomacea canaliculata were studied. Luminal protease activity was found in the crop, the style sac and the coiled gut and was significantly higher in the coiled gut. Several protease bands and their apparent molecular weights were identified in both tissue extracts and luminal contents by gel zymography: (1 a 125 kDa protease in salivary gland extracts and in the crop content; (2 a 30 kDa protease throughout all studied luminal contents and in extracts of the midgut gland and of the endosymbionts isolated from this gland; (3 two proteases of 145 and 198 kDa in the coiled gut content. All these proteases were inhibited by aprotinin, a serine-protease inhibitor, and showed maximum activity between 30°C and 35°C and pH between 8.5 and 9.5. Tissue L-alanine-N-aminopeptidase activity was determined in the wall of the crop, the style sac and the coiled gut and was significantly higher in the coiled gut. Our findings show that protein digestion in P. canaliculata is carried out through a battery of diverse proteases originated from the salivary glands and the endosymbionts lodged in the midgut gland and by proteases of uncertain origin that occur in the coiled gut lumen.

  17. Diversity evaluation based on morphological, physiological and isozyme variation in genetic resources of garlic (Allium sativum L.) collected worldwide.

    Science.gov (United States)

    Hirata, Sho; Abdelrahman, Mostafa; Yamauchi, Naoki; Shigyo, Masayoshi

    2016-11-26

    The aim of this study was to obtain primary information about the global diversity of garlic (Allium sativum L.) by evaluating morphological, physiological and isozyme variation. A total of 107 garlic accessions collected worldwide were grown in Yamaguchi, Japan. Five morphological traits (bulb weight, bulb diameter, number of cloves per bulb, number of bulbils and scape length) and one physiological trait (bolting period) of the collected garlic showed wide variation. Meanwhile, a total of 140 garlic accessions, including the 107 mentioned above, were characterized by leucine aminopeptidase (LAP) and phosphoglucoisomerase (PGI) isozyme analyses; they clearly showed polymorphisms in putative isozyme loci (Lap-1, Lap-2 and Pgi-1). Allelic frequencies were estimated in each group of accessions categorized by their geographical origin, and the observed (Ho) and expected (He) heterozygosities were calculated. The allelic frequencies differed between groups. A principal component analysis based on morpho-physiological data indicated a grouping of the garlic accessions into Central Asian and Northern Mediterranean groups as well as others. We discuss the roles of artificial and natural selection that may have caused differentiation in these traits, on the assumption that ancestral domesticated garlic populations have adapted in various regions using standing variation or mutations that accumulated during expansion, and have evolved along with human-preferred traits over a long history of cultivation.

  18. 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.

  19. Proteomic Analysis of GLUT4 Storage Vesicles Reveals Tumor Suppressor Candidate 5 (TUSC5) as a Novel Regulator of Insulin Action in Adipocytes.

    Science.gov (United States)

    Fazakerley, Daniel J; Naghiloo, Sheyda; Chaudhuri, Rima; Koumanov, Françoise; Burchfield, James G; Thomas, Kristen C; Krycer, James R; Prior, Matthew J; Parker, Ben L; Murrow, Beverley A; Stöckli, Jacqueline; Meoli, Christopher C; Holman, Geoffrey D; James, David E

    2015-09-25

    Insulin signaling augments glucose transport by regulating glucose transporter 4 (GLUT4) trafficking from specialized intracellular compartments, termed GLUT4 storage vesicles (GSVs), to the plasma membrane. Proteomic analysis of GSVs by mass spectrometry revealed enrichment of 59 proteins in these vesicles. We measured reduced abundance of 23 of these proteins following insulin stimulation and assigned these as high confidence GSV proteins. These included established GSV proteins such as GLUT4 and insulin-responsive aminopeptidase, as well as six proteins not previously reported to be localized to GSVs. Tumor suppressor candidate 5 (TUSC5) was shown to be a novel GSV protein that underwent a 3.7-fold increase in abundance at the plasma membrane in response to insulin. siRNA-mediated knockdown of TUSC5 decreased insulin-stimulated glucose uptake, although overexpression of TUSC5 had the opposite effect, implicating TUSC5 as a positive regulator of insulin-stimulated glucose transport in adipocytes. Incubation of adipocytes with TNFα caused insulin resistance and a concomitant reduction in TUSC5. Consistent with previous studies, peroxisome proliferator-activated receptor (PPAR) γ agonism reversed TNFα-induced insulin resistance. TUSC5 expression was necessary but insufficient for PPARγ-mediated reversal of insulin resistance. These findings functionally link TUSC5 to GLUT4 trafficking, insulin action, insulin resistance, and PPARγ action in the adipocyte. Further studies are required to establish the exact role of TUSC5 in adipocytes.

  20. Improved recovery of proteome-informative, protein N-terminal peptides by combined fractional diagonal chromatography (COFRADIC).

    Science.gov (United States)

    Staes, An; Van Damme, Petra; Helsens, Kenny; Demol, Hans; Vandekerckhove, Joël; Gevaert, Kris

    2008-04-01

    We previously described a proteome-wide, peptide-centric procedure for sorting protein N-terminal peptides and used these peptides as readouts for protease degradome and xenoproteome studies. This procedure is part of a repertoire of gel-free techniques known as COmbined FRActional DIagonal Chromatography (COFRADIC) and highly enriches for alpha-amino-blocked peptides, including alpha-amino-acetylated protein N-terminal peptides. Here, we introduce two additional steps that significantly increase the fraction of such proteome-informative, N-terminal peptides: strong cation exchange (SCX) segregation of alpha-amino-blocked and alpha-amino-free peptides and an enzymatic step liberating pyroglutamyl peptides for 2,4,6-trinitrobenzenesulphonic acid (TNBS) modification and thus COFRADIC sorting. The SCX step reduces the complexity of the analyte mixture by enriching N-terminal peptides and depleting alpha-amino-free internal peptides as well as proline-starting peptides prior to COFRADIC. The action of pyroglutamyl aminopeptidases prior to the first COFRADIC peptide separation results in greatly diminishing numbers of contaminating pyroglutamyl peptides in peptide maps. We further show that now close to 95% of all COFRADIC-sorted peptides are alpha-amino-acetylated and, using the same amount of starting material, our novel procedure leads to an increased number of protein identifications.

  1. In-Silico Determination of Insecticidal Potential of Vip3Aa-Cry1Ac Fusion Protein Against Lepidopteran Targets Using Molecular Docking.

    Science.gov (United States)

    Ahmad, Aftab; Javed, Muhammad R; Rao, Abdul Q; Khan, Muhammad A U; Ahad, Ammara; Din, Salah Ud; Shahid, Ahmad A; Husnain, Tayyab

    2015-01-01

    Study and research of Bt (Bacillus thuringiensis) transgenic plants have opened new ways to combat insect pests. Over the decades, however, insect pests, especially the Lepidopteran, have developed tolerance against Bt delta-endotoxins. Such issues can be addressed through the development of novel toxins with greater toxicity and affinity against a broad range of insect receptors. In this computational study, functional domains of Bacillus thuringiensis crystal delta-endotoxin (Cry1Ac) insecticidal protein and vegetative insecticidal protein (Vip3Aa) have been fused to develop a broad-range Vip3Aa-Cry1Ac fusion protein. Cry1Ac and Vip3Aa are non-homologous insecticidal proteins possessing receptors against different targets within the midgut of insects. The insecticidal proteins were fused to broaden the insecticidal activity. Molecular docking analysis of the fusion protein against aminopeptidase-N (APN) and cadherin receptors of five Lepidopteran insects (Agrotis ipsilon, Helicoverpa armigera, Pectinophora gossypiella, Spodoptera exigua, and Spodoptera litura) revealed that the Ser290, Ser293, Leu337, Thr340, and Arg437 residues of the fusion protein are involved in the interaction with insect receptors. The Helicoverpa armigera cadherin receptor, however, showed no interaction, which might be due to either loss or burial of interactive residues inside the fusion protein. These findings revealed that the Vip3Aa-Cry1Ac fusion protein has a strong affinity against Lepidopteran insect receptors and hence has a potential to be an efficient broad-range insecticidal protein.

  2. 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.

  3. Proteolytic degradation of neuropeptide Y (NPY) from head to toe: Identification of novel NPY-cleaving peptidases and potential drug interactions in CNS and Periphery.

    Science.gov (United States)

    Wagner, Leona; Wolf, Raik; Zeitschel, Ulrike; Rossner, Steffen; Petersén, Åsa; Leavitt, Blair R; Kästner, Florian; Rothermundt, Matthias; Gärtner, Ulf-Torsten; Gündel, Daniel; Schlenzig, Dagmar; Frerker, Nadine; Schade, Jutta; Manhart, Susanne; Rahfeld, Jens-Ulrich; Demuth, Hans-Ulrich; von Hörsten, Stephan

    2015-12-01

    The bioactivity of neuropeptide Y (NPY) is either N-terminally modulated with respect to receptor selectivity by dipeptidyl peptidase 4 (DP4)-like enzymes or proteolytic degraded by neprilysin or meprins, thereby abrogating signal transduction. However, neither the subcellular nor the compartmental differentiation of these regulatory mechanisms is fully understood. Using mass spectrometry, selective inhibitors and histochemistry, studies across various cell types, body fluids, and tissues revealed that most frequently DP4-like enzymes, aminopeptidases P, secreted meprin-A (Mep-A), and cathepsin D (CTSD) rapidly hydrolyze NPY, depending on the cell type and tissue under study. Novel degradation of NPY by cathepsins B, D, L, G, S, and tissue kallikrein could also be identified. The expression of DP4, CTSD, and Mep-A at the median eminence indicates that the bioactivity of NPY is regulated by peptidases at the interphase between the periphery and the CNS. Detailed ex vivo studies on human sera and CSF samples recognized CTSD as the major NPY-cleaving enzyme in the CSF, whereas an additional C-terminal truncation by angiotensin-converting enzyme could be detected in serum. The latter finding hints to potential drug interaction between antidiabetic DP4 inhibitors and anti-hypertensive angiotensin-converting enzyme inhibitors, while it ablates suspected hypertensive side effects of only antidiabetic DP4-inhibitors application. The bioactivity of neuropeptide Y (NPY) is either N-terminally modulated with respect to receptor selectivity by dipeptidyl peptidase 4 (DP4)-like enzymes or proteolytic degraded by neprilysin or meprins, thereby abrogating signal transduction. However, neither the subcellular nor the compartmental differentiation of these regulatory mechanisms is fully understood. Using mass spectrometry, selective inhibitors and histochemistry, studies across various cell types, body fluids, and tissues revealed that most frequently DP4-like enzymes

  4. Cloning and sequence analysis of the isoforms H11-4 of the vaccine candidate antigen H11 from Haemonchus contortus%捻转血矛线虫ZJ株疫苗候选抗原H11亚型基因H11-4的克隆及序列分析

    Institute of Scientific and Technical Information of China (English)

    段丽君; 周前进; 张红丽; 杨怡; 闫宝龙; 杜爱芳

    2013-01-01

    微粒体氨肽酶H11天然提取物是目前捻转血矛线虫(Haemonchus contortus)防治研究中最好的疫苗候选抗原之一,但其重组形式均不能提供有效的免疫保护效果;同时报道H11蛋白存在多种亚型,推测其某种亚型或亚型组合可能在天然提取物参与免疫保护中起了关键作用.本试验参考NCBI公布的H.contortus H11-4基因序列,设计2对特异性引物,以H.contortus ZJ株总RNA为模板,利用RT-PCR技术分段扩增出该基因的部分片段,并进行T-A克隆.测序正确后,利用含有不同片段的阳性质粒经BamH Ⅰ/Nco Ⅰ消化,连接后获得H11-4基因的全长cDNA序列.测序结果显示成功获得H11 4基因,开放阅读框大小为2 916 bp,与NCBI中公布的核苷酸序列同源性为97.8%,氨基酸序列同源性为97.6%.生物信息学分析,已获得的H11-4与H11 (H11-3)亚型氨基酸序列高度同源,且具有保守糖基化位点、相对保守的跨膜区与微粒体氨肽酶活性中心锌指基序.为进一步分析H11天然提取物各亚型在参与免疫保护的机制和角色分工奠定了基础.%The recombinant microsomal aminopeptidase H11 antigen has not shown highly protective efficacy against Haemonchus contortus compared to its native extract which is considered to be the most efficient vaccine candidate antigen. It is reported that there exist several isoforms of native H11 and it was supposed that one or combined isoforms of native H11 play a key role in immune protection. Fragments of H11-4 gene were amplified by reverse transcription polymerase chain reaction (RT-PCR) with two pairs of primers designed according to the published gene sequence from NCBI. Then the fragments of H11-4 gene were ligated to the T-A cloning vector pMD18-T and sequenced. Positive clones representative the different two fragments were digested with BamH Ⅰ / Nco Ⅰ and then ligated to obtain the full-length cDNA. Sequence analysis shows that with 2 916 bp, H11-4 gene

  5. Determinação da compatibilidade de desenvolvimento de culturas bacteriocinogênicas e fermento láctico Determination of the growth compatibility between bacteriocinogenic and starter cultures

    Directory of Open Access Journals (Sweden)

    Maristela da Silva do Nascimento

    2009-03-01

    Full Text Available Além da utilização como bioconservantes de alimentos, algumas culturas bacteriocinogênicas estão sendo empregadas para acelerar a maturação de queijos. Porém a compatibilidade de desenvolvimento destas culturas com o fermento láctico é essencial para a obtenção de produtos característicos. O objetivo deste estudo foi avaliar a compatibilidade de desenvolvimento de Lactococcus lactis subsp. lactis ATCC 11454, Lactobacillus plantarum ALC 01 e Enterococcus faecium FAIR-E 198 com duas marcas comerciais de fermentos lácticos. Inicialmente, foi determinada a sensibilidade in vitro dos fermentos às culturas bacteriocinogênicas, somente Lc. lactis subsp. lactis ATCC 11454 foi capaz de promover a inibição de ambos os fermentos. Durante desenvolvimento associativo em leite a 35 ºC, as culturas bacteriocinogênicas não afetaram significativamente a produção de ácido láctico pelos fermentos. Estes, por sua vez proporcionaram aumento significativo da atividade de pediocina AcH e enterocina FAIR-E 198 e supressão da atividade da nisina. Dentre todas as culturas lácticas, Lb. plantarum ALC 01 apresentou a maior atividade de aminopeptidases (0,226 a 0,390. Portanto, baseado nos resultados em questão, Lb. plantarum ALC 01 e E. faecium FAIR-E 198 apresentam características de compatibilidade de desenvolvimento com o fermento mesofílico tipo O para serem empregadas como adjuntas no processamento de queijos.In addition to being used as food bioconservants, some bacteriocinogenic cultures have been employed to accelerate cheese ripening. However, the compatibility between their growth and starter cultures is essential to obtain the characteristic products. The purpose of this study was to evaluate the growth compatibility between Lactococcus lactis subsp. lactis ATCC 11454, Lactobacillus plantarum ALC 01, and Enterococcus faecium FAIR-E 198 and two commercial starter cultures. Initially, the sensibility in vitro of the starter to

  6. Identificação de parentais e híbridos entre Vitis vinifera e Vitis rotundifolia utilizando polimorfismo enzimático e marcador RAPD Identification of parents and hybrids among Vitis vinifera and Vitis rotundifolia using isozyme polymorphism and rapd marker

    Directory of Open Access Journals (Sweden)

    Haiko Enok Sawazaki

    1996-01-01

    Full Text Available Identificaram-se parentais e híbridos entre Vitis vinifera (videiras comuns e V. rotundifolia (muscadínias, utilizando-se o polimorfismo enzimático e marcador RAPD (Random Amplified Polymorphic DNA. Os sistemas GOT (glutamato-oxalo-acetato-transaminase, IDH (isocitrato desidrogenase e PGI (fosfoglucose isomerase diferenciaram a muscadínia, sendo observadas cinco aloenzimas para o GOT, duas para o LAP (leucina aminopeptidase e quatro para o EST (esterase. Os sistemas PGI e IDH apresentaram-se como diméricos com o fenótipo de quatro aloenzimas em duas regiões e três em uma região respectivamente. O marcador RAPD apresentou polimorfismo que permitiu a diferenciação entre todos os cultivares. Os dendrogramas UPGMA (unweighted pair-group method with aritmetic mean obtidos pelas isoenzimas e pelo marcador RAPD foram semelhantes, sendo a aproximação mais forte entre 'Itália' e 'Rubi' que se ligaram aos cultivares Patrícia e A Dona. Os cultivares Piratininga e Eugênio, também bastante próximos, foram os seguintes a se ligarem às demais viníferas. Pelo polimorfismo enzimático e marcador RAPD, a muscadínia ficou bastante isolada dos outros grupos. Pelo método RAPD, aplicado às muscadínias, ao híbrido da Carolina do Norte NC66 C203-9, a um possível híbrido e seu parental feminino, observou-se o seguinte: os híbridos foram intermediários às muscadínias e viníferas, porém o possível híbrido se assemelhou ao parental feminino, enquanto o NC66203-9 apresentou bandas provenientes das muscadínias e viníferas, comprovando sua origem híbrida.Aiming to identify parents and hybrids among Vitis vinifera (bunch grapes and Vitis rotundifolia (muscadine grapes isozyme polymorphism and RAPD marker were used. GOT (glutamate oxaloacetate transaminase, IDH (isocitrate dehydrogenase and PGI (phosphoglucoisomerase systems could differentiate the muscadine, being identified five allozymes for GOT, two allozymes for LAP (leucine

  7. Caracterização de cultivares de pessegueiro e de nectarineira por marcadores moleculares Characterization of peach and nectarine cultivars through molecular markers

    Directory of Open Access Journals (Sweden)

    Marli Rocha Lima

    2003-03-01

    Full Text Available Em espécies de estreita base genética, como o pessegueiro e a nectarineira (Prunus persica (L. Batsch, a utilização de marcadores moleculares para a caracterização de cultivares é de grande importância, além do potencial de uso para fins de proteção. As técnicas de eletroforese em gel e RAPD foram empregadas com o objetivo de caracterizar as cultivares de pessegueiro Granada, Esmeralda, Jade, Eldorado, Riograndense, Capdeboscq, Aldrighi, Precocinho, Diamante, Turmalina, Maciel, BR-1, Pepita, Coral, Chinoca, Marfim, Chiripá, Della Nona e Planalto, e as de nectarineira Dulce e Anita. Foram analisadas isoenzimas de 6-fosfogluconato desidrogenase e fosfatase ácida em pólen, peroxidase, fosfoglucoisomerase, aspartato transaminase e isocitrato desidrogenase em folhas, e malato desidrogenase, leucina aminopeptidase e fosfoglucomutase em pólen e folhas. Dos 50 primers testados, 11 foram escolhidos para análise de RAPD em folhas. As análises de similaridade e de agrupamento entre os genótipos foram feitas empregando-se o coeficiente de Jaccard e o método da média aritmética não ponderada. Apesar das diferenças detectadas nas isoenzimas de malato desidrogenase em pólen e folhas de pessegueiro e nectarineira, o baixo polimorfismo apresentado pelos demais sistemas não permitiu a caracterização de todas as cultivares por essa técnica. Os marcadores RAPD, associados ou não à eletroforese de isoenzimas, foram eficientes para caracterizar as cultivares de pessegueiro e nectarineira.In species with a narrow genetic basis, such as peach and nectarine (Prunus persica (L. Batsch, the utilization of molecular markers in cultivar characterization is very important, besides the potential of use for protection. Gel electrophoresis and RAPD techniques were used to characterize peach cultivars Granada, Esmeralda, Jade, Eldorado, Riograndense, Capdeboscq, Aldrighi, Precocinho, Diamante, Turmalina, Maciel, BR-1, Pepita, Coral, Chinoca, Marfim

  8. Isoenzymatic variability of cassava accessions from different regions in Brazil Variabilidade isoenzimática de acessos de mandioca de diferentes regiões do Brasil

    Directory of Open Access Journals (Sweden)

    Betânia Lúcia Rocha Cabral

    2002-09-01

    Full Text Available Cassava (Manihot esculenta Crantz belongs to the Euphorbiaceae family, and is widely cultivated in Brazil. The objective of this study was to evaluate the isoenzymatic variability of 200 cassava accessions from the germplasm bank of Embrapa Amazonia Oriental. Seven groups were formed according to their origin: 1-Amazonas, 2-Amapa, 3-Bahia, 4-Para, 5-Rondonia, 6-Various, for accessions with a maximum of three individuals per place of origin, and 7 - Accessions of indefinite origin. The accessions were also evaluated as a whole. For the electrophoretic analyses, samples of young leaves were used in a 12% starch gel. Eight isoenzymatic systems were evaluated: acid phosphatase (ACP, leucine aminopeptidase (LAP, glucose-6-phosphate dehydrogenase (G6PDH, malate dehydrogenase (MDH, shikimate dehydrogenase (SKDH, malic enzyme (ME, glutamate dehydrogenase (GTDH and isocitrate dehydrogenase (IDH. Analysis revealed a polymorphic locus for each system and high isoenzymatic variability among accessions. The average number of alleles per locus varied from 2.3 to 2.5. Average observed heterozigosity varied from 0.381 to 0.615 and the diversity index varied from 0.479 to 0.559. Genetic variability within groups was greater than among groups, suggesting a distribution pattern similar to what can be expected for natural populations of outcrossing plants.A mandioca (Manihot esculenta Crantz pertence à família Euphorbiaceae, gênero Manihot, cultivada em todo o país. É a única do gênero utilizada na alimentação. O presente trabalho teve como objetivo o estudo da variabilidade isoenzimática de 200 acessos de mandioca obtidos junto ao banco de germoplasma da Embrapa Amazônia Oriental. Os acessos foram agrupados de acordo com o local de origem, obtendo-se desta forma sete grupos: 1-Amazonas, 2-Amapa, 3-Bahia, 4-Para, 5-Rondonia, 6-Diversos, incluindo-se neste grupo os acessos que se apresentavam em pequena quantidade por local de origem (um ou no m

  9. Hotsphere illumination

    Science.gov (United States)

    Razavi, Bahar S.; Kuzyakov, Yakov

    2016-04-01

    root demonstrated plant specific patterns of enzyme distribution: it was uniform and homogenous along the lentil roots, whereas the enzyme activities in maize rhizosphere were higher at the apical or proximal root parts. The activity of leucine-aminopeptidase was higher at the apical parts and β-glucosidase activity was higher at both apical and proximal part of individual maize roots. Much higher activity of leucine-aminopeptidase and β-glucosidase per mm2 of hotspots were found for rhizosphere (12-5 fold), drilosphere (10-4), spermosphere (9-4), biopore (9-1), hyphasphere (8-3) and detritusphere (5-2) compared to the bulk soil. Despite the transient nature of spermosphere, its microbial activities had long-lasting impact. We conclude that improved zymography is promising in situ technique to identify, analyze, visualize and quantify temporal-spatial distribution of enzyme activities in the various hotspheres. Key words: hotsphere, enzyme distribution, temporal-spatial, zymography Reference: Kuzyakov Y, Blagodatskaya E (2015) Microbial hotspots and hot moments in soil: Concept & review. Soil Biology and Biochemistry 83: 184-199. Spohn M, Kuzyakov Y (2014) Spatial and temporal dynamics of hotspots of enzyme activity in soil as affected by living and dead roots- a soil zymography analysis, Plant Soil 379: 67-77.

  10. Serum specific vasopressin-degrading activity is related to blood total cholesterol levels in men but not in women.

    Science.gov (United States)

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

    2012-07-01

    The role of vasopressin (AVP) in the pathophysiology of cardiovascular disease is controversial, but this peptide hormone is elevated in heart failure and some forms of hypertension. Also, AVP has vasoconstrictor, mitogenic, hyperplasic and renal fluid retaining properties which, by analogy with angiotensin II, may have deleterious effects when present in chronic excess. Furthermore, cholesterol blood levels are also associated with hypertension, although the underlying mechanism is not known. Here we analyze the relationship between blood total cholesterol levels and serum vasopressin- degrading cystyl-aminopeptidase activity (AVP-DA) in healthy humans, and the differences between men and women. Linear correlation coefficients were calculated to test relationships between AVP-DA and blood total cholesterol levels. Sex differences were observed for AVP-DA, being this activity higher in men than in women. According to the linear model of the regression analysis, AVP-DA showed a significant negative correlation with blood total cholesterol levels in men, whereas no correlation was observed in women. Several studies in humans demonstrate the existence of greater plasma AVP concentrations in normal men compared to normal women, which could explain the gender-differences observed in the present work in relation with AVP-DA. However, AVP-DA is related to blood cholesterol levels only in men, although in our hands, women showed higher blood cholesterol levels than men. This could indicate that the risk of high cholesterol-related hypertension is more probable in men than in women. Although AVP-DA misregulation could be involved in the pathogenesis of hypertension, its relation with cholesterol levels appears only in men, but not in women.

  11. [Neonatal hypoxic-ischemic nephropathy and urinary diagnostic indices: the utility of measuring tubular enzymes (NAG and AAP)].

    Science.gov (United States)

    Bertotti, A; De Marchi, S; Brovedani, P; Gaeta, G; Peratoner, L; Mangiarotti, M A

    1990-01-01

    Feto-neonatal hypoxia can cause a functional kidney impairment, which is often temporary and not clinically overt, but sometimes leading to acute renal failure. Hypoxic stress may result in a tubulo-interstitial damage, and kidney tubular enzymes determination has proved to be an easy, early, and non invasive method to define a tubular interstitial lesion. A major target of nephrotoxicity is the proximal tubular cell: alterations in brush-border membrane and cytoplasm result in increased turnover processes in the kidney cortex, following by a corresponding increased excretion of alanine-aminopeptidase (AAP) and N-acetyl-glucosaminidase (NAG) from the proximal tubular cells, long before glomerular or tubular functions are impaired. AAP and NAG excretion is directly correlated with the strength and the duration of toxic alteration of the proximal tubule. NAG and AAP have been already studied in the adults and the children; they have been chosen for this investigation with a double aim: 1) to define the amount of their urinary excretion in relation with gestational age at birth; 2) to evaluate if in the newborn, independently of the gestational age, their urinary concentration may be increased by ischaemic conditions caused by hypoxia. We studied 52 healthy newborns (7 preterm of 33-36 weeks and 45 full-term) and 16 newborns with feto-neonatal hypoxia (8 preterm of 26-36 weeks and full-term) at the forth day of life. Urinary NAG and AAP were assayed by colorimetric methods and the results expressed as mU/mg. creatininuria.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Temperature Sensitivity as a Microbial Trait Using Parameters from Macromolecular Rate Theory

    Directory of Open Access Journals (Sweden)

    Charlotte Jean Alster

    2016-11-01

    Full Text Available The activity of soil microbial extracellular enzymes is strongly controlled by temperature, yet the degree to which temperature sensitivity varies by microbe and enzyme type is unclear. Such information would allow soil microbial enzymes to be incorporated in a traits-based framework to improve prediction of ecosystem response to global change. If temperature sensitivity varies for specific soil enzymes, then determining the underlying causes of variation in temperature sensitivity of these enzymes will provide fundamental insights for predicting nutrient dynamics belowground. In this study, we characterized how both microbial taxonomic variation as well as substrate type affects temperature sensitivity. We measured β-glucosidase, leucine aminopeptidase, and phosphatase activities at six temperatures: 4, 11, 25, 35, 45, and 60°C, for seven different soil microbial isolates. To calculate temperature sensitivity, we employed two models, Arrhenius, which predicts an exponential increase in reaction rate with temperature, and Macromolecular Rate Theory (MMRT, which predicts rate to peak and then decline as temperature increases. We found MMRT provided a more accurate fit and allowed for more nuanced interpretation of temperature sensitivity in all of the enzyme × isolate combinations tested. Our results revealed that both the enzyme type and soil isolate type explain variation in parameters associated with temperature sensitivity. Because we found temperature sensitivity to be an inherent and variable property of an enzyme, we argue that it can be incorporated as a microbial functional trait, but only when using the MMRT definition of temperature sensitivity. We show that the Arrhenius metrics of temperature sensitivity are overly sensitive to test conditions, with activation energy changing depending on the temperature range it was calculated within. Thus, we propose the use of the MMRT definition of temperature sensitivity for accurate

  13. Temperature Sensitivity as a Microbial Trait Using Parameters from Macromolecular Rate Theory.

    Science.gov (United States)

    Alster, Charlotte J; Baas, Peter; Wallenstein, Matthew D; Johnson, Nels G; von Fischer, Joseph C

    2016-01-01

    The activity of soil microbial extracellular enzymes is strongly controlled by temperature, yet the degree to which temperature sensitivity varies by microbe and enzyme type is unclear. Such information would allow soil microbial enzymes to be incorporated in a traits-based framework to improve prediction of ecosystem response to global change. If temperature sensitivity varies for specific soil enzymes, then determining the underlying causes of variation in temperature sensitivity of these enzymes will provide fundamental insights for predicting nutrient dynamics belowground. In this study, we characterized how both microbial taxonomic variation as well as substrate type affects temperature sensitivity. We measured β-glucosidase, leucine aminopeptidase, and phosphatase activities at six temperatures: 4, 11, 25, 35, 45, and 60°C, for seven different soil microbial isolates. To calculate temperature sensitivity, we employed two models, Arrhenius, which predicts an exponential increase in reaction rate with temperature, and Macromolecular Rate Theory (MMRT), which predicts rate to peak and then decline as temperature increases. We found MMRT provided a more accurate fit and allowed for more nuanced interpretation of temperature sensitivity in all of the enzyme × isolate combinations tested. Our results revealed that both the enzyme type and soil isolate type explain variation in parameters associated with temperature sensitivity. Because we found temperature sensitivity to be an inherent and variable property of an enzyme, we argue that it can be incorporated as a microbial functional trait, but only when using the MMRT definition of temperature sensitivity. We show that the Arrhenius metrics of temperature sensitivity are overly sensitive to test conditions, with activation energy changing depending on the temperature range it was calculated within. Thus, we propose the use of the MMRT definition of temperature sensitivity for accurate interpretation of

  14. Constitutive activation of the midgut response to Bacillus thuringiensis in Bt-resistant Spodoptera exigua.

    Directory of Open Access Journals (Sweden)

    Patricia Hernández-Martínez

    Full Text Available Bacillus thuringiensis is the most effective microbial control agent for controlling numerous species from different insect orders. The main threat for the long term use of B. thuringiensis in pest control is the ability of insects to develop resistance. Thus, the identification of insect genes involved in conferring resistance is of paramount importance. A colony of Spodoptera exigua (Lepidoptera: Noctuidae was selected for 15 years in the laboratory for resistance to Xentari™, a B. thuringiensis-based insecticide, reaching a final resistance level of greater than 1,000-fold. Around 600 midgut ESTs were analyzed by DNA-macroarray in order to find differences in midgut gene expression between susceptible and resistant insects. Among the differentially expressed genes, repat and arylphorin were identified and their increased expression was correlated with B. thuringiensis resistance. We also found overlap among genes that were constitutively over-expressed in resistant insects with genes that were up-regulated in susceptible insects after exposure to Xentari™, suggesting a permanent activation of the response to Xentari™ in resistant insects. Increased aminopeptidase activity in the lumen of resistant insects in the absence of exposure to Xentari™ corroborated the hypothesis of permanent activation of response genes. Increase in midgut proliferation has been proposed as a mechanism of response to pathogens in the adult from several insect species. Analysis of S. exigua larvae revealed that midgut proliferation was neither increased in resistant insects nor induced by exposure of susceptible larvae to Xentari™, suggesting that mechanisms other than midgut proliferation are involved in the response to B. thuringiensis by S. exigua larvae.

  15. Renal metabolism of calcitonin

    Energy Technology Data Exchange (ETDEWEB)

    Simmons, R.E.; Hjelle, J.T.; Mahoney, C.; Deftos, L.J.; Lisker, W.; Kato, P.; Rabkin, R.

    1988-04-01

    The kidneys account for approximately two-thirds of the metabolism of calcitonin, but relatively little is known regarding the details thereof. To further characterize this process, we examined the renal handling and metabolism of human calcitonin (hCT) by the isolated perfused rat kidney. We also studied the degradation of radiolabeled salmon calcitonin (sCT) by subcellular fractions prepared from isolated rabbit proximal tubules. The total renal (organ) clearance of immunoreactive hCT by the isolated kidney was 1.96 +/- 0.18 ml/min. This was independent of the perfusate total calcium concentration from 5.5 to 10.2 mg/dl. Total renal clearance exceeded the glomerular filtration rate (GFR, 0.68 +/- 0.05 ml/min), indicating filtration-independent removal. Urinary calcitonin clearance as a fraction of GFR averaged 2.6%. Gel filtration chromatography of medium from isolated kidneys perfused with /sup 125/I-labeled sCT showed the principal degradation products to be low molecular weight forms eluting with monoiodotyrosine. Intermediate size products were not detected. In the subcellular fractionation experiments, when carried out at pH 5.0, calcitonin hydrolysis exclusively followed the activities of the lysosomal enzyme N-acetyl-beta-glucosaminidase. Typically, at pH 7.5, 42% of total degradation occurred in the region of the brush-border enzyme alanyl aminopeptidase and 29% occurred in the region of the cytosolic enzyme phosphoglucomutase. Although 9% of the calcitonin-degrading activity was associated with basolateral membrane fractions, most of this activity could be accounted for by the presence of brush-border membranes.

  16. Bioremediation (bioaugmentation/biostimulation) trials of oil polluted seawater: a mesocosm simulation study.

    Science.gov (United States)

    Hassanshahian, Mehdi; Emtiazi, Giti; Caruso, Gabriella; Cappello, Simone

    2014-04-01

    Bioaugmentation (amendment with selected bacterial strains) and/or biostimulation (nutrients addition and/or air supply) are relatively new fields in environmental microbiology for preventing pollution and cleanup contamination. In this study, the efficiency of application of bioaugmentation/biostimulation treatments, for recovery of crude oil-polluted seawater, was evaluated. Three different series of experiments were performed in a "Mesocosm Facility" (10.000 L). Natural seawater was artificially polluted with crude oil (1000 ppm) and was amended with inorganic nutrients (Mesocosm 1, M1), inorganic nutrient and an inoculum of Alcanivorax borkumensis SK2(T) (Mesocosm 2, M2) and inorganic nutrient and an inoculum of A. borkumensis SK2(T) and Thalassolituus oleivorans MIL-1(T) (Mesocosm 3, M3), respectively. During the experimental period (20 days) bacterial abundance (DAPI count), culturable heterotrophic bacteria (CFU count), MPN, microbial metabolic activity [Biochemical Oxygen Demand and enzymatic activity (leucine aminopeptidase LAP, β-glucosidase BG, alkaline phosphatase AP)] and quali-, quantitative analysis of the composition of total extracted and resolved hydrocarbons and their derivates (TERHCs) were carried out. The microbiological and physiological analysis of marine microbial community found during the three different biostimulation and bioaugmentation assays performed in mesocosms show that the load of crude oil increases total microbial abundance, inhibits the activity of some enzymes such as LAP while stimulates both AP and BG activities. The biodegradation results show that bioaugmentation with A. borkumensis SK2(T) alone is able to produce the highest percentage of degradation (95%) in comparison with the biostimulation treatment (80%) and bioaugmentation using an Alcanivorax-Thalassolituus bacterial consortium (70%). This result highlights the reduced biodegradation capability of the consortium used in this study, suggesting an unfavourable

  17. Sorting signals, N-terminal modifications and abundance of the chloroplast proteome.

    Directory of Open Access Journals (Sweden)

    Boris Zybailov

    Full Text Available Characterization of the chloroplast proteome is needed to understand the essential contribution of the chloroplast to plant growth and development. Here we present a large scale analysis by nanoLC-Q-TOF and nanoLC-LTQ-Orbitrap mass spectrometry (MS of ten independent chloroplast preparations from Arabidopsis thaliana which unambiguously identified 1325 proteins. Novel proteins include various kinases and putative nucleotide binding proteins. Based on repeated and independent MS based protein identifications requiring multiple matched peptide sequences, as well as literature, 916 nuclear-encoded proteins were assigned with high confidence to the plastid, of which 86% had a predicted chloroplast transit peptide (cTP. The protein abundance of soluble stromal proteins was calculated from normalized spectral counts from LTQ-Obitrap analysis and was found to cover four orders of magnitude. Comparison to gel-based quantification demonstrates that 'spectral counting' can provide large scale protein quantification for Arabidopsis. This quantitative information was used to determine possible biases for protein targeting prediction by TargetP and also to understand the significance of protein contaminants. The abundance data for 550 stromal proteins was used to understand abundance of metabolic pathways and chloroplast processes. We highlight the abundance of 48 stromal proteins involved in post-translational proteome homeostasis (including aminopeptidases, proteases, deformylases, chaperones, protein sorting components and discuss the biological implications. N-terminal modifications were identified for a subset of nuclear- and chloroplast-encoded proteins and a novel N-terminal acetylation motif was discovered. Analysis of cTPs and their cleavage sites of Arabidopsis chloroplast proteins, as well as their predicted rice homologues, identified new species-dependent features, which will facilitate improved subcellular localization prediction. No evidence

  18. [The Qualitative Analysis of the Amide Derivative of HLDF-6 Peptide and Its Metabolites with the Use of Tritium- and Deuterium-Labeled Derivatives].

    Science.gov (United States)

    Zolotarev, A; Dadayan, A K; Kost, N V; Voevodina, M E; Sokolov, O Y; Kozik, V S; Shram, S I; Azev, V N; Bocharov, E V; Bogachouk, A P; Lipkin, V M; Myasoedov, N F

    2015-01-01

    The goal of the study was to elaborate the pharmacokinetics methods of the amide derivative of peptide HLDF-6 (TGENHR-NH2) and its range of nootropic and neuroprotective activity is wide. The hexapeptide 41TGENHR46 is a fragment of the HDLF differentiation factor. It forms the basis for the development of preventive and therapeutic preparations for treating cerebrovascular and neurodegenerative conditions. Pharmacokinetic and molecular mechanisms of the action of the HLDF-6 peptide were studied using tritium- and deuterium-labeled derivatives of this peptide, produced with the use of the high-temperature solid-state catalytic isotope exchange reaction (HSCIE). This reaction was employed to produce the tritium-labeled peptide [3H]TGENHR-NH2 with a molar radioactivity of 230 Ci/mmol and the deuterium-labeled peptide [2H]TGENHR-NH2 with an average deuterium incorporation equal to 10.5 atoms. It was shown by the NMR spectroscopy that the isotope label distribution over the labeled peptide's molecule was uniform, which allowed qualitative analysis ofboth the peptide itself and its fragments in the organism's tissues to be conducted. The newly developed pharmacokinetics method makes it possible to avoid almost completely losses of the peptides under study due to biodegradation during the analysis of tissues. These labeled peptides were used in mice, rats and rabbits to study the pharmacokinetics of the peptide and to calculate the values of its principal pharmacokinetic parameters. Characteristics of its pharmacokinetic profile in the blood were obtained, the hypothesis of pharmacokinetics linearity tested, its metabolism analyzed and its bioavailability value, 34%, calculated. It has been shown that the studied TGENHR-NH2 peptide shows high resistance to hydrolysis in the blood plasma, with dipeptidyl aminopeptidases making the largest contribution to its hydrolysis.

  19. Clinical application of the multi-joint testing vaginitis in diagnosis of gynecological vaginal diseases%阴道炎多联检测在妇科阴道疾病诊断中的临床应用

    Institute of Scientific and Technical Information of China (English)

    唐子平

    2011-01-01

    目的:探讨阴道炎多联检测在妇科阴道疾病的诊断中的临床应用.方法:对1516例阴道分泌物进行多联检测(pH值、过氧化氢浓度、白细胞脂酶、唾液酸苷酶、脯氨酸氨基肽酶、N-乙酰氨基葡萄糖苷酶)和用Amsel方法检测,并对其中正常阴道组、霉菌性阴道炎、滴虫性阴道炎和BV组进行对照比较.结果:根据临床表现和多联检测,BV的发病率高达27.2%,滴虫性阴道炎占12.5%,霉菌性阴道炎占9.0%.结论:利用多项目联合检测法检测阴道分泌物,能快速、简单、客观而有效的对阴道疾病进行辅助诊断.%Objective: To discuss the clinical application of the multi - joint testing vaginitis in diagnosis of gynecological vaginal diseases. Methods: More than 1516 cases of vaginal secretions of joint detection ( pH value, hydrogen peroxide concentration, white blood lipase, neuraminidase, proline aminopeptidase, gan enzyme acetyl glucosamine )and detected by Amsel and on which normal vaginal group, fungal vaginitis and trichomonas vaginitis and BV group for comparison. Results: Based on clinical manifestations and multi -joint testing, BV incidence rate as high as 27.2%, 12.5% trichomoniasis vaginitis, fungal vaginitis 9.0%. Conclusion: The use of multi - project joint detection assay of vaginal secretions can rapid,simple,objective and effective diagnosis vaginal diseases.

  20. [Effect of injection of enkephalin and bestatin in caudate-putamen on operant conditioning in rats].

    Science.gov (United States)

    Zhang, S Y; Zhang, Y P; Zhang, M L; Qi, H X; Wang, B

    1992-10-01

    Female Wistar rats were trained in a Skinner-box, 30 trials per day in a dark room to establish operant defence conditioning. Training started with a light (15 s), then combined with footshock for further 8 s. When the rats learned to press the key to avoid footshock within 15 s, conditioned response was considered established. After the rats reached a conditioning rate (CR) above 80% for 5 days, cannulae were implanted into caudate-putamen. Two to three days later, Met-enkephalin (MEK) or bestatin (an aminopeptidase inhibitor) was injected bilaterally into caudate-putamen. 30 min, 2 h, 24 h and 48 h after injection, conditioning tests were conducted, with each session consisting of 30 trials. Control experiments were done when 0.9% NaCl (NS) was injected. After injection of NS, CR maintained above 80% in all 4 test sessions. MEK (60 ng/rat) or bestatin (10 micrograms/rat) significantly lowered the CR during the 30 min and 2 h test session. In the latter case, the latency (L) was also prolonged. However both CR and L returned to the control level in the 24 h and 48 h test sessions. Naloxone (2 mg/kg, i.p.) blocked the conditioning-depression effect of bestatin. No significant alteration was seen in locomotor activity after MEK or bestatin injection. The results suggest that enkephalin in caudate-putamen may be involved in the regulation of retrieval of conditioning. Bestatin mimics the effect of MEK on conditioning reflex probably by increasing production of endogenous enkephalin.

  1. Identification of a short region on chromosome 6 affecting direct calving ease in Piedmontese cattle breed.

    Directory of Open Access Journals (Sweden)

    Silvia Bongiorni

    Full Text Available Calving in cattle is affected by calf morphology and by dam characteristics. It is described by two different traits: maternal calving ease, which is the ability to generate dams with good physiological predisposition to calving, and direct calving ease, which is the ability to generate calves that are easily born. The aim of this study was to identify regions of cattle genome harboring genes possibly affecting direct calving ease in the Piedmontese cattle breed. A population of 323 bulls scored for direct calving ease (EBV was analyzed by a medium-density SNP marker panel (54,001 SNPs to perform a genome-wide scan. The strongest signal was detected on chromosome 6 between 37.8 and 38.7 Mb where 13 SNPs associated to direct calving ease were found. Three genes are located in this region: LAP3, encoding for a leucine aminopeptidase involved in the oxytocin hydrolysis; NCAPG, encoding for a non-SMC condensin I complex, which has been associated in cattle with fetal growth and carcass size; and LCORL, which has been associated to height in humans and cattle. To further confirm the results of the genome-wide scan we genotyped additional SNPs within these genes and analyzed their association with direct calving ease. The results of this additional analysis fully confirmed the findings of the GWAS and particularly indicated LAP3 as the most probable gene involved. Linkage Disequilibrium (LD analysis showed high correlation between SNPs located within LAP3 and LCORL indicating a possible selection signature due either to increased fitness or breeders' selection for the trait.

  2. Comparing MALDI-TOF Mass Spectrometry with Molecular and Biochemical Methods in Identifying Enterococcus Faecium and Enterococcus Faecalis Isolated from Clinical

    Directory of Open Access Journals (Sweden)

    Samadi Kafil,H.

    2013-01-01

    Full Text Available Abstract Background and Objective: Enterococci are Gram-positive members of human gastrointestinal flora, in Dairy products and environment. they have emerged as important causes of opportunistic nosocomial infections in recent years. In this study we aimed to investigat and compare the efficiency of MALDI-TOF mass spectroscopy method through Biochemical and Molecular methods for detecting Enterococcus faecalis and Enterococcus faecium.Materials and Methods: seventhy five clinical samples were collected for biochemical, molecular and mass spectroscopy investigations. Samples were treated with Esculin hydrolysis, Catalase, Pyrrolidonyl aminopeptidase, 6.5% NaCl solution, motility, 0.04% Tellurite, L-Arabinose and Sorbitol. Using specific primes allele specific PCR was used.The samples were then analyzed by MALDI-TOF mass spectroscopy and Biotyper 3 software.Results: Enterococcus faecium and Enterococcus faecalis were detected in thirty and forty two samples, respectively whereas three samples showed both bacterial infections. Using biochemical analysis, two E. faecium isolates were Arabinose negative and one E. faecalis isolates was Telliurite negative. All samples were showed correct bands in PCR results but two of them didn't show clear bands(on agarose gel. In mass spectroscopy analysis all strains were correctly detected and well defined.Conclusion: According to our results, MALDI-TOF mass spectrometry in comparison with Molecular and Biochemical Methods could be a reliable and accurate method that can easily and quickly identify and differentiate Enterococcus faecium and Enterococcus faecalis in clinical samples.Key words: Enterococcus faecalis, Enterococcus faecium, MALDI-TOF mass spectrometry, PCR

  3. "Mixed inhibitor-prodrug" as a new approach toward systemically active inhibitors of enkephalin-degrading enzymes.

    Science.gov (United States)

    Fournié-Zaluski, M C; Coric, P; Turcaud, S; Lucas, E; Noble, F; Maldonado, R; Roques, B P

    1992-06-26

    In order to evaluate the possible advantages of potentiating the effects of the endogenous enkephalins, to obtain analgesia without the serious drawbacks of morphine, it was essential to design systemically active compounds which inhibit the two metabolizing enzymes, aminopeptidase N (APN) and neutral endopeptidase 24.11 (NEP). A new concept combining the idea of "prodrug" and "mixed inhibitor" was therefore developed. Given the high efficiency of beta-mercaptoalkylamines as APN inhibitors and of N-(mercaptoacyl) amino acids as NEP inhibitors, compounds associating these molecules through disulfide or thioester bonds, which are known to increase lipophilicity and to favor passage across the blood-brain barrier, have been synthesized. An HPLC study indicated that the disulfide bridge was resistant to serum enzymes but was cleaved by brain membrane homogenates, suggesting that the active inhibitors were released in the central nervous system. The validity of the approach was verified by the efficient antinociceptive responses obtained in the hot plate test in mice after iv administration of disulfide-containing inhibitors (ED50s of from 4 to 26 mg/kg on the jump latency time). The analgesic potencies of the "mixed inhibitor-prodrug" RB 101 [H2NCH(CH2CH2SCH3)CH2SSCH2CH(CH2Ph)CONHCH( CH2Ph)COOCH2Ph] after iv administration were three times greater than those of a similar combined dose of its two constitutive moieties. The separation of the two diastereoisomers constituting RB 101 showed that the analgesia has a stereochemical dependence, the (S,S,S)-isomer being more active than the (S,R,S)-isomer. Furthermore, in the tail flick test in the rat, RB 101 gave 38% analgesia at a dose of 80 mg/kg. Due to its high efficiency and its longer pharmacological effect, RB 101 was selected for a complete study of its analgesic properties.

  4. Chronic low vitamin intake potentiates cisplatin-induced intestinal epithelial cell apoptosis in WNIN rats

    Institute of Scientific and Technical Information of China (English)

    Bodiga Vijayalakshmi; Boindala Sesikeran; Putcha Udaykumar; Subramaniam Kalyanasundaram; Manchala Raghunath

    2006-01-01

    AIM: To investigate if cisplatin alters vitamin status and if VR modulates cisplatin induced intestinal apoptosis and oxidative stress in Wistar/NIN (WNIN) male rats.METHODS: Weanling, WNIN male rats (n = 12 per group) received adlibitum for 17 wk: control diet (20%protein) or the same with 50% vitamin restriction. They were then sub-divided into two groups of six rats each and administered cisplatin (2.61 mg/kg bodyweight)once a week for three wk or PBS (vehicle control).Intestinal epithelial cell (IEC) apoptosis was monitored by morphometry, Annexin-V binding, M30 cytodeath assay and DNA fragmentation. Structural and functional integrity of the villus were assessed by villus height /crypt depth ratio and activities of alkaline phosphatase,lys, ala-dipeptidyl amino-peptidase, respectively. To assess the probable mechanism(s) of altered apoptosis,oxidative stress parameters, caspase-3 activity, and expression of Bcl-2 and Bax were determined.RESULTS: Cisplatin per se decreased plasma vitamin levels and they were the lowest in VR animals treated with cisplatin. As expected VR increased only villus apoptosis, whereas cisplatin increased stem cell apoptosis in the crypt. However, cisplatin treatment of VR rats increased apoptosis both in villus and crypt regions and was associated with higher levels of TBARS,protein carbonyls and caspase-3 activity, but lower GSH concentrations. VR induced decrease in Bcl-2 expression was further lowered by cisplatin. Bax expression,unaffected by VR was increased on cisplatin treatment.Mucosal functional integrity was severely compromised in cisplatin treated VR-rats.CONCLUSION: Low intake of vitamins increases the sensitivity of rats to cisplatin and promotes intestinal epithelial cell apoptosis.

  5. Biochar affects soil organic matter cycling and microbial functions but does not alter microbial community structure in a paddy soil.

    Science.gov (United States)

    Tian, Jing; Wang, Jingyuan; Dippold, Michaela; Gao, Yang; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2016-06-15

    The application of biochar (BC) in conjunction with mineral fertilizers is one of the most promising management practices recommended to improve soil quality. However, the interactive mechanisms of BC and mineral fertilizer addition affecting microbial communities and functions associated with soil organic matter (SOM) cycling are poorly understood. We investigated the SOM in physical and chemical fractions, microbial community structure (using phospholipid fatty acid analysis, PLFA) and functions (by analyzing enzymes involved in C and N cycling and Biolog) in a 6-year field experiment with BC and NPK amendment. BC application increased total soil C and particulate organic C for 47.4-50.4% and 63.7-74.6%, respectively. The effects of BC on the microbial community and C-cycling enzymes were dependent on fertilization. Addition of BC alone did not change the microbial community compared with the control, but altered the microbial community structure in conjunction with NPK fertilization. SOM fractions accounted for 55% of the variance in the PLFA-related microbial community structure. The particulate organic N explained the largest variation in the microbial community structure. Microbial metabolic activity strongly increased after BC addition, particularly the utilization of amino acids and amines due to an increase in the activity of proteolytic (l-leucine aminopeptidase) enzymes. These results indicate that microorganisms start to mine N from the SOM to compensate for high C:N ratios after BC application, which consequently accelerate cycling of stable N. Concluding, BC in combination with NPK fertilizer application strongly affected microbial community composition and functions, which consequently influenced SOM cycling.

  6. Modulation of histamine release from human colon mast cells by protease inhibitors

    Institute of Scientific and Technical Information of China (English)

    Shao-Heng He; Hua Xie

    2004-01-01

    AIM: To investigate the ability of protease inhibitors to modulate histamine release from human colon mast cells.METHODS: Enzymatically dispersed cells from human colon were challenged with anti-IgE or calcium ionophore A23187 in the absence or presence of tryptase and chymase inhibitors, and histamine release was determined.RESULTS: IgE dependent histamine release from colon mast cells was inhibited by up to approximately 37%, 26% and 36.8% by chymase inhibitors Z-Ile-Glu-Pro-Phe-CO2Me (ZIGPFM), N-Tosyl-L-phenylalanyl-chloromethyl ketone (TPCK), and α1-antitrypsin, respectively. Similarly, inhibitors of tryptase leupeptin, N-tosyl-L-lysine chloromethyl ketone (TLCK), lactoferrin and protamine were also able to inhibit anti-IgE induced histamine release by a maximum of some 48%, 37%, 40% and 34%, respectively. Preincubation of these inhibitors with cells for 20 min before challenged with anti-IgE had small effect on the inhibitory actions of these inhibitors on colon mast cells. A specific inhibitor of aminopeptidase amastatin had no effect on anti-IgE induced histamine release. The significant inhibition of calcium ionophore induced histamine release was also observed with the inhibitors of tryptase and chymase examined. Apart from leupeptin and protamine, the inhibitors tested by themselves did not stimulate colon mast cells.CONCLUSION: It was demonstrated that both tryptase and chymase inhibitors could inhibit IgE dependent and calcium ionophore induced histamine release from dispersed colon mast cells in a concentration dependent of manner, which suggest that they are likely to be developed as a novel class of anti-inflammatory drugs to treat chronic of colitis in man.

  7. Proteomic analysis of Mecistocirrus digitatus and Haemonchus contortus intestinal protein extracts and subsequent efficacy testing in a vaccine trial.

    Directory of Open Access Journals (Sweden)

    Alison J Dicker

    2014-06-01

    Full Text Available BACKGROUND: Gastrointestinal nematode infections, such as Haemonchus contortus and Mecistocirrus digitatus, are ranked in the top twenty diseases affecting small-holder farmers' livestock, yet research into M. digitatus, which infects cattle and buffalo in Asia is limited. Intestine-derived native protein vaccines are effective against Haemonchus, yet the protective efficacy of intestine-derived M. digitatus proteins has yet to be determined. METHODOLOGY/PRINCIPAL FINDINGS: A simplified protein extraction protocol (A is described and compared to an established method (B for protein extraction from H. contortus. Proteomic analysis of the H. contortus and M. digitatus protein extracts identified putative vaccine antigens including aminopeptidases (H11, zinc metallopeptidases, glutamate dehydrogenase, and apical gut membrane polyproteins. A vaccine trial compared the ability of the M. digitatus extract and two different H. contortus extracts to protect sheep against H. contortus challenge. Both Haemonchus fractions (A and B were highly effective, reducing cumulative Faecal Egg Counts (FEC by 99.19% and 99.89% and total worm burdens by 87.28% and 93.64% respectively, compared to the unvaccinated controls. There was no effect on H. contortus worm burdens following vaccination with the M. digitatus extract and the 28.2% reduction in cumulative FEC was not statistically significant. However, FEC were consistently lower in the M. digitatus extract vaccinates compared to the un-vaccinated controls from 25 days post-infection. CONCLUSIONS/SIGNIFICANCE: Similar, antigenically cross-reactive proteins are found in H. contortus and M. digitatus; this is the first step towards developing a multivalent native vaccine against Haemonchus species and M. digitatus. The simplified protein extraction method could form the basis for a locally produced vaccine against H. contortus and, possibly M. digitatus, in regions where effective cold chains for vaccine

  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

    2012-08-01

    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 bacterioplankton communities responded to rising chlorophyll a concentrations after a lag phase of only a few days with increasing protein production and extracellular enzyme activity and revealed a close coupling of heterotrophic bacterial activity to phytoplankton productivity in this experiment. The natural extracellular enzyme assemblages showed increased activity in response to moderate acidification. A decrease in seawater pH of 0.5 units roughly doubled rates of β-glucosidase and leucine-aminopeptidase. Activities of extracellular enzymes in the mesocosms were directly related to both seawater pH and primary production. Also primary production and bacterial protein production in the mesocosms at different pCO2 were positively correlated. Therefore, it can be suggested that the efficient heterotrophic carbon utilization in this Arctic microbial food web had the potential to counteract increased phytoplankton production that was achieved under elevated pCO2 in this study. However, our results also show that the transfer of beneficial pCO2-related effects on the cellular bacterial metabolism to the scale of community activity and organic matter degradation can be mitigated by the top-down control of bacterial abundances in natural microbial communities.

  9. Intestinal morphology and enzymatic activity in newly weaned pigs fed contrasting fiber concentrations and fiber properties.

    Science.gov (United States)

    Hedemann, M S; Eskildsen, M; Laerke, H N; Pedersen, C; Lindberg, J E; Laurinen, P; Knudsen, K E Bach

    2006-06-01

    The main objective of this study was to determine the effect of fiber source and concentration on morphological characteristics, mucin staining pattern, and mucosal enzyme activities in the gastrointestinal tract of pigs. The experiment included 50 pigs from 10 litters weaned at 4 wk of age (BW 8.6 +/- 1.4 kg) and divided into 5 treatment groups. Diets containing fiber of various physico-chemical properties and concentrations were formulated to contain 73, 104, or 145 g of dietary fiber/kg of DM. The diets were based on raw wheat and barley flours. Pectin and barley hulls, representing soluble and insoluble fiber sources, respectively, were used to increase the fiber concentration. The pigs were fed the experimental diets for 9 d, and then the pigs were euthanized and the entire gastrointestinal tract was removed. Tissue samples were taken from the mid and distal small intestine and from the mid colon. Inclusion of pectin in the diets significantly decreased (P fiber content, whereas sucrase activity was increased in pigs fed the pectin-containing diets. The activity of the peptidases, aminopeptidase N and dipeptidylpeptidase IV, was increased when feeding high fiber diets, whereas the activity of gamma-glutamyl transpeptidase remained unaffected by the experimental diets. In conclusion, the reduced feed intake observed with the pectin-containing diets could explain the lower villous height and crypt depth observed in this study. However, direct effects of pectin also are possible, and thus further study is warranted. Feeding pigs high insoluble fiber diets improved gut morphology by increasing villi length and increased mucosal enzyme activity when compared with pigs fed pectin-containing diets. The mucin content as determined by staining characteristics suggests that pigs fed high insoluble fiber diets might be better protected against pathogenic bacteria than pigs fed diets high in soluble fiber.

  10. Inhibition of serine-peptidase activity enhances the generation of a survivin-derived HLA-A2-presented CTL epitope in colon-carcinoma cells.

    Science.gov (United States)

    Preta, G; Marescotti, D; Fortini, C; Carcoforo, P; Castelli, C; Masucci, M; Gavioli, R

    2008-12-01

    Cytotoxic T lymphocytes eliminate tumor cells expressing antigenic peptides in the context of MHC-I molecules. Peptides are generated during protein degradation by the proteasome and resulting products, surviving cytosolic amino-peptidases activity, may be presented by MHC-I molecules. The MHC-I processing pathway is altered in a large number of malignancies and modulation of antigen generation is one strategy employed by cells to evade immune control. In this study we analyzed the generation and presentation of a survivin-derived CTL epitope in HLA-A2-positive colon-carcinoma cells. Although all cell lines expressed the anti-apoptotic protein survivin, some tumors were poorly recognized by ELTLGEFLKL (ELT)-specific CTL cultures. The expression of MHC-I or TAP molecules was similar in all cell lines suggesting that tumors not recognized by CTLs may present defects in the generation of the ELT-epitope which could be due either to lack of generation or to subsequent degradation of the epitope. The cells were analyzed for the expression and the activity of extra-proteasomal peptidases. A significant overexpression and higher activity of TPPII was observed in colon-carcinoma cells which are not killed by ELT-specific CTLs, suggesting a possible role of TPPII in the degradation of the ELT-epitope. To confirm the role of TPPII in the degradation of the ELT-peptide, we showed that treatment of colon-carcinoma cells with a TPPII inhibitor resulted in a dose-dependent increased sensitivity to ELT-specific CTLs. These results suggest that TPPII is involved in degradation of the ELT-peptide, and its overexpression may contribute to the immune escape of colon-carcinoma cells.

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

    Directory of Open Access Journals (Sweden)

    Junfeng Fan

    2013-01-01

    Full Text Available 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 their inhibitory effects on DPP-IV by using a luminescence assay and computational modeling. ANC from blueberry-blackberry wine blends strongly inhibited DPP-IV activity (IC50, 0.07 ± 0.02 to >300 μM. Of the twenty-seven phenolics tested, the most potent DPP-IV inhibitors were resveratrol (IC50, 0.6 ± 0.4 nM, luteolin (0.12 ± 0.01 μM, apigenin (0.14 ± 0.02 μM, and flavone (0.17 ± 0.01 μM, with IC50 values lower than diprotin A (4.21 ± 2.01 μM, a reference standard inhibitory compound. Analyses of computational modeling showed that resveratrol and flavone were competitive inhibitors which could dock directly into all three active sites of DPP-IV, while luteolin and apigenin docked in a noncompetitive manner. Hydrogen bonding was the main binding mode of all tested phenolic compounds with DPP-IV. These results indicate that flavonoids, particularly luteolin, apigenin, and flavone, and the stilbenoid resveratrol can act as naturally occurring DPP-IV inhibitors.

  12. Sugarcane giant borer transcriptome analysis and identification of genes related to digestion.

    Directory of Open Access Journals (Sweden)

    Fernando Campos de Assis Fonseca

    Full Text Available Sugarcane is a widely cultivated plant that serves primarily as a source of sugar and ethanol. Its annual yield can be significantly reduced by the action of several insect pests including the sugarcane giant borer (Telchin licus licus, a lepidopteran that presents a long life cycle and which efforts to control it using pesticides have been inefficient. Although its economical relevance, only a few DNA sequences are available for this species in the GenBank. Pyrosequencing technology was used to investigate the transcriptome of several developmental stages of the insect. To maximize transcript diversity, a pool of total RNA was extracted from whole body insects and used to construct a normalized cDNA database. Sequencing produced over 650,000 reads, which were de novo assembled to generate a reference library of 23,824 contigs. After quality score and annotation, 43% of the contigs had at least one BLAST hit against the NCBI non-redundant database, and 40% showed similarities with the lepidopteran Bombyx mori. In a further analysis, we conducted a comparison with Manduca sexta midgut sequences to identify transcripts of genes involved in digestion. Of these transcripts, many presented an expansion or depletion in gene number, compared to B. mori genome. From the sugarcane giant borer (SGB transcriptome, a number of aminopeptidase N (APN cDNAs were characterized based on homology to those reported as Cry toxin receptors. This is the first report that provides a large-scale EST database for the species. Transcriptome analysis will certainly be useful to identify novel developmental genes, to better understand the insect's biology and to guide the development of new strategies for insect-pest control.

  13. Heme protein-induced tubular cytoresistance: expression at the plasma membrane level.

    Science.gov (United States)

    Zager, R A

    1995-05-01

    Following experimental rhabdomyolysis, animals become resistant to heme protein-induced acute renal failure (ARF). The goals of this study were to: (a) ascertain whether this resistance, previously documented only in vivo, is expressed directly at the proximal tubular cell level; (b) determine whether heme proteinuria (vs. other consequences of rhabdomyolysis) is its trigger; and (c) ascertain some of its subcellular determinants. Rats were injected with a borderline toxic dose of glycerol and 24 hours later proximal tubular segments (PTS) were isolated for study. Their vulnerability to diverse forms of injury (FeSO4-induced oxidant stress, hypoxia, Ca2+ ionophore, cytochalasin D, PLA2) was compared to that found in normal PTS. Post-glycerol PTS manifested significant resistance to each insult (decreased lactate dehydrogenase +/- N-acetyl-beta-D-glucosaminidase release). Protection against FeSO4 was virtually complete and it was associated with a 50% decrease in membrane lipid peroxidation. No decrease in hydroxyl radical generation was noted during the FeSO4 challenge (salicylate trap assessment), suggesting a primary increase in membrane resistance to attack. That PLA2 addition caused less deacylation, plasma membrane enzyme (alanine aminopeptidase) release, and LDH leakage from post-glycerol versus normal tubules supported this hypothesis. To test whether cytoresistance was specifically triggered by heme proteins (vs. being a non-specific filtered protein effect, or a result of endotoxin cascade activation), rats were injected with purified myoglobin, non-heme containing filterable proteins, or endotoxin. Only myoglobin induced cytoresistance. In vivo heme oxygenase inhibition (tin-protoporphyrin) did not block the emergence of cytoresistance and it was expressed despite Na,K-ATPase inhibition (ouabain) or cytoskeletal disruption (cytochalasin D). In vivo heat shock failed to protect. In conclusion, (1) rhabdomyolysis induces broad based proximal tubular

  14. Responses of soil enzyme activity and microbial community compositions to nitrogen addition in bulk and microaggregate soil in the temperate steppe of Inner Mongolia

    Science.gov (United States)

    Shi, Yao; Sheng, Lianxi; Wang, Zhongqiang; Zhang, Xinyu; He, Nianpeng; Yu, Qiang

    2016-10-01

    In order to explore the responses of soil enzyme activities and microbial community compositions to long-term nitrogen (N) addition in both bulk soil and microaggregate of chestnut soil, we conducted a 7-year urea addition experiment with N treatments at 6 levels (0, 56, 112, 224, 392 and 560 kg N ha-1 yr-1) in a temperate steppe of Inner Mongolia in China. Soil properties and the activities of four enzymes involved in carbon (C), nitrogen (N) and phosphorus (P) cycling were measured in both bulk soil and microaggregate, and phospholipid fatty acids (PLFAs) were measured in bulk soil. The results indicated that: 1) in bulk soil, N addition significantly decreased β-1,4-glucosidase (BG) and leucine aminopeptidase (LAP) activities at the treatment amounts of 224, 392 and 560 kg N ha-1 yr-1, and obviously suppressed β-1,4-N-acetylglucosaminidase (NAG) activity at the treatment amount of 560 kg N ha-1 yr-1. N addition enhanced total PLFAs (totPLFAs) and bacterial PLFAs (bacPLFAs) at the treatment amounts of 392 and 560 kg N ha-1 yr-1, respectively, but fungal PLFAs showed no response to N addition. The activities of BG, NAG and LAP were positively correlated with soil pH, but negatively correlated with the concentration of NH 4 + -N; 2) in microaggregate (53-250 μm), the activities of BG, NAG and AP showed no response to increased addition of N, but the significantly decreased LAP activity was observed at the treatment amount of 392 kg N ha-1 yr-1. These results suggested that enzyme activities were more sensitive to N addition than PLFA biomarkers in soil, and LAP activity in microaggregate may be a good indicator for evaluating N cycle response to long-term N addition.

  15. Loss of chloride channel ClC-5 impairs endocytosis by defective trafficking of megalin and cubilin in kidney proximal tubules.

    Science.gov (United States)

    Christensen, Erik I; Devuyst, Olivier; Dom, Geneviève; Nielsen, Rikke; Van der Smissen, Patrick; Verroust, Pierre; Leruth, Michèle; Guggino, William B; Courtoy, Pierre J

    2003-07-08

    Loss of the renal endosome-associated chloride channel, ClC-5, in Dent's disease and knockout (KO) mice strongly inhibits endocytosis of filtered proteins by kidney proximal tubular cells (PTC). The underlying mechanism remains unknown. We therefore tested whether this endocytic failure could primarily reflect a loss of reabsorption by the multiligand receptors, megalin, and cubilin, caused by a trafficking defect. Impaired protein endocytosis in PTC of ClC-5 KO mice was demonstrated by (i) a major decreased uptake of injected 125I-beta 2-microglobulin, but not of the fluid-phase tracer, FITC-dextran, (ii) reduced labeling of endosomes by injected peroxidase and for the endogenous megalin/cubilin ligands, vitamin D- and retinol-binding proteins, and (iii) urinary appearance of low-molecular-weight proteins and the selective cubilin ligand, transferrin. Contrasting with preserved mRNA levels, megalin and cubilin abundance was significantly decreased in kidney extracts of KO mice. Percoll gradients resolving early and late endosomes (Rab5a, Rab7), brush border (villin, aminopeptidase M), and a dense peak comprising lysosomes (acid hydrolases) showed a disappearance of the brush border component for megalin and cubilin in KO mice. Quantitative ultrastructural immunogold labeling confirmed the overall decrease of megalin and cubilin in PTC and their selective loss at the brush border. In contrast, total contents of the rate-limiting endocytic catalysts, Rab5a and Rab7, were unaffected. Thus, impaired protein endocytosis caused by invalidation of ClC-5 primarily reflects a trafficking defect of megalin and cubilin in PTC.

  16. Detection of renal tissue and urinary tract proteins in the human urine after space flight.

    Directory of Open Access Journals (Sweden)

    Lyudmila Kh Pastushkova

    Full Text Available The urine protein composition samples of ten Russian cosmonauts (male, aged of 35 up to 51 performed long flight missions and varied from 169 up to 199 days on the International Space Station (ISS were analyzed. As a control group, urine samples of six back-up cosmonauts were analyzed. We used proteomic techniques to obtain data and contemporary bioinformatics approaches to perform the analysis. From the total number of identified proteins (238 in our data set, 129 were associated with a known tissue origin. Preflight samples contained 92 tissue-specific proteins, samples obtained on Day 1 after landing had 90 such proteins, while Day 7 samples offered 95 tissue-specific proteins. Analysis showed that consistently present proteins in urine (under physiological conditions and after space flight are cubilin, epidermal growth factor, kallikrein-1, kininogen-1, megalin, osteopontin, vitamin K-dependent protein Z, uromodulin. Variably present proteins consists of: Na(+/K(+ ATPase subunit gamma, β-defensin-1, dipeptidyl peptidase 4, maltasa-glucoamilasa, cadherin-like protein, neutral endopeptidase and vascular cell adhesion protein 1. And only three renal proteins were related to the space flight factors. They were not found in the pre-flight samples and in the back-up cosmonaut urine, but were found in the urine samples after space flight: AFAM (afamin, AMPE (aminopeptidase A and AQP2 (aquaporin-2. This data related with physiological readaptation of water-salt balance. The proteomic analysis of urine samples in different phases of space missions with bioinformation approach to protein identification provides new data relative to biomechemical mechanism of kidney functioning after space flight.

  17. Effects of warming on stream biofilm organic matter use capabilities.

    Science.gov (United States)

    Ylla, Irene; Canhoto, Cristina; Romaní, Anna M

    2014-07-01

    The understanding of ecosystem responses to changing environmental conditions is becoming increasingly relevant in the context of global warming. Microbial biofilm communities in streams play a key role in organic matter cycling which might be modulated by shifts in flowing water temperature. In this study, we performed an experiment at the Candal stream (Portugal) longitudinally divided into two reaches: a control half and an experimental half where water temperature was 3 °C above that of the basal stream water. Biofilm colonization was monitored during 42 days in the two stream halves. Changes in biofilm function (extracellular enzyme activities and carbon substrate utilization profiles) as well as chlorophyll a and prokaryote densities were analyzed. The biofilm in the experimental half showed a higher capacity to decompose cellulose, hemicellulose, lignin, and peptidic compounds. Total leucine-aminopeptidase, cellobiohydrolase and β-xylosidase showed a respective 93, 66, and 61% increase in activity over the control; much higher than would be predicted by only the direct temperature physical effect. In contrast, phosphatase and lipase activity showed the lowest sensitivity to temperature. The biofilms from the experimental half also showed a distinct functional fingerprint and higher carbon usage diversity and richness, especially due to a wider use of polymers and carbohydrates. The changes in the biofilm functional capabilities might be indirectly affected by the higher prokaryote and chlorophyll density measured in the biofilm of the experimental half. The present study provides evidence that a realistic stream temperature increase by 3 °C changes the biofilm metabolism to a greater decomposition of polymeric complex compounds and peptides but lower decomposition of lipids. This might affect stream organic matter cycling and the transfer of carbon to higher trophic levels.

  18. Degradation capability of the coastal environment adjacent to the Itata River in central Chile (36.5° S

    Directory of Open Access Journals (Sweden)

    P. Ampuero

    2011-08-01

    Full Text Available The response of the coastal ocean influenced by both river discharges and inputs of photosynthetically derived organic carbon product of upwelling, was evaluated by estimating rates of microbial hydrolysis of macromolecules with the goal of estimating the potential degradation capability of the coastal ecosystem off central Chile. Extracellular enzymatic activity (EEA in seawater was dominated by aminopeptidase activity on substrate L-leucine-4-methyl-7-coumarinylamide (MCA-leu (1.2 to 182 nmol l−1 h−1 followed by 4-methylumbelliferyl-ß-D-glucoside (MUF-glu (0.08–61 nmol l−1 h−1 and 4-methylumbelliferyl-ß-D-cellobiose (MUF-cel (0.15–7 nmol l−1 h−1, with the highest rates measured during spring-summer. In riverine waters, extracellular enzymatic hydrolysis remained within the range of 45 to 131 nmol l−1 h−1 for MCA-leu and ca. 20 nmol l−1 h−1 for glucosidic substrates, year-round. Contrary to the EEA observed for the marine water column, surface sediment extracellular enzymatic hydrolysis of MCA-leu (0.04 to 6.13 nmol g−1 dw h−1 was in the same order of magnitude as the rates observed for MUF-cel (0.004 to 5.1 nmol g−1 dw h−1 and MUF-glu (0.007 to 10.5 nmol g−1 dw h−1. Moreover, hydrolysis in sediments was characterized by higher rates during winter compared with spring-summer in the coastal and estuarine zone. The five years of data allowed us to evaluate the potential capability of microbial processing of organic carbon in the coastal area adjacent to the Itata river discharge where the increase in primary production in the productive seasons is accompanied by the increase in hydrolysis of macromolecules.

  19. [Effects of applying different kind fertilizers on enzyme activities related to carbon, nitrogen, and phosphorus cycles in reddish paddy soil].

    Science.gov (United States)

    Xu, Li-Li; Wang, Qiu-Bing; Zhang, Xin-Yu; Sun, Xiao-Min; Dai, Xiao-Qin; Yang, Feng-Ting; Bu, Jin-Feng; Wang, Hui-min

    2013-04-01

    Based on the long-term fixed position experimental data from Qianyanzhou Ecological Experiment Station, Chinese Academy of Sciences in 1998, this paper analyzed the effects of applying different kind fertilizers (straw, ST; pig manure, OM; and chemical fertilizer, NPK) on the nutrients (C, N, and P) status and the activities of related enzymes ( beta-1,4-glucosidase, betaG; beta-1,4-N-acetylglucosaminidase, NAG; L-leucine aminopeptidase, LAP; and acid phosphatase, AP) in reddish paddy soil. With the application of OM, the activities of soil betaG, NAG, and LAP increased significantly, as compared with other treatments, and were 1.4, 2. 6, and 1.9 times higher than the control (CK) , respectively. Applying OM also improved the ratio of soil organic carbon to total nitrogen (C/N), but decreased the soil betaG/(NAG+LAP) ratio, suggesting that pig manure could benefit the degradation of soil cellulose and the accumulation of soil organic carbon. Applying NPK increased the activities of soil betaG, NAG, and LAP, but decreased the AP activity, with a decrement of 34% as compared with CK. Under the application of NPK, the soilbetaG/AP and (NAG+ LAP)/AP ratios increased, but the ratios of soil organic carbon to total phosphorus (C/P) and of soil total nitrogen to total phosphorus (N/P) decreased, indicating that chemical fertilizers could induce the accumulation of soil inorganic phosphorus, and inhibit the microbial functions of degrading polysaccharides and phosphate phospholipids.

  20. Carbohydrate maldigestion induces necrotizing enterocolitis in preterm pigs.

    Science.gov (United States)

    Thymann, Thomas; Møller, Hanne K; Stoll, Barbara; Støy, Ann Cathrine F; Buddington, Randal K; Bering, Stine B; Jensen, Bent B; Olutoye, Oluyinka O; Siggers, Richard H; Mølbak, Lars; Sangild, Per T; Burrin, Douglas G

    2009-12-01

    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 that a formula containing maltodextrin vs. a formula containing lactose as the principal source of carbohydrate would predispose preterm pigs to a higher NEC incidence. Cesarean-derived preterm pigs were given total parenteral nutrition for 48 h followed by total enteral nutrition with a lactose-based (n = 11) or maltodextrin-based (n = 11) formula for 36 h. A higher incidence (91% vs. 27%) and severity (score of 3.3 vs. 1.8) of NEC were observed in the maltodextrin than in the lactose group. This higher incidence of NEC in the maltodextrin group was associated with significantly lower activities of lactase, maltase, 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 acids were observed in the maltodextrin group. In a second study, we quantified net portal absorption of aldohexoses (glucose and galactose) during acute jejunal infusion of a maltodextrin- or a lactose-based formula (n = 8) into preterm pigs. We found lower net portal aldohexose absorption (4% vs. 42%) and greater intestinal recovery of undigested carbohydrate (68% vs. 27%) in pigs acutely perfused with the maltodextrin-based formula than those perfused with the lactose-based formula. The higher digestibility of the lactose than the maltodextrin in the formulas can be attributed to a 5- to 20-fold higher hydrolytic activity of tissue-specific lactase than maltases. We conclude that carbohydrate maldigestion is sufficient to increase the incidence and severity of NEC in preterm pigs.

  1. New drug therapies interfering with the renin-angiotensin-aldosterone system for resistant hypertension.

    Science.gov (United States)

    Monge, Matthieu; Lorthioir, Aurélien; Bobrie, Guillaume; Azizi, Michel

    2013-12-01

    There is a persistent need for the development of new antihypertensive drugs, because the control of blood pressure is still not achievable in a significant proportion of hypertensive patients. Since the approval in 2007 of aliskiren, no other new antihypertensive based on new mechanism(s) of action have been approved. In fact, the development of promising novel drugs has been stopped for safety, efficacy or marketing reasons. Despite these difficulties, the pipeline is not dry and different new antihypertensive strategies targeting the renin-angiotensin-aldosterone pathway, are in clinical development stage. The dual angiotensin II receptor-neprilysin inhibitor LCZ696, a single molecule synthetized by cocrystallisation of valsartan and the neprilysin inhibitor prodrug AHU377 is in development for resistant hypertension and for heart failure. Daglutril is a dual neprylisin-endothelin converting enzyme inhibitor which was shown to decrease BP in patients with type 2 diabetic nephropathy. Aldosterone synthase inhibitors and the third and fourth generation non-steroidal dihydropyridine based mineralocorticoid receptors blockers are new ways to target the multiple noxious effects of aldosterone in the kidney, vessels and heart. Centrally acting aminopeptidase A inhibitors block brain angiotensin III formation, one of the main effector peptides of the brain renin angiotensin system. However, a long time will be still necessary to evaluate extensively the efficacy and safety of these new approaches. In the mean time, using appropriate and personalized daily doses of available drugs, decreasing physician inertia, improving treatment adherence, improving access to healthcare and reducing treatment costs remain major objectives to reduce the incidence of resistant hypertension.

  2. Identification of Neisseria spp., Haemophilus spp., and other fastidious gram-negative bacteria with the MicroScan Haemophilus-Neisseria identification panel.

    Science.gov (United States)

    Janda, W M; Bradna, J J; Ruther, P

    1989-01-01

    The Haemophilus-Neisseria identification (HNID) panel (American MicroScan, Sacramento, Calif.) is a 4-h microdilution format system for identification of Haemophilus and Neisseria spp., Branhamella (Moraxella) catarrhalis, and Gardnerella vaginalis. The HNID panel was evaluated by using 423 clinical isolates and stock strains of these organisms, and HNID identifications were compared with those obtained by conventional methods. In addition, 32 isolates representing six genera not included in the HNID data base were tested to determine whether these organisms would produce unique biotype numbers for possible inclusion in the data base. The HNID panel correctly identified 95.3% of 86 Neisseria gonorrhoeae strains, 96% of 25 G. vaginalis strains, and 100% of 28 Neisseria lactamica strains and 48 B. catarrhalis strains. Only 64.7% of 68 Neisseria meningitidis isolates were identified correctly owing to false-negative or equivocal carbohydrate and/or aminopeptidase reactions. Among the Haemophilus spp., 98.8% of 83 H. influenzae strains, 97.1% of 34 H. parainfluenzae strains, and 80% of 15 H. aphrophilus and H. paraphrophilus strains were correctly identified. Eight strains of Neisseria cinerea, a species not included in the data base, produced profiles identical with those for B. catarrhalis and N. gonorrhoeae. Isolates of other species not included in the data base, including Eikenella corrodens, Kingella spp., and Cardiobacterium hominis, produced unique biochemical reaction patterns on the panel. Modification of interpretative criteria for certain tests, expansion of the data base to include other species, and suggestions for additional confirmatory tests will increase the accuracy and utility of the HNID panel. PMID:2501351

  3. Toxicity of Cry1A toxins from Bacillus thuringiensis to CF1 cells does not involve activation of adenylate cyclase/PKA signaling pathway.

    Science.gov (United States)

    Portugal, Leivi; Muñóz-Garay, Carlos; Martínez de Castro, Diana L; Soberón, Mario; Bravo, Alejandra

    2017-01-01

    Bacillus thuringiensis (Bt) bacteria produce Cry toxins that are able to kill insect pests. Different models explaining the mode of action of these toxins have been proposed. The pore formation model proposes that the toxin creates pores in the membrane of the larval midgut cells after interaction with different receptors such as cadherin, aminopeptidase N and alkaline phosphatase and that this pore formation activity is responsible for the toxicity of these proteins. The alternative model proposes that interaction with cadherin receptor triggers an intracellular cascade response involving protein G, adenylate cyclase (AC) and protein kinase A (PKA). In addition, it was shown that Cry toxins induce a defense response in the larvae involving the activation of mitogen-activated kinases such as MAPK p38 in different insect orders. Here we analyzed the mechanism of action of Cry1Ab and Cry1Ac toxins and a collection of mutants from these toxins in the insect cell line CF1 from Choristoneura fumiferana, that is naturally sensitive to these toxins. Our results show that both toxins induced permeability of K(+) ions into the cells. The initial response after intoxication with Cry1Ab and Cry1Ac toxins involves the activation of a defense response that involves the phosphorylation of MAPK p38. Analysis of activation of PKA and AC activities indicated that the signal transduction involving PKA, AC and cAMP was not activated during Cry1Ab or Cry1Ac intoxication. In contrast we show that Cry1Ab and Cry1Ac activate apoptosis. These data indicate that Cry toxins can induce an apoptotic death response not related with AC/PKA activation. Since Cry1Ab and Cry1Ac toxins affected K(+) ion permeability into the cells, and that mutant toxins affected in pore formation are not toxic to CF1, we propose that pore formation activity of the toxins is responsible of triggering cell death response in CF1cells.

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

    Directory of Open Access Journals (Sweden)

    George Janssen

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

  5. Midgut proteins released by microapocrine secretion in Spodoptera frugiperda.

    Science.gov (United States)

    Silva, Walciane; Cardoso, Christiane; Ribeiro, Alberto F; Terra, Walter R; Ferreira, Clélia

    2013-01-01

    Microapocrine vesicles bud from the lepidopteran midgut microvilli as double membrane vesicles. To identify the proteins secreted by this process, antibodies raised against isolated microapocrine vesicles from Spodoptera frugiperda were used for screening a midgut cDNA expression library. Positive clones were sequenced, assembled and N blasted against S. frugiperda sequences obtained by pyrosequencing midgut mRNA. This procedure led to the extension of microapocrine sequences that were annotated. A similar procedure was used to identify midgut microvillar proteins that necessarily are part of the microapocrine vesicle. Forty-eight proteins were associated with microvillar membranes. They pertain to 8 functional groups: digestive enzymes, peritrophic membrane, protection, transporters, receptors, secretory machinery, cytoskeleton and signaling, and unknown. Twenty-eight proteins are putatively secreted by microapocrine secretion. Most of them are digestive enzymes, but the list also includes proteins involved in protection and in peritrophic membrane formation. Among the identified digestive enzymes, aminopeptidases are typically microvillar and group into the classes 1, 2, 3, 5, and 6. There are two amylases secreted by microapocrine secretion: one is a digestive enzyme and the other is a transporter-like amylase with no clear function. One lipase has a predicted transmembrane loop, whereas the others are supposed to be secreted by microapocrine secretion and be digestive. Trypsin is membrane bound and is delivered by microapocrine secretion, but has no predicted features to bind membranes. It may remain bound through the signal peptide till be delivered into the midgut lumen. Proteins supposed to be involved in the microapocrine secretory machinery were: calmodulin, annexin, myosin 7a, and gelsolin 1. Their putative roles are discussed, but more research is necessary to settle this subject.

  6. Evidence of multiple/cross resistance to Bt and organophosphate insecticides in Puerto Rico population of the fall armyworm, Spodoptera frugiperda.

    Science.gov (United States)

    Zhu, Yu Cheng; Blanco, Carlos A; Portilla, Maribel; Adamczyk, John; Luttrell, Randall; Huang, Fangneng

    2015-07-01

    Fall armyworm (FAW) is a damaging pest of many economic crops. Long-term use of chemical control prompted resistance development to many insecticide classes. Many populations were found to be significantly less susceptible to major Bt toxins expressed in transgenic crops. In this study, a FAW strain collected from Puerto Rico (PR) with 7717-fold Cry1F-resistance was examined to determine if it had also developed multiple/cross resistance to non-Bt insecticides. Dose response assays showed that the PR strain developed 19-fold resistance to acephate. Besides having a slightly smaller larval body weight and length, PR also evolved a deep (2.8%) molecular divergence in mitochondrial oxidase subunit II. Further examination of enzyme activities in the midgut of PR larvae exhibited substantial decreases of alkaline phosphatase (ALP), aminopeptidase (APN), 1-NA- and 2-NA-specific esterase, trypsin, and chymotrypsin activities, and significant increases of PNPA-specific esterase and glutathione S-transferase (GST) activities. When enzyme preparations from the whole larval body were examined, all three esterase, GST, trypsin, and chymotrypsin activities were significantly elevated in the PR strain, while ALP and APN activities were not significantly different from those of susceptible strain. Data indicated that multiple/cross resistances may have developed in the PR strain to both Bt toxins and conventional insecticides. Consistently reduced ALP provided evidence to support an ALP-mediated Bt resistance mechanism. Esterases and GSTs may be associated with acephate resistance through elevated metabolic detoxification. Further studies are needed to clarify whether and how esterases, GSTs, and other enzymes (such as P450s) are involved in cross resistance development to Bt and other insecticide classes.

  7. Boar seminal plasma exosomes: effect on sperm function and protein identification by sequencing.

    Science.gov (United States)

    Piehl, Lidia L; Fischman, M Laura; Hellman, Ulf; Cisale, Humberto; Miranda, Patricia V

    2013-04-15

    Mammalian seminal plasma contains membranous vesicles (exosomes), with a high content of cholesterol and sphingomyelin and a complex protein composition. Their physiological role is uncertain because sperm stabilization and activation effects have been reported. To analyze a putative modulatory role for semen exosomes on sperm activity in the boar, the effects of these vesicles on several sperm functional parameters were examined. Additionally, boar exosome proteins were sequenced and their incorporation into sperm was explored. Boar sperm were incubated under conditions that induce capacitation, manifested as increased tyrosine phosphorylation, cholesterol loss and greater fluidity in apical membranes, and the ability to undergo the lysophosphatidylcholine-induced acrosome reaction. After establishing this cluster of capacitation-dependent functional parameters, the effect produced by exosomes when present during or after sperm capacitation was analyzed. Exosomes inhibited the capacitation-dependent cholesterol efflux and fluidity increase in apical membranes, and the disappearance of a 14-kD phosphorylated polypeptide. In contrast, the acrosome reaction (spontaneous and lysophosphatidylcholine-induced) was not affected, and sperm binding to the oocyte zona pellucida was reduced only when vesicles were present during gamete coincubation. Liposomes with a lipid composition similar to that present in exosomes mimicked these effects, except the one on zona pellucida binding. Interaction between exosomes and sperm was confirmed by transfer of aminopeptidase activity. In addition, the major exosome protein, identified as actin, appeared to associate with sperm after coincubation. Exosome composition had a predominance for structural proteins (actin, plastin, ezrin, and condensin), enzymes, and several porcine seminal plasma-specific polypeptides (e.g., spermadhesins). Transfer of proteins from exosome to sperm and their ability to block cholesterol efflux supports a

  8. [A21-Asparaginimide] insulin. Saponification of insulin hexamethyl ester, I.

    Science.gov (United States)

    Gattner, H G; Schmitt, E W

    1977-01-01

    [Asn A21]Insulin is formed as the main product during alkaline saponification of insulin hexamethyl ester. Purification was achieved by gel chromatography followed by ion-exchange chromatography on carboxymethyl cellulose at pH 4 or by preparative isoelectric focusing in a granulated gel over a narrow pH range. Two main products could be isolated. One of them showed the electrophoretic behaviour of insulin (A), whilst the other corresponded to insulin with a blocked carboxyl function (B). Incubation of this product B with carboxypeptidase A liberated only the C-terminal alanine of the B-chain, but not the asparagine of the C-terminus of the A-chain. Chymotryptic digestion of the isolated S-sulfonate A-chain derivative (C) followed by high-voltage electrophoresis confirmed that the carboxyl function of asparagine A21 was blocked. In order to determine the free carboxyl functions of the A-chain derivative C, it was coupled with glycine methyl ester yielding D. Amino acid analysis of the chymotryptic peptides of D showed that the carboxyl functions of glutamic acid A4 and A17 had been free prior to coupling. The amino acid analysis of the enzymatic hydrolysate (subtilisin, aminopeptidase M) of the A-chain derivative C showed an additional peak with an elution position identical to the model compound aminosuccinimide. The biological activity of the [Asm A21[insulin was found to be about 40% in the fat cell test and 13.2 units/mg measured by the mouse convulsion method.

  9. Characterization of certain bacterial strains for potential use as starter or probiotic cultures in dairy products.

    Science.gov (United States)

    Monteagudo-Mera, A; Caro, I; Rodríguez-Aparicio, L B; Rúa, J; Ferrero, M A; García-Armesto, M R

    2011-08-01

    The present work was aimed at characterizing 12 strains of lactic acid bacteria (LAB) to obtain improved potential starter or probiotic cultures that could be used for making dairy products from ewe's milk and cow's milk. Eight strains with antimicrobial properties, isolated from ewe's milk and from cheese made from ewe's and/or cow's milk, were studied. They were identified as Enterococcus faecalis (five strains), Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides, and Lactobacillus paracasei subsp. paracasei (one strain of each species). Additionally, four strains were obtained from the American Type Culture Collection: Lactobacillus casei 393 (isolated from cheese), L. lactis subsp. lactis 11454 (origin nonspecified and a producer of nisin), and two strains isolated from human feces (L. paracasei subsp. paracasei 27092 and Lactobacillus rhamnosus 53103, antibacterial agent producer). All E. faecalis strains showed at least one virulence factor (either hemolysin or gelatinase), which emphasizes the importance of these studies in this species. Both L. lactis strains and most Lactobacillus spp. were good acidifiers in ewe's milk and cow's milk at 30°C. High β-galactosidase activity, as well as aminopeptidase activities that favor the development of desirable flavors in cheese, were detected in all Lactobacillus spp. strains. Furthermore, L. rhamnosus ATCC 53103 showed α-fucosidase activity (thought to help colonization of the intestine) and lack of α-glucosidase activity (a trait considered positive for diabetic and obese humans). This last enzymatic activity was also lacking in L. lactis ATCC 11454. L. mesenteroides was the only strain D(2)-lactic acid producer. The selection of any particular strain for probiotic or dairy cultures should be performed according to the technological and/or functional abilities needed.

  10. Characterization of new class III lantibiotics--erythreapeptin, avermipeptin and griseopeptin from Saccharopolyspora erythraea, Streptomyces avermitilis and Streptomyces griseus demonstrates stepwise N-terminal leader processing.

    Science.gov (United States)

    Völler, Ginka H; Krawczyk, Joanna M; Pesic, Alexander; Krawczyk, Bartlomiej; Nachtigall, Jonny; Süssmuth, Roderich D

    2012-05-29

    Lantibiotics are a large group of ribosomally synthesized peptides post-translationally modified to incorporate the amino acid lanthionine. They are classified, according to their biosynthetic pathway and bioactivity, into three major subtypes. Of Actinomycetes type III lantibiotics, only four peptides (SapB, SapT, LabA1, and LabA2) have been described and structurally characterized, although homologous gene clusters are abundant in other Actinomycetes. All these gene clusters share a similar architecture with a characteristic Ser/Ser/Cys motif in precursor peptides, which has previously been suggested to act as a precursor for lanthionine (SapB) and labionin (LabA2) rings. Mass spectrometry screening led to the discovery and characterization of three new representatives of type III lantibiotics: Avermipeptin (Avi), Erythreapeptin (Ery), and Griseopeptin (Gri) from Streptomyces avermitilis DSM 46492, Saccharopolyspora erythraea NRRL 2338, and Streptomyces griseus DSM 40236, respectively. Apart from the assignment of these peptides to their corresponding gene clusters, additional investigations on Avi, Ery and Gri peptides indicate stepwise leader processing by putative aminopeptidase-like protease(s), thus yielding mixtures of differently N-terminal-processed lantibiotic peptides. Similar peptide processing was observed for a heterologously expressed eryth biosynthetic gene cluster expressed in a Streptomyces host system. Remarkably, all isolates of the new type III lantibiotics contain both the amino acids lanthionine and labionin, thus implying dual-mode cyclase activity of the processing lyase-kinase-cyclase enzymes. These findings have implications for the structures and maturation of other type III lantibiotics from Actinomycetes.

  11. Podridão peduncular em manga: patogenicidade, agressividade e caracterização de isolados pela análise isoenzimática

    Directory of Open Access Journals (Sweden)

    Severina Rodrigues de Oliveira Lins

    2013-12-01

    Full Text Available Trinta e quatro isolados de Lasiodiplodia theobromae coletados de diferentes órgãos de mangueira, em duas épocas sazonais diferentes, em distintas regiões geográficas, foram avaliados quanto à patogenicidade, agressividade e produção de enzimas em substratos sólidos específicos e em sistemas de géis de poliacrilamida. Independente do órgão do qual foram isolados, todos foram patogênicos quando inoculados em manga, diferindo no grau de agressividade, sendo separados em três grupos: altamente, medianamente e fracamente agressivos. As atividades amilolítica, celulolítica, lipolítica e proteolítica foram estimadas por meio da difusão enzimática em meio sólido específico e mensuração do halo de degradação do substrato. As atividades das enzimas á-esterase, â-esterase, leucina-aminopeptidase, fosfatase ácida e proteínas totais foram analisadas em géis de poliacrilamida com meios de revelação específicos para cada análise. Os padrões eletroforéticos bem como as análises das enzimas extracelulares apresentaram polimorfismo, demonstrando a diversidade na base genética dos isolados, independente da época de coleta, região geográfica e órgão de isolamento.

  12. Inhibition of tryptase release from human colon mast cells by protease inhibitors

    Institute of Scientific and Technical Information of China (English)

    Shao-Heng He; Hua Xie

    2004-01-01

    AIM: To investigate the ability of protease inhibitors to modulate tryptase release from human colon mast cells.METHODS: Enzymatically dispersed cells from human colon were challenged with anti-IgE or calcium ionophore A23187 in the absence or presence of tryptase and chymase inhibitors,and tryptase release was determined.RESULTS: IgE dependent tryptase release from colon mast cells was inhibited by up to approximately 37%, 40% and 36.6% by chymase inhibitors Z-Ile-Glu-Pro-Phe-CO2Me (ZIGPFM), N-tosyl-L-phenylalanyl-chloromethyl ketone (TPCK), and α1-antitrypsin, respectively. Similarly, the inhibitors of tryptase leupeptin, N-tosyl-L-lysine chloromethyl ketone (TLCK) and lactoferrin were also able to inhibit anti-IgE induced tryptase release by a maximum of 39.4%,47.6% and 36.6%, respectively. The inhibitory actions of chymase inhibitors, but not tryptase inhibitors on colon mast cells were enhanced by preincubation of them with cells for 20 min before challenged with anti-IgE. At a concentration of 10 μg/mL, protamine was able to inhibit anti-IgE and calcium ionophore induced tryptase release. However, at 100 μg/mL, protamine elevated tryptase levels in supernatants.A specific inhibitor of aminopeptidase amastatin had no effect on anti-IgE induced tryptase release. The significant inhibition of calcium ionophore induced tryptase release was also observed with the inhibitors of tryptase and chymase examined. The inhibitors tested by themselves did not stimulate tryptase release from colon mast cells.CONCLUSION: It was demonstrated for the first time that both tryptase and chymase inhibitors could inhibit IgE dependent and calcium ionophore induced tryptase release from dispersed colon mast cells in a concentration dependent of manner, which suggest that they are likely to be developed as a novel class of anti-inflammatory drugs to treat chronic of colitis in man.

  13. Chemical and organic fertilizers affect physiological performance and antioxidant

    Directory of Open Access Journals (Sweden)

    M Mardani-Talaee1

    2016-04-01

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

  14. A genome-wide survey for host response of silkworm, Bombyx mori during pathogen Bacillus bombyseptieus infection.

    Directory of Open Access Journals (Sweden)

    Lulin Huang

    Full Text Available Host-pathogen interactions are complex relationships, and a central challenge is to reveal the interactions between pathogens and their hosts. Bacillus bombysepticus (Bb which can produces spores and parasporal crystals was firstly separated from the corpses of the infected silkworms (Bombyx mori. Bb naturally infects the silkworm can cause an acute fuliginosa septicaemia and kill the silkworm larvae generally within one day in the hot and humid season. Bb pathogen of the silkworm can be used for investigating the host responses after the infection. Gene expression profiling during four time-points of silkworm whole larvae after Bb infection was performed to gain insight into the mechanism of Bb-associated host whole body effect. Genome-wide survey of the host genes demonstrated many genes and pathways modulated after the infection. GO analysis of the induced genes indicated that their functions could be divided into 14 categories. KEGG pathway analysis identified that six types of basal metabolic pathway were regulated, including genetic information processing and transcription, carbohydrate metabolism, amino acid and nitrogen metabolism, nucleotide metabolism, metabolism of cofactors and vitamins, and xenobiotic biodegradation and metabolism. Similar to Bacillus thuringiensis (Bt, Bb can also induce a silkworm poisoning-related response. In this process, genes encoding midgut peritrophic membrane proteins, aminopeptidase N receptors and sodium/calcium exchange protein showed modulation. For the first time, we found that Bb induced a lot of genes involved in juvenile hormone synthesis and metabolism pathway upregulated. Bb also triggered the host immune responses, including cellular immune response and serine protease cascade melanization response. Real time PCR analysis showed that Bb can induce the silkworm systemic immune response, mainly by the Toll pathway. Anti-microorganism peptides (AMPs, including of Attacin, Lebocin, Enbocin, Gloverin

  15. Tomato pathogenesis-related protein genes are expressed in response to Trialeurodes vaporariorum and Bemisia tabaci biotype B feeding.

    Science.gov (United States)

    Puthoff, David P; Holzer, Frances M; Perring, Thomas M; Walling, Linda L

    2010-11-01

    The temporal and spatial expression of tomato wound- and defense-response genes to Bemisia tabaci biotype B (the silverleaf whitefly) and Trialeurodes vaporariorum (the greenhouse whitefly) feeding were characterized. Both species of whiteflies evoked similar changes in tomato gene expression. The levels of RNAs for the methyl jasmonic acid (MeJA)- or ethylene-regulated genes that encode the basic β-1,3-glucanase (GluB), basic chitinase (Chi9), and Pathogenesis-related protein-1 (PR-1) were monitored. GluB and Chi9 RNAs were abundant in infested leaves from the time nymphs initiated feeding (day 5). In addition, GluB RNAs accumulated in apical non-infested leaves. PR-1 RNAs also accumulated after whitefly feeding. In contrast, the ethylene- and salicylic acid (SA)-regulated Chi3 and PR-4 genes had RNAs that accumulated at low levels and GluAC RNAs that were undetectable in whitefly-infested tomato leaves. The changes in Phenylalanine ammonia lyase5 (PAL5) were variable; in some, but not all infestations, PAL5 RNAs increased in response to whitefly feeding. PAL5 RNA levels increased in response to MeJA, ethylene, and abscisic acid, and declined in response to SA. Transcripts from the wound-response genes, leucine aminopeptidase (LapA1) and proteinase inhibitor 2 (pin2), were not detected following whitefly feeding. Furthermore, whitefly infestation of transgenic LapA1:GUS tomato plants showed that whitefly feeding did not activate the LapA1 promoter, although crushing of the leaf lamina increased GUS activity up to 40 fold. These studies indicate that tomato plants perceive B. tabaci and T. vaporariorum in a manner similar to baterical pathogens and distinct from tissue-damaging insects.

  16. A highly sensitive and simply operated protease sensor toward point-of-care testing.

    Science.gov (United States)

    Park, Seonhwa; Shin, Yu Mi; Seo, Jeongwook; Song, Ji-Joon; Yang, Haesik

    2016-04-21

    Protease sensors for point-of-care testing (POCT) require simple operation, a detection period of less than 20 minutes, and a detection limit of less than 1 ng mL(-1). However, it is difficult to meet these requirements with protease sensors that are based on proteolytic cleavage. This paper reports a highly reproducible protease sensor that allows the sensitive and simple electrochemical detection of the botulinum neurotoxin type E light chain (BoNT/E-LC), which is obtained using (i) low nonspecific adsorption, (ii) high signal-to-background ratio, and (iii) one-step solution treatment. The BoNT/E-LC detection is based on two-step proteolytic cleavage using BoNT/E-LC (endopeptidase) and l-leucine-aminopeptidase (LAP, exopeptidase). Indium-tin oxide (ITO) electrodes are modified partially with reduced graphene oxide (rGO) to increase their electrocatalytic activities. Avidin is then adsorbed on the electrodes to minimize the nonspecific adsorption of proteases. Low nonspecific adsorption allows a highly reproducible sensor response. Electrochemical-chemical (EC) redox cycling involving p-aminophenol (AP) and dithiothreitol (DTT) is performed to obtain a high signal-to-background ratio. After adding a C-terminally AP-labeled oligopeptide, DTT, and LAP simultaneously to a sample solution, no further treatment of the solution is necessary during detection. The detection limits of BoNT/E-LC in phosphate-buffered saline are 0.1 ng mL(-1) for an incubation period of 15 min and 5 fg mL(-1) for an incubation period of 4 h. The detection limit in commercial bottled water is 1 ng mL(-1) for an incubation period of 15 min. The developed sensor is selective to BoNT/E-LC among the four types of BoNTs tested. These results indicate that the protease sensor meets the requirements for POCT.

  17. Spatial and temporal population genetic structure of four northeastern Pacific littorinid gastropods: the effect of mode of larval development on variation at one mitochondrial and two nuclear DNA markers.

    Science.gov (United States)

    Lee, Hyuk Je; Boulding, Elizabeth G

    2009-05-01

    We investigated the effect of development mode on the spatial and temporal population genetic structure of four littorinid gastropod species. Snails were collected from the same three sites on the west coast of Vancouver Island, Canada in 1997 and again in 2007. DNA sequences were obtained for one mitochondrial gene, cytochrome b (Cyt b), and for up to two nuclear genes, heat shock cognate 70 (HSC70) and aminopeptidase N intron (APN54). We found that the mean level of genetic diversity and long-term effective population sizes (N(e)) were significantly greater for two species, Littorina scutulata and L. plena, that had a planktotrophic larval stage than for two species, Littorina sitkana and L. subrotundata, that laid benthic egg masses which hatched directly into crawl-away juveniles. Predictably, two poorly dispersing species, L. sitkana and L. subrotundata, showed significant spatial genetic structure at an 11- to 65-km geographical scale that was not observed in the two planktotrophic species. Conversely, the two planktotrophic species had more temporal genetic structure over a 10-year interval than did the two direct-developing species and showed highly significant temporal structure for spatially pooled samples. The greater temporal genetic variation of the two planktotrophic species may have been caused by their high fecundity, high larval dispersal, and low but spatially correlated early survivorship. The sweepstakes-like reproductive success of the planktotrophic species could allow a few related females to populate hundreds of kilometres of coastline and may explain their substantially larger temporal genetic variance but lower spatial genetic variance relative to the direct-developing species.

  18. 烟夜蛾幼虫中肠Bt Cry1Ac毒素受体蛋白cDNA片段的克隆和序列测定%Molecular Cloning and Sequencing of cDNA Fragment Encoding Bt Cry1Ac Toxin Binding Protein from the Midgut of Helicoverpa assulta Guenée Larva

    Institute of Scientific and Technical Information of China (English)

    安世恒; 郭线茹; 罗梅浩; 蒋金炜; 马继盛

    2005-01-01

    利用RT-PCR技术扩增烟夜蛾(Helicoverpa assulta Guenée)幼虫中肠Bt毒素Cry1Ac受体蛋白APN(N-氨基肽酶,aminopeptidase N,APN)基因片段,克隆和测序结果表明,测序得到的812 bp的片段编码270个氨基酸残基,且该片段在阅读框内.通过同源性分析发现,其核苷酸序列与棉铃虫(H. armigera)、澳洲棉铃虫(H. punctigera)、烟芽夜蛾(H. virescens)、舞毒蛾(Lymantria dispar)、小菜蛾(Plutella xylostella)、印度谷螟(Plodia interpunctella)RC688品系和HD198品系、烟草天蛾(Manduca sexta)和家蚕(Bombyx mori)的Cry1Ac受体蛋白基因的同源性分别为97.0%,90.0%,78.0%,63.5%,55.0%,60.3%,61.2%,55.0%和59.0%.推导的烟夜蛾Cry1Ac受体蛋白基因的氨基酸序列与棉铃虫、烟芽夜蛾、斑实夜蛾、舞毒蛾的氨基酸序列同源性分别为95.6%,81.0%,82.7%和55.7%.该片段编码的氨基酸属于氨肽酶家族,与烟夜蛾对Bt Cry1Ac毒素的抗性有关.

  19. Identification of Neisseria spp., Haemophilus spp., and other fastidious gram-negative bacteria with the MicroScan Haemophilus-Neisseria identification panel.

    Science.gov (United States)

    Janda, W M; Bradna, J J; Ruther, P

    1989-05-01

    The Haemophilus-Neisseria identification (HNID) panel (American MicroScan, Sacramento, Calif.) is a 4-h microdilution format system for identification of Haemophilus and Neisseria spp., Branhamella (Moraxella) catarrhalis, and Gardnerella vaginalis. The HNID panel was evaluated by using 423 clinical isolates and stock strains of these organisms, and HNID identifications were compared with those obtained by conventional methods. In addition, 32 isolates representing six genera not included in the HNID data base were tested to determine whether these organisms would produce unique biotype numbers for possible inclusion in the data base. The HNID panel correctly identified 95.3% of 86 Neisseria gonorrhoeae strains, 96% of 25 G. vaginalis strains, and 100% of 28 Neisseria lactamica strains and 48 B. catarrhalis strains. Only 64.7% of 68 Neisseria meningitidis isolates were identified correctly owing to false-negative or equivocal carbohydrate and/or aminopeptidase reactions. Among the Haemophilus spp., 98.8% of 83 H. influenzae strains, 97.1% of 34 H. parainfluenzae strains, and 80% of 15 H. aphrophilus and H. paraphrophilus strains were correctly identified. Eight strains of Neisseria cinerea, a species not included in the data base, produced profiles identical with those for B. catarrhalis and N. gonorrhoeae. Isolates of other species not included in the data base, including Eikenella corrodens, Kingella spp., and Cardiobacterium hominis, produced unique biochemical reaction patterns on the panel. Modification of interpretative criteria for certain tests, expansion of the data base to include other species, and suggestions for additional confirmatory tests will increase the accuracy and utility of the HNID panel.

  20. First Evidence of an Important Organic Matter Trophic Pathway between Temperate Corals and Pelagic Microbial Communities.

    Directory of Open Access Journals (Sweden)

    J A Fonvielle

    Full Text Available Mucus, i.e., particulate and dissolved organic matter (POM, DOM released by corals, acts as an important energy carrier in tropical ecosystems, but little is known on its ecological role in temperate environments. This study assessed POM and DOM production by the temperate coral Cladocora caespitosa under different environmental conditions. The subsequent enzymatic degradation, growth of prokaryotes and virus-like particles (VLPs as well as changes in the structure of the prokaryotic communities were also monitored. C. caespitosa produced an important quantity of mucus, which varied according to the environmental conditions (from 37.8 to 67.75 nmol carbon h-1 cm-2, but remained higher or comparable to productions observed in tropical corals. It has an important nutritional value, as highlighted by the high content in dissolved nitrogen (50% to 90% of the organic matter released. Organic matter was rapidly degraded by prokaryotes' enzymatic activities, and due to its nitrogen content, aminopeptidase activity was 500 fold higher than the α-glucosidase activity. Prokaryotes, as well as VLPs, presented a rapid growth in the mucus, with prokaryote production rates as high as 0.31 μg h-1 L-1. Changes in bacterial and archaeal communities were observed in the ageing mucus and between mucus and the water column, suggesting a clear impact of mucus on microorganism diversity. Overall, our results show that the organic matter released by temperate corals, such as C. caespitosa, which can form reef structures in the Mediterranean Sea, stimulates microbial activity and thereby functions as a significant carbon and nitrogen supplier to the microbial loop.

  1. Identification and analysis of the acetylated status of poplar proteins reveals analogous N-terminal protein processing mechanisms with other eukaryotes.

    Directory of Open Access Journals (Sweden)

    Chang-Cai Liu

    Full Text Available BACKGROUND: The N-terminal protein processing mechanism (NPM including N-terminal Met excision (NME and N-terminal acetylation (N(α-acetylation represents a common protein co-translational process of some eukaryotes. However, this NPM occurred in woody plants yet remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: To reveal the NPM in poplar, we investigated the N(α-acetylation status of poplar proteins during dormancy by combining tandem mass spectrometry with TiO2 enrichment of acetylated peptides. We identified 58 N-terminally acetylated (N(α-acetylated proteins. Most proteins (47, >81% are subjected to N(α-acetylation following the N-terminal removal of Met, indicating that N(α-acetylation and NME represent a common NPM of poplar proteins. Furthermore, we confirm that poplar shares the analogous NME and N(α-acetylation (NPM to other eukaryotes according to analysis of N-terminal features of these acetylated proteins combined with genome-wide identification of the involving methionine aminopeptidases (MAPs and N-terminal acetyltransferase (Nat enzymes in poplar. The N(α-acetylated reactions and the involving enzymes of these poplar proteins are also identified based on those of yeast and human, as well as the subcellular location information of these poplar proteins. CONCLUSIONS/SIGNIFICANCE: This study represents the first extensive investigation of N(α-acetylation events in woody plants, the results of which will provide useful resources for future unraveling the regulatory mechanisms of N(α-acetylation of proteins in poplar.

  2. Bacterial community composition and extracellular enzyme activity in temperate streambed sediment during drying and rewetting.

    Directory of Open Access Journals (Sweden)

    Elisabeth Pohlon

    Full Text Available Droughts are among the most important disturbance events for stream ecosystems; they not only affect stream hydrology but also the stream biota. Although desiccation of streams is common in Mediterranean regions, phases of dryness in headwaters have been observed more often and for longer periods in extended temperate regions, including Central Europe, reflecting global climate change and enhanced water withdrawal. The effects of desiccation and rewetting on the bacterial community composition and extracellular enzyme activity, a key process in the carbon flow of streams and rivers, were investigated in a typical Central European stream, the Breitenbach (Hesse, Germany. Wet streambed sediment is an important habitat in streams. It was sampled and exposed in the laboratory to different drying scenarios (fast, intermediate, slow for 13 weeks, followed by rewetting of the sediment from the fast drying scenario via a sediment core perfusion technique for 2 weeks. Bacterial community structure was analyzed using CARD-FISH and TGGE, and extracellular enzyme activity was assessed using fluorogenic model substrates. During desiccation the bacterial community composition shifted toward composition in soil, exhibiting increasing proportions of Actinobacteria and Alphaproteobacteria and decreasing proportions of Bacteroidetes and Betaproteobacteria. Simultaneously the activities of extracellular enzymes decreased, most pronounced with aminopeptidases and less pronounced with enzymes involved in the degradation of polymeric carbohydrates. After rewetting, the general ecosystem functioning, with respect to extracellular enzyme activity, recovered after 10 to 14 days. However, the bacterial community composition had not yet achieved its original composition as in unaffected sediments within this time. Thus, whether the bacterial community eventually recovers completely after these events remains unknown. Perhaps this community undergoes permanent changes

  3. Kinetics and spatial distribution of enzymes of carbon, nitrogen and phosphorus cycles in earthworm biopores

    Science.gov (United States)

    Hoang Thi Thu, Duyen; Razavi, Bahar S.

    2016-04-01

    Earthworms boost microbial activities and consequently form hotspots in soil. The distribution of enzyme activities inside the earthworm biopores is completely unknown. For the first time, we analyzed enzyme kinetics and visualized enzyme distribution inside and outside biopores by in situ soil zymography. Kinetic parameters (Vmax and Km) of 6 enzymes β-glucosidase (GLU), cellobiohydrolase (CBH), xylanase (XYL), chitinase (NAG), leucine aminopeptidase (LAP) and acid phosphatase (APT) were determined in biopores formed by Lumbricus terrestris L.. The spatial distributions of GLU, NAG and APT become visible via zymograms in comparison between earthworm-inhabited and earthworm-free soil. Zymography showed heterogeneous distribution of hotspots in the rhizosphere and biopores. The hotspot areas were 2.4 to 14 times larger in the biopores than in soil without earthworms. The significantly higher Vmax values for GLU, CBH, XYL, NAG and APT in biopores confirmed the stimulation of enzyme activities by earthworms. For CBH, XYL and NAG, the 2- to 3-fold higher Km values in biopores indicated different enzyme systems with lower substrate affinity compared to control soil. The positive effects of earthworms on Vmax were cancelled by the Km increase for CBH, XYL and NAG at a substrate concentration below 20 μmol g-1 soil. The change of enzyme systems reflected a shift in dominant microbial populations toward species with lower affinity to holo-celluloses and to N-acetylglucosamine, and with higher affinity to proteins as compared to the biopores-free soil. We conclude that earthworm biopores are microbial hotspots with much higher and dense distribution of enzyme activities compared to bulk soil. References Spohn M, Kuzyakov Y. (2014) Spatial and temporal dynamics of hotspots of enzyme activity in soil as affected by living and dead roots - a soil zymography analysis, Plant Soil 379: 67-77. Blagodatskaya, E., Kuzyakov, Y., 2013. Review paper: Active microorganisms in soil

  4. Genome-wide expression profiling of complex regional pain syndrome.

    Directory of Open Access Journals (Sweden)

    Eun-Heui Jin

    Full Text Available Complex regional pain syndrome (CRPS is a chronic, progressive, and devastating pain syndrome characterized by spontaneous pain, hyperalgesia, allodynia, altered skin temperature, and motor dysfunction. Although previous gene expression profiling studies have been conducted in animal pain models, there genome-wide expression profiling in the whole blood of CRPS patients has not been reported yet. Here, we successfully identified certain pain-related genes through genome-wide expression profiling in the blood from CRPS patients. We found that 80 genes were differentially expressed between 4 CRPS patients (2 CRPS I and 2 CRPS II and 5 controls (cut-off value: 1.5-fold change and p<0.05. Most of those genes were associated with signal transduction, developmental processes, cell structure and motility, and immunity and defense. The expression levels of major histocompatibility complex class I A subtype (HLA-A29.1, matrix metalloproteinase 9 (MMP9, alanine aminopeptidase N (ANPEP, l-histidine decarboxylase (HDC, granulocyte colony-stimulating factor 3 receptor (G-CSF3R, and signal transducer and activator of transcription 3 (STAT3 genes selected from the microarray were confirmed in 24 CRPS patients and 18 controls by quantitative reverse transcription-polymerase chain reaction (qRT-PCR. We focused on the MMP9 gene that, by qRT-PCR, showed a statistically significant difference in expression in CRPS patients compared to controls with the highest relative fold change (4.0±1.23 times and p = 1.4×10(-4. The up-regulation of MMP9 gene in the blood may be related to the pain progression in CRPS patients. Our findings, which offer a valuable contribution to the understanding of the differential gene expression in CRPS may help in the understanding of the pathophysiology of CRPS pain progression.

  5. Pore formation by Cry toxins.

    Science.gov (United States)

    Soberón, Mario; Pardo, Liliana; Muñóz-Garay, Carlos; Sánchez, Jorge; Gómez, Isabel; Porta, Helena; Bravo, Alejandra

    2010-01-01

    Bacillus thuringiensis (Bt) bacteria produce insecticidal Cry and Cyt proteins used in the biological control of different insect pests. In this review, we will focus on the 3d-Cry toxins that represent the biggest group of Cry proteins and also on Cyt toxins. The 3d-Cry toxins are pore-forming toxins that induce cell death by forming ionic pores into the membrane of the midgut epithelial cells in their target insect. The initial steps in the mode of action include ingestion of the protoxin, activation by midgut proteases to produce the toxin fragment and the interaction with the primary cadherin receptor. The interaction of the monomeric CrylA toxin with the cadherin receptor promotes an extra proteolytic cleavage, where helix alpha-1 of domain I is eliminated and the toxin oligomerization is induced, forming a structure of 250 kDa. The oligomeric structure binds to a secondary receptor, aminopeptidase N or alkaline phosphatase. The secondary receptor drives the toxin into detergent resistant membrane microdomains formingpores that cause osmotic shock, burst of the midgut cells and insect death. Regarding to Cyt toxins, these proteins have a synergistic effect on the toxicity of some Cry toxins. Cyt proteins are also proteolytic activated in the midgut lumen of their target, they bind to some phospholipids present in the mosquito midgut cells. The proposed mechanism of synergism between Cry and Cyt toxins is that Cyt1Aa function as a receptor for Cry toxins. The Cyt1A inserts into midgut epithelium membrane and exposes protein regions that are recognized by Cry11Aa. It was demonstrated that this interaction facilitates the oligomerization of Cry11Aa and also its pore formation activity.

  6. Midgut Transcriptome of the Cockroach Periplaneta americana and Its Microbiota: Digestion, Detoxification and Oxidative Stress Response.

    Directory of Open Access Journals (Sweden)

    Jianhua Zhang

    Full Text Available The cockroach, Periplaneta americana, is an obnoxious and notorious pest of the world, with a strong ability to adapt to a variety of complex environments. However, the molecular mechanism of this adaptability is mostly unknown. In this study, the genes and microbiota composition associated with the adaptation mechanism were studied by analyzing the transcriptome and 16S rDNA pyrosequencing of the P. americana midgut, respectively. Midgut transcriptome analysis identified 82,905 unigenes, among which 64 genes putatively involved in digestion (11 genes, detoxification (37 genes and oxidative stress response (16 genes were found. Evaluation of gene expression following treatment with cycloxaprid further revealed that the selected genes (CYP6J1, CYP4C1, CYP6K1, Delta GST, alpha-amylase, beta-glucosidase and aminopeptidase were upregulated at least 2.0-fold at the transcriptional level, and four genes were upregulated more than 10.0-fold. An interesting finding was that three digestive enzymes positively responded to cycloxaprid application. Tissue expression profiles further showed that most of the selected genes were midgut-biased, with the exception of CYP6K1. The midgut microbiota composition was obtained via 16S rDNA pyrosequencing and was found to be mainly dominated by organisms from the Firmicutes phylum, among which Clostridiales, Lactobacillales and Burkholderiales were the main orders which might assist the host in the food digestion or detoxification of noxious compounds. The preponderant species, Clostridium cellulovorans, was previously reported to degrade lignocellulose efficiently in insects. The abundance of genes involved in digestion, detoxification and response to oxidative stress, and the diversity of microbiota in the midgut might provide P. americana high capacity to adapt to complex environments.

  7. Regulation of split anergy in natural killer cells by inhibition of cathepsins C and H and cystatin F.

    Science.gov (United States)

    Magister, Špela; Tseng, Han-Ching; Bui, Vickie T; Kos, Janko; Jewett, Anahid

    2015-09-01

    Freshly isolated human primary NK cells induce preferential lysis of Oral Squamous Carcinoma Stem Cells (OSCSCs) when compared to differentiated Oral Squamous Carcinoma Cells (OSCCs), while anti-CD16 antibody and monocytes induce functional split anergy in primary NK cells by decreasing the cytotoxic function of NK cells and increasing the release of IFN-γ. Since NK92 cells have relatively lower levels of cytotoxicity when compared to primary NK cells, and have the ability to increase secretion of regulatory cytokines IL-10 and IL-6, we used these cells as a model of NK cell anergy to identify and to study the upstream regulators of anergy. We demonstrate in this paper that the levels of truncated monomeric cystatin F, which is known to inhibit the functions of cathepsins C and H, is significantly elevated in NK92 cells and in anergized primary NK cells. Furthermore, cystatin F co-localizes with cathepsins C and H in the lysosomal/endosomal vesicles of NK cells. Accordingly, the mature forms of aminopeptidases cathepsins C and H, which regulate the activation of effector granzymes in NK cells, are significantly decreased, whereas the levels of pro-cathepsin C enzyme is increased in anergized NK cells after triggering of the CD16 receptor. In addition, the levels of granzyme B is significantly decreased in anti-CD16mAb and target cell anergized primary NK cells and NK92 cells. Our study provides the cellular and molecular mechanisms by which target cells may utilize to inhibit the cytotoxic function of NK cells.

  8. 尿L-FABP与LAP对2型糖尿病肾病早期诊断的意义

    Institute of Scientific and Technical Information of China (English)

    王瑾堃; 刘中柱; 刘艳姝; 史为伍; 王振

    2016-01-01

    目的:探讨测定尿肝脏型脂肪酸结合蛋白(liver fatty acid binding proteins,L-FABP)和亮氨酸氨基肽酶(Leucine aminopeptidase,LAP)在糖尿病肾病(diabetic nephropathy,DN)早期诊断的意义及对比性研究.方法:收集正常组、单纯2型糖尿病组、早期2型糖尿病肾病组、临床2型糖尿病肾病组各30例,通过ELISA检测各组尿L-FABP、尿LAP的浓度,并观察其与尿蛋白排泄率(urinary albumin excretion rates,UAER)相关性,比较两个肾小管标记物在DN早期诊断的价值.结果:随着DN肾脏损害程度的加重,尿L-FABP和尿LAP水平逐渐增高(P<0.05);尿L-FABP与UAER呈正相关(r=0.64,P<0.01);尿LAP与UAER呈正相关(r=0.31,P<0.01).结论:尿L-FABP与尿LAP水平可反映DN早期肾脏损伤,尿L-FABP与尿LAP相比可较好的作为早期DN诊断指标,为指导临床DN早期诊断提供理论依据.

  9. A proteomic insight into the effects of the immunomodulatory hydroxynaphthoquinone lapachol on activated macrophages.

    Science.gov (United States)

    Oliveira, Renato A S; Correia-Oliveira, Janaina; Tang, Li-Jun; Garcia, Rodolfo C

    2012-09-01

    We report the effect of an immunomodulatory and anti-mycobacterial naphthoquinone, lapachol, on the bi-dimensional patterns of protein expression of toll-like receptor 2 (TLR2)-agonised and IFN-γ-treated THP-1 macrophages. This non-hypothesis driven proteomic analysis intends to shed light on the cellular functions lapachol may be affecting. Proteins of both cytosol and membrane fractions were analysed. After quantification of the protein spots, the protein levels corresponding to macrophages activated in the absence or presence of lapachol were compared. A number of proteins were identified, the levels of which were appreciably and significantly increased or decreased as a result of the action of lapachol on the activated macrophages: cofilin-1, fascin, plastin-2, glucose-6-P-dehydrogenase, adenylyl cyclase-associated protein 1, pyruvate kinase, sentrin-specific protease 6, cathepsin B, cathepsin D, cytosolic aminopeptidase, proteasome β type-4 protease, tryptophan-tRNA ligase, DnaJ homolog and protein disulphide isomerase. Altogether, the comparative analysis performed indicates that lapachol could be hypothetically causing an impairment of cell migration and/or phagocytic capacity, an increase in NADPH availability, a decrease in pyruvate concentration, protection from proteosomal protein degradation, a decrease in lysosomal protein degradation, an impairment of cytosolic peptide generation, and an interference with NOS2 activation and grp78 function. The present proteomic results suggest issues that should be experimentally addressed ex- and in-vivo, to establish more accurately the potential of lapachol as an anti-infective drug. This study also constitutes a model for the pre-in-vivo evaluation of drug actions.

  10. The Human Leukocyte Antigen (HLA)-B27 Peptidome in Vivo, in Spondyloarthritis-susceptible HLA-B27 Transgenic Rats and the Effect of Erap1 Deletion.

    Science.gov (United States)

    Barnea, Eilon; Melamed Kadosh, Dganit; Haimovich, Yael; Satumtira, Nimman; Dorris, Martha L; Nguyen, Mylinh T; Hammer, Robert E; Tran, Tri M; Colbert, Robert A; Taurog, Joel D; Admon, Arie

    2017-04-01

    HLA-B27 is a class I major histocompatibility (MHC-I) allele that confers susceptibility to the rheumatic disease ankylosing spondylitis (AS) by an unknown mechanism. ERAP1 is an aminopeptidase that trims peptides in the endoplasmic reticulum for binding to MHC-I molecules. ERAP1 shows genetic epistasis with HLA-B27 in conferring susceptibility to AS. Male HLA-B27 transgenic rats develop arthritis and serve as an animal model of AS, whereas female B27 transgenic rats remain healthy. We used large scale quantitative mass spectrometry to identify over 15,000 unique HLA-B27 peptide ligands, isolated after immunoaffinity purification of the B27 molecules from the spleens of HLA-B27 transgenic rats. Heterozygous deletion of Erap1, which reduced the Erap1 level to less than half, had no qualitative or quantitative effects on the B27 peptidome. Homozygous deletion of Erap1 affected approximately one-third of the B27 peptidome but left most of the B27 peptidome unchanged, suggesting the possibility that some of the HLA-B27 immunopeptidome is not processed in the presence of Erap1. Deletion of Erap1 was permissive for the AS-like phenotype, increased mean peptide length and increased the frequency of C-terminal hydrophobic residues and of N-terminal Ala, Ser, or Lys. The presence of Erap1 increased the frequency of C-terminal Lys and Arg, of Glu and Asp at intermediate residues, and of N-terminal Gly. Several peptides of potential interest in AS pathogenesis, previously identified in human cell lines, were isolated. However, rats susceptible to arthritis had B27 peptidomes similar to those of non-susceptible rats, and no peptides were found to be uniquely associated with arthritis. Whether specific B27-bound peptides are required for AS pathogenesis remains to be determined. Data are available via ProteomeXchange with identifier PXD005502.

  11. Prion Aggregates Are Recruited to the Insoluble Protein Deposit (IPOD) via Myosin 2-Based Vesicular Transport.

    Science.gov (United States)

    Kumar, Rajesh; Nawroth, Peter P; Tyedmers, Jens

    2016-09-01

    Aggregation of amyloidogenic proteins is associated with several neurodegenerative diseases. Sequestration of misfolded and aggregated proteins into specialized deposition sites may reduce their potentially detrimental properties. Yeast exhibits a distinct deposition site for amyloid aggregates termed "Insoluble PrOtein Deposit (IPOD)", but nothing is known about the mechanism of substrate recruitment to this site. The IPOD is located directly adjacent to the Phagophore Assembly Site (PAS) where the cell initiates autophagy and the Cytoplasm-to-Vacuole Targeting (CVT) pathway destined for delivery of precursor peptidases to the vacuole. Recruitment of CVT substrates to the PAS was proposed to occur via vesicular transport on Atg9 vesicles and requires an intact actin cytoskeleton and "SNAP (Soluble NSF Attachment Protein) Receptor Proteins (SNARE)" protein function. It is, however, unknown how this vesicular transport machinery is linked to the actin cytoskeleton. We demonstrate that recruitment of model amyloid PrD-GFP and the CVT substrate precursor-aminopeptidase 1 (preApe1) to the IPOD or PAS, respectively, is disturbed after genetic impairment of Myo2-based actin cable transport and SNARE protein function. Rather than accumulating at the respective deposition sites, both substrates reversibly accumulated often together in the same punctate structures. Components of the CVT vesicular transport machinery including Atg8 and Atg9 as well as Myo2 partially co-localized with the joint accumulations. Thus we propose a model where vesicles, loaded with preApe1 or PrD-GFP, are recruited to tropomyosin coated actin cables via the Myo2 motor protein for delivery to the PAS and IPOD, respectively. We discuss that deposition at the IPOD is not an integrated mandatory part of the degradation pathway for amyloid aggregates, but more likely stores excess aggregates until downstream degradation pathways have the capacity to turn them over after liberation by the Hsp104

  12. Prion Aggregates Are Recruited to the Insoluble Protein Deposit (IPOD via Myosin 2-Based Vesicular Transport.

    Directory of Open Access Journals (Sweden)

    Rajesh Kumar

    2016-09-01

    Full Text Available Aggregation of amyloidogenic proteins is associated with several neurodegenerative diseases. Sequestration of misfolded and aggregated proteins into specialized deposition sites may reduce their potentially detrimental properties. Yeast exhibits a distinct deposition site for amyloid aggregates termed "Insoluble PrOtein Deposit (IPOD", but nothing is known about the mechanism of substrate recruitment to this site. The IPOD is located directly adjacent to the Phagophore Assembly Site (PAS where the cell initiates autophagy and the Cytoplasm-to-Vacuole Targeting (CVT pathway destined for delivery of precursor peptidases to the vacuole. Recruitment of CVT substrates to the PAS was proposed to occur via vesicular transport on Atg9 vesicles and requires an intact actin cytoskeleton and "SNAP (Soluble NSF Attachment Protein Receptor Proteins (SNARE" protein function. It is, however, unknown how this vesicular transport machinery is linked to the actin cytoskeleton. We demonstrate that recruitment of model amyloid PrD-GFP and the CVT substrate precursor-aminopeptidase 1 (preApe1 to the IPOD or PAS, respectively, is disturbed after genetic impairment of Myo2-based actin cable transport and SNARE protein function. Rather than accumulating at the respective deposition sites, both substrates reversibly accumulated often together in the same punctate structures. Components of the CVT vesicular transport machinery including Atg8 and Atg9 as well as Myo2 partially co-localized with the joint accumulations. Thus we propose a model where vesicles, loaded with preApe1 or PrD-GFP, are recruited to tropomyosin coated actin cables via the Myo2 motor protein for delivery to the PAS and IPOD, respectively. We discuss that deposition at the IPOD is not an integrated mandatory part of the degradation pathway for amyloid aggregates, but more likely stores excess aggregates until downstream degradation pathways have the capacity to turn them over after liberation by the

  13. Growth factor TGF-β induces intestinal epithelial cell (IEC-6) differentiation: miR-146b as a regulatory component in the negative feedback loop.

    Science.gov (United States)

    Liao, Yalin; Zhang, Man; Lönnerdal, Bo

    2013-01-01

    TGF-β is a potent pleiotropic factor that promotes small intestinal cell differentiation. The role of microRNAs in the TGF-β induction of intestinal epithelial phenotype is largely unknown. We hypothesized that microRNAs are functionally involved in TGF-β-induced intestinal cell growth. In this study, TGF-β caused a morphological change of IEC-6 cells and stimulated expression of the epithelial cell markers alkaline phosphatase, villin, and aminopeptidase N. By global microRNA profiling during TGF-β-induced intestinal crypt cell (IEC-6) differentiation, we identified 19 differentially expressed microRNAs. We showed by real-time Q-PCR that miR-146b expression increased rapidly after TGF-β treatment; sequence analysis and in vitro assays revealed that miR-146b targets SIAH2, an E3 ubiquitin ligase, with decreased protein expression upon IEC-6 cell differentiation. Transfection of miR-146b inhibitor before TGF-β treatment blocked the down-regulation of SIAH2 in response to TGF-β. Moreover, SIAH2 over-expression during TGF-β treatment caused a significant decrease in Smad7 protein expression in IEC-6 cells. Furthermore, activation of the ERK1/2 pathway is active in the up-regulation of miR-146b by TGF-β. These findings suggest a novel mechanism whereby TGF-β signaling during IEC-6 cell differentiation may be modulated in part by microRNAs, and we propose a key role for miR-146b in the homeostasis of growth factor TGF-β signaling through a negative feedback regulation involving down-regulation of SIAH2 repressed Smad7 activities.

  14. A tomato bZIP transcription factor, SlAREB, is involved in water deficit and salt stress response.

    Science.gov (United States)

    Hsieh, Tsai-Hung; Li, Chia-Wen; Su, Ruey-Chih; Cheng, Chiu-Ping; Sanjaya; Tsai, Yi-Chien; Chan, Ming-Tsair

    2010-05-01

    Abiotic stresses such as cold, water deficit, and salt stresses severely reduce crop productivity. Tomato (Solanum lycopersicum) is an important economic crop; however, not much is known about its stress responses. To gain insight into stress-responsive gene regulation in tomato plants, we identified transcription factors from a tomato cDNA microarray. An ABA-responsive element binding protein (AREB) was identified and named SlAREB. In tomato protoplasts, SlAREB transiently transactivated luciferase reporter gene expression driven by AtRD29A (responsive to dehydration) and SlLAP (leucine aminopeptidase) promoters with exogenous ABA application, which was suppressed by the kinase inhibitor staurosporine, indicating that an ABA-dependent post-translational modification is required for the transactivation ability of SlAREB protein. Electrophoretic mobility shift assays showed that the recombinant DNA-binding domain of SlAREB protein is able to bind AtRD29A and SlLAP promoter regions. Constitutively expressed SlAREB increased tolerance to water deficit and high salinity stresses in both Arabidopsis and tomato plants, which maintained PSII and membrane integrities as well as water content in plant bodies. Overproduction of SlAREB in Arabidopsis thaliana and tomato plants regulated stress-related genes AtRD29A, AtCOR47, and SlCI7-like dehydrin under ABA and abiotic stress treatments. Taken together, these results show that SlAREB functions to regulate some stress-responsive genes and that its overproduction improves plant tolerance to water deficit and salt stress.

  15. Brugia malayi excreted/secreted proteins at the host/parasite interface: stage- and gender-specific proteomic profiling.

    Directory of Open Access Journals (Sweden)

    Sasisekhar Bennuru

    Full Text Available Relatively little is known about the filarial proteins that interact with the human host. Although the filarial genome has recently been completed, protein profiles have been limited to only a few recombinants or purified proteins of interest. Here, we describe a large-scale proteomic analysis using microcapillary reverse-phase liquid chromatography-tandem-mass spectrometry to identify the excretory-secretory (ES products of the L3, L3 to L4 molting ES, adult male, adult female, and microfilarial stages of the filarial parasite Brugia malayi. The analysis of the ES products from adult male, adult female, microfilariae (Mf, L3, and molting L3 larvae identified 852 proteins. Annotation suggests that the functional and component distribution was very similar across each of the stages studied; however, the Mf contributed a higher proportion to the total number of identified proteins than the other stages. Of the 852 proteins identified in the ES, only 229 had previous confirmatory expressed sequence tags (ESTs in the available databases. Moreover, this analysis was able to confirm the presence of 274 "hypothetical" proteins inferred from gene prediction algorithms applied to the B. malayi (Bm genome. Not surprisingly, the majority (160/274 of these "hypothetical" proteins were predicted to be secreted by Signal IP and/or SecretomeP 2.0 analysis. Of major interest is the abundance of previously characterized immunomodulatory proteins such as ES-62 (leucyl aminopeptidase, MIF-1, SERPIN, glutathione peroxidase, and galectin in the ES of microfilariae (and Mf-containing adult females compared to the adult males. In addition, searching the ES protein spectra against the Wolbachia database resulted in the identification of 90 Wolbachia-specific proteins, most of which were metabolic enzymes that have not been shown to be immunogenic. This proteomic analysis extends our knowledge of the ES and provides insight into the host-parasite interaction.

  16. Maize toxin degrades peritrophic matrix proteins and stimulates compensatory transcriptome responses in fall armyworm midgut.

    Science.gov (United States)

    Fescemyer, Howard W; Sandoya, Germán V; Gill, Torrence A; Ozkan, Seval; Marden, James H; Luthe, Dawn S

    2013-03-01

    Understanding the molecular mechanisms underlying insect compensatory responses to plant defenses could lead to improved plant resistance to herbivores. The Mp708 inbred line of maize produces the maize insect resistant 1-cysteine protease (Mir1-CP) toxin. Reduced feeding and growth of fall armyworm larvae fed on Mp708 was previously linked to impairment of nutrient utilization and degradation of the midgut (MG) peritrophic matrix (PM) by Mir1-CP. Here we examine the biochemical and transcriptional responses of fall armyworm larvae to Mir1-CP. Insect Intestinal Mucin (IIM) was severely depleted from pure PMs treated in vitro with recombinant Mir1-CP. Larvae fed on Mp708 midwhorls excrete frass largely depleted of IIM. Cracks, fissures and increased porosity previously observed in the PM of larvae fed on Mp708 midwhorls could ensue when Mir1-CP degrades the IIM that cross-links chitin fibrils in the PM. Both targeted and global transcriptome analyses were performed to determine how complete dissolution of the structure and function of the PM is prevented, enabling larvae to continue growing in the presence of Mir1-CP. The MGs from fall armyworm fed on Mp708 upregulate expression of genes encoding proteins involved in PM production as an apparent compensation to replace the disrupted PM structure and restore appropriate counter-current MG gradients. Also, several families of digestive enzymes (endopeptidases, aminopeptidases, lipases, amylase) were more highly expressed in MGs from larvae fed on Mp708 than MGs from larvae fed on diets lacking Mir1-CP (artificial diet, midwhorls from Tx601 or B73 maize). Impaired growth of larvae fed on Mp708 probably results from metabolic costs associated with higher production of PM constituents and digestive enzymes in a compensatory attempt to maintain MG function.

  17. Characterization and functional analysis ofβ-1,3-galactosyltransferase involved in Cry1Ac resistance from Helicoverpa armigera (Hübner)

    Institute of Scientific and Technical Information of China (English)

    ZHANG Li-li; LIANG Ge-mei; GAO Xi-wu; CAO Guang-chun; GUO Yu-yuan

    2015-01-01

    Carbohydrate chains are the principal antigens by which Bacil us thuringiensis (Bt) identify receptor proteins. The interaction between the antigen and Bt causes a pore in the membrane of midgut epithelial cel s of insects. Receptor proteins, such as aminopeptidase N and alkaline phosphatase, are glycoproteins. Cadherin is another cel surface receptor protein which has potential glycosylation sites. Glycosyltransferase is very important for the synthesis and modiifcation of receptor proteins. It can indirectly inlfuence the function of Bt. The 1 950 bp ful-length cDNA encodingβ-1,3-galactosyltransferase was cloned from the the midgut of Helicoverpa armigera by degenerative PCR combined with RACE techniques (GAL-Harm, GenBank accession no.:GQ904195.1) with two potential N-glycosylation sites (157NNTI160 and 272NKTL275). Protein sequence alignments revealed that H. armigeraβ-1,3-galactosyltransferase shared high identity withβ-1,3-galactosyltransferase in other insect species. The expression level of theβ-1,3-galactosyltransferase gene in Cry1Ac-resistant H. armigera larvae was 9.2-fold higher than that in susceptible strain. The function ofβ-1,3-galactosyltransferase was investigated using RNAi technique. The result showed Cry1Ac enhanced the toxicity against the siRNA-treated larvae compared with non-siRNA-treated ones, which indicatedβ-1,3-galactosyltransferase played an important role for the insecticidal toxicity of Cry1Ac in H. armigera.

  18. Microbial communities and biogeochemistry in an area of Engraulis encrasicolus spawning in the Sicilian Channel

    Directory of Open Access Journals (Sweden)

    Rosabruna La Ferla

    2014-06-01

    The rates of production, hydrolysis and degradation activities were quite low, in agreement with the general oligotrophy of the area and in agreement with this findings, picophytoplankton contribution to total production amounted to over than 65%. In terms of metabolic activities, different spatial distributions were observed between the autotrophic and heterotrophic components. In particular, heterotrophic metabolism showed high values in the layer located below the thermocline and above the DCM. Concerning the enzymatic activities, total leucine aminopeptidase activity showed the highest rates, followed by alkaline phosphatase and beta-Glucosidase (Caruso et al., 2014. Distribution of the dissolved enzymatic activities acting on proteins varied widely along the column, accounting for 5 to 90% of the total enzymatic activity and always prevailed on dissolved b-GLU. Total enzymatic activity rates were comparable to those obtained in the oligotrophic zones of Ionian and Mediterranean Sea (Zaccone et al., 2012, while the analysis of the dissolved fraction was the first report for the Mediterranean. In conclusion, the multidisciplinary scientific approach used in this study depicted a complex picture of the study area evidencing a high heterogeneity and dynamism of plankton communities, probably associated to peculiar hydrology of this ecosystem. On the whole, the study area appeared to be characterized by a relatively efficient microbial food web. However, low trophic conditions were stressed by all the biological and biochemical patterns, suggesting an important role of heterotrophic processes in this area in the examined summer period. Consequently, how and how much the microbial web sustains fish reproduction and larval survival need a more comprehensive analysis and will be focused in further research.

  19. Enzymatic activity in extracts of allergy-causing astigmatid mites.

    Science.gov (United States)

    Morgan, Marjorie S; Arlian, Larry G

    2006-11-01

    Many of the previously characterized allergens of house dust mites are known to be proteases, and this enzymatic activity is thought to contribute to their allergenicity. Other astigmatid mites, including stored-product mites and the ectoparasitic itch mite, Sarcoptes scabiei De Geer, are also known to be allergenic, but little or nothing is known about their enzymatic activities. The purpose of this study was to characterize the enzymatic activities present in extracts of the parasitic itch mite and from eight other species of free-living astigmatid mites. Extracts were prepared from one parasitic mite (S. scabiei), five stored-product mites (Chortoglyphus arcuatus (Troupeau), Lepidoglyphus destructor (Schrank), Blomia tropicalis Bronswijk, Cock, Oshima, Tyrophagus putrescentiae (Schrank), and Acarus siro L.), and three house dust mites [Dermatophagoidesfarinae Hughes, Dermatophagoides pteronyssinus (Troussart), and Euroglyphus maynei (Cooreman) ]. ApiZym strips were used to screen for the presence of 19 individual enzyme activities. Digestion of nine other substrates was evaluated by spectrophotometric or electrophoretic methods. All mite extracts exhibited some form of phosphatase, esterase, aminopeptidase, and glycosidase activity, although their substrate specificities varied considerably. Itch mite extract did not possess detectable serine peptidase activity nor was it able to hydrolyze gelatin or casein, whereas all other mite extracts exhibited these activities. Storage mite extracts possessed enzymes capable of degrading the widest range of substrates, whereas itch mite extract had the most limited proteolytic capacity. Extracts of nine species of allergy-causing astigmatid mites contain wide and diverse repertoires of enzymatic activities. These catalytic activities may be important contributors to the induction and manifestation of inflammatory and immune responses to mites in patients.

  20. 血清前白蛋白与相关酶类检测在肝病诊断中的比较

    Institute of Scientific and Technical Information of China (English)

    刘春利; 陈旭虞; 陈樱君; 张素华; 李佳; 梁有祥

    2008-01-01

    本研究通过比较不同类型肝病患者血清前白蛋白(preal-bumin,PAB)、血清丙氨酸氨基转移酶(alanine aminotrans-ferase,ALT)、天冬氨酸氨基转移酶(aspartate aminotransferase,AST)、γ-谷氨酰转肽酶(γ-glutamyltransferase,GGT)、腺苷脱氨酶(adenosine deaminase,ADA)、甘氨酰脯氨酸二肽氨基肽酶(glycylproline dipeptidyl aminopeptidase,GPDA)和α-L-岩藻糖苷酶(α-L-fucosidase,AFU)的测定结果,探讨这些检测指标在肝病患者中的改变及其诊断价值。对象和方法标本来源:肝病患者组:220例均为我院2006年9月~2007年10月门诊或住院患者,其中男150例,女70例,年龄14~77岁,按病因分为急性肝炎(44例)、慢性肝炎(70例)、酒精性肝炎(44例)、肝硬化(37例)和肝癌(25例)组。正常对照组:57例健康体检者,其中男39例,女18例,年龄21~64岁。方法:采集清晨空腹静脉血,使用AU-400全自动生化分析仪,ALT、AST、GGT、AD...