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Sample records for americanum proteinase inhibitor

  1. [Proteinase-proteinase inhibitor complex in rats under oxidative stress caused by administration of cobalt chloride].

    Science.gov (United States)

    Kaliman, P A; Samokhin, A A; Samokhina, L M

    2000-01-01

    Mechanisms of proteinase-inhibitor proteinase system response was estimated following of cobalt chloride injection. The increase proteinase activity, which led to significant decrease of alpha-2-macroglobulin (alpha-2-MG) level was established that indicated to the removal of the proteinase in complex with alpha-2-MG from the organism. Increase of alpha-1-proteinase inhibitor (alpha-1-PI) trypsin-inhibitory activity in the kidneys testify about removal of oxidative alpha-1-PI. PMID:10979565

  2. Molecular dynamic and docking interaction study of Heterodera glycines serine proteinase with Vigna mungo proteinase inhibitor.

    Science.gov (United States)

    Prasad, C V S Siva; Gupta, Saurabh; Gaponenko, Alex; Tiwari, Murlidhar

    2013-08-01

    Many plants do produce various defense proteins like proteinase inhibitors (PIs) to protect them against various pests. PIs function as pseudosubstrates of digestive proteinase, which inhibits proteolysis in pests and leads to amino acid deficiency-based mortality. This work reports the structural interaction studies of serine proteinase of Heterodera glycines (SPHG) with Vigna mungo proteinase inhibitor (VMPI). 3D protein structure modeling, validation of SPHG and VMPI, and their putative protein-protein binding sites were predicted. Protein-protein docking followed by molecular dynamic simulation was performed to find the reliable confirmation of SPHG-VMPI complex. Trajectory analysis of each successive conformation concludes better interaction of first loop in comparison with second loop. Lysine residues of first loop were actively participating in complex formation. Overall, this study discloses the structural aspects and interaction mechanisms of VMPI with SPHG, and it would be helpful in the development of pest-resistant genetically modified crops.

  3. [Effect of adrenal stress on activity of proteinase and alpha-1-proteinase inhibitor in rats].

    Science.gov (United States)

    Samokhina, L M; Kaliman, P A

    1994-01-01

    The effect of adrenal stress on the proteinase and alpha-1-proteinase inhibitor activities in blood serum and cytosols of the rat organs were investigated. The reliable change was marked only in the alpha-1-PI level research of lung tissue cytosol. The proteolysis suppression was revealed in the heart and kidney tissue, while the proteolysis activation was revealed in serum and less in the lung tissue cytosol. Changes in proteinase level in the myocardium and kidney tissue play the primary role in respect to those of the other research liquids under study. PMID:7747353

  4. [Effect of pentoxyphylline on certain indicators of the proteinase-proteinase inhibitor system in rats upon administration of cycloheximide].

    Science.gov (United States)

    Samokhin, A A; Kaliman, P A; Samokhinka, L M

    2001-01-01

    The pentoxifylline influence on neutral proteinase, alpha-2-macroglobulin, trypsin-alpha-1-proteinase inhibitor and elastaseinhibitory activity under cycloheximide injection has been investigated. Two hours after cycloheximide injection the activity of neutral proteinases increases in rats serum, lungs, heart, liver and kidneys. The preliminary injection of pentoxifylline prevents increase of neutral proteinases activity. Cycloheximide also decreases alpha-2-macroglobulin activity in serum and liver and trypsin-, elastaseinhibitory activity of alpha-1-proteinase inhibitor in all investigated organs. At using pentoxifylline the alpha-2-macroglobulin activity doesn't change in liver and increases in serum in comparison with only cycloheximide and there are no observed any alpha-1 inhibitor proteinase activity changes in rats serum and organs. PMID:12035527

  5. An electroblotting, two-step procedure for the detection of proteinases and the study of proteinase/inhibitor complexes in gelatin-containing polyacrylamide gels.

    Science.gov (United States)

    Visal-Shah, S; Vrain, T C; Yelle, T C; Nguyen-Quoc, B; Michaud, D

    2001-08-01

    A two-step gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE) procedure was devised for the detection of proteinases and the study of proteinase/inhibitor interactions in complex biological extracts. The proteins are first resolved by sodium dodecyl sulfate (SDS)-PAGE under reducing or nonreducing conditions, and electrotransferred into a 0.75 mm-thick accompanying polyacrylamide slab gel containing 0.1% w/v porcine gelatin. The active proteinase bands are developed by a gelatin proteolysis step in the accompanying gel in the presence or absence of diagnostic proteinase inhibitors, allowing the assessment of proteinase classes and the visual discrimination of inhibitor-'sensitive' and -'insensitive' proteinases in complex extracts. Alternatively, protein extracts are preincubated with specific reversible inhibitors before electrophoresis, allowing a rapid discrimination of strong and weak interactions implicating proteinases and reversible inhibitors. In comparison with the standard gelatin/PAGE procedure, that involves copolymerization of gelatin with acrylamide in the resolving gel, this new procedure simplifies proteinase patterns, avoids overestimation of proteinase numbers in complex extracts, and allows in certain conditions the estimation of proteinase molecular weights. Stem bromelain (EC 3.4.22.32), bovine trypsin (EC 3.4.21.4), papain (EC 3.4.22.2), and the extracellular (digestive) cysteine proteinases of five herbivorous pests are used as model enzymes to illustrate the usefulness of this approach in detecting proteinases and in studying their interactions with specific proteinaceous inhibitors potentially useful in biotechnology.

  6. [Effect of quercetin on some indicators of the proteinase-proteinase inhibitor system in rats upon administration of cobalt chloride to them].

    Science.gov (United States)

    Kaliman, P A; Samokhin, A A; Samokhina, L M

    2001-01-01

    The results of quercetin effect on some changes of proteinase--proteinase inhibitor system parameters in rats under cobalt chloride injection are shown. It was established that preliminary quercetin administration prevened neutral proteinase activation and alpha-2-macroglobulin activity decreasing after 2 h of cobalt chloride influence. PMID:12199071

  7. Gelatinases and serine proteinase inhibitors of seminal plasma and the reproductive tract of turkey (Meleagris gallopavo).

    Science.gov (United States)

    Kotłowska, M; Kowalski, R; Glogowski, J; Jankowski, J; Ciereszko, A

    2005-04-01

    This study examined proteolytic enzymes and serine proteinase inhibitors in turkey seminal plasma with relation to their distribution within the reproductive tract and to yellow semen syndrome (YSS). Proteases of blood plasma, extracts from the reproductive tract, and seminal plasma were analyzed by gelatin zymography. We found a clear regional distribution of proteolytic enzymes in the turkey reproductive tract. Each part was characterized by a unique profile of serine proteolytic enzymes of molecular weights ranging from 29 to 88 kDa. The ductus deferens was found to be a site of very intense proteolytic activity. Two metalloproteases of 58 and 66 kDa were detected in all parts of the reproductive tract and seminal plasma. Using electrophoretic methods for detection of anti-trypsin activity, we found three serine proteinase inhibitors in turkey seminal plasma. Two inhibitors were found in the testis and epididymis and a third in the ductus deferens and seminal plasma. Blood plasma was characterized by the presence of two metalloproteinases and one serine proteinase inhibitor (of low migration rate) that were also detected in the reproductive tract. Amidase and anti-trypsin activities (expressed per gram of protein) differed for yellow and white seminal plasma. We concluded that turkey seminal plasma contains metalloproteases, serine proteinases, and serine proteinase inhibitors. The metalloproteases and one proteinase inhibitor are related to blood proteinases but the other two inhibitors and serine proteinases seem to be unique for the reproductive tract.

  8. Digestive duet: Midgut digestive proteinases of Manduca sexta ingesting Nicotiana attenuata with manipulated trypsin proteinase inhibitor expression

    NARCIS (Netherlands)

    Zavala, J.A.; Giri, A.P.; Jongsma, M.A.; Baldwin, I.T.

    2008-01-01

    The defensive effect of endogenous trypsin proteinase inhibitors (NaTPIs) on the herbivore Manduca sexta was demonstrated by genetically altering NaTPI production in M. sexta's host plant, Nicotiana attenuata. To understand how this defense works, we studied the effects of NaTPI on M. sexta gut prot

  9. Digestive duet: midgut digestive proteinases of Manduca sexta ingesting Nicotiana attenuata with manipulated trypsin proteinase inhibitor expression.

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    Jorge A Zavala

    Full Text Available BACKGROUND: The defensive effect of endogenous trypsin proteinase inhibitors (NaTPIs on the herbivore Manduca sexta was demonstrated by genetically altering NaTPI production in M. sexta's host plant, Nicotiana attenuata. To understand how this defense works, we studied the effects of NaTPI on M. sexta gut proteinase activity levels in different larval instars of caterpillars feeding freely on untransformed and transformed plants. METHODOLOGY/ PRINCIPAL FINDINGS: Second and third instars larvae that fed on NaTPI-producing (WT genotypes were lighter and had less gut proteinase activity compared to those that fed on genotypes with either little or no NaTPI activity. Unexpectedly, NaTPI activity in vitro assays not only inhibited the trypsin sensitive fraction of gut proteinase activity but also halved the NaTPI-insensitive fraction in third-instar larvae. Unable to degrade NaTPI, larvae apparently lacked the means to adapt to NaTPI in their diet. However, caterpillars recovered at least part of their gut proteinase activity when they were transferred from NaTPI-producing host plants to NaTPI-free host plants. In addition extracts of basal leaves inhibited more gut proteinase activity than did extracts of middle stem leaves with the same protein content. CONCLUSIONS/ SIGNIFICANCE: Although larvae can minimize the effects of high NaTPI levels by feeding on leaves with high protein and low NaTPI activity, the host plant's endogenous NaTPIs remain an effective defense against M. sexta, inhibiting gut proteinase and affecting larval performance.

  10. Stress inducible proteinase inhibitor diversity in Capsicum annuum

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    Mishra Manasi

    2012-11-01

    Full Text Available Abstract Background Wound-inducible Pin-II Proteinase inhibitors (PIs are one of the important plant serine PIs which have been studied extensively for their structural and functional diversity and relevance in plant defense against insect pests. To explore the functional specialization of an array of Capsicum annuum (L. proteinase inhibitor (CanPIs genes, we studied their expression, processing and tissue-specific distribution under steady-state and induced conditions. Inductions were performed by subjecting C. annuum leaves to various treatments, namely aphid infestation or mechanical wounding followed by treatment with either oral secretion (OS of Helicoverpa armigera or water. Results The elicitation treatments regulated the accumulation of CanPIs corresponding to 4-, 3-, and 2-inhibitory repeat domains (IRDs. Fourty seven different CanPI genes composed of 28 unique IRDs were identified in total along with those reported earlier. The CanPI gene pool either from uninduced or induced leaves was dominated by 3-IRD PIs and trypsin inhibitory domains. Also a major contribution by 4-IRD CanPI genes possessing trypsin and chymotrypsin inhibitor domains was specifically revealed in wounded leaves treated with OS. Wounding displayed the highest number of unique CanPIs while wounding with OS treatment resulted in the high accumulation of specifically CanPI-4, -7 and −10. Characterization of the PI protein activity through two dimensional gel electrophoresis revealed tissue and induction specific patterns. Consistent with transcript abundance, wound plus OS or water treated C. annuum leaves exhibited significantly higher PI activity and isoform diversity contributed by 3- and 4-IRD CanPIs. CanPI accumulation and activity was weakly elicited by aphid infestation yet resulted in the higher expression of CanPI-26, -41 and −43. Conclusions Plants can differentially perceive various kinds of insect attacks and respond appropriately through activating

  11. Secretory leukocyte proteinase inhibitor is a major leukocyte elastase inhibitor in human neutrophils.

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    Sallenave, J M; Si Tahar, M; Cox, G; Chignard, M; Gauldie, J

    1997-06-01

    Secretory leukocyte proteinase inhibitor (SLPI) is the main neutrophil elastase (HLE) inhibitor found in the upper airways during pulmonary inflammation. It has been shown to be synthesized and secreted in vitro by epithelial cells and has been localized in tracheal glands and bronchiolar epithelial cells by immunocytochemistry. In this study, using immunodetection and immunopurification techniques with specific anti-SLPI immunoglobulin G (IgG), we show that SLPI is present as a native 14-kDa molecule in neutrophil cytosol. In addition, we demonstrate that SLPI is the major inhibitor of HLE present in neutrophil cytosol because pre-incubation with specific anti-SLPI IgG was able to inhibit completely the anti-HLE activity of the cytosol. SLPI can be secreted (probably in an inactive form) by neutrophils and its secretion is enhanced when the cells are stimulated with phorbol myristate acetate (PMA). Elafin, an elastase-specific inhibitor, is also present in minute amounts in neutrophil cytosol and its secretion can be up-regulated. The presence of SLPI in the cytosol of neutrophils may serve as a protective screen against proteinases spilling from azurophilic granules. An alternative or supplementary role may be the maintenance of a differentiated phenotype. PMID:9201260

  12. Cloning of a serine proteinase inhibitor from bovine brain: expression in the brain and characterization of its target proteinases.

    Science.gov (United States)

    Nakaya, N; Nishibori, M; Kawabata, M; Saeki, K

    1996-12-01

    A cDNA encoding of the serine proteinase inhibitor (serpin), B-43, was cloned from the cDNA library of the bovine brain. It encoded 378 amino acids, and the MW of the protein was estimated to be 42.6 kDa, which is consistent with that of the native B-43 purified from the bovine brain. The homology search revealed that B-43 belongs to the ovalbumin branch of the serpin superfamily. Among them, B-43 was most homologous to human placental thrombin inhibitor (PI-6) and its murine counterpart, with the amino acid identity of 76% and 71%, respectively. Northern blot analysis showed that the size of the transcript was 1.4 kb, and that the expression of B-43 in the bovine brain varied depending on the brain regions, i.e. a lower level of expression was observed in the cerebral cortex and the hippocampus compared to the level of expression that was observed in the medulla oblongata. [35S]-labeled B-43 protein was synthesized in vitro by using a rabbit reticulocyte lysate system, which formed complexes with proteinases such as thrombin, trypsin, alpha-chymotrypsin, and 7S nerve growth factor (NGF), but not with urokinase or plasmin. These results, together with the immunohistochemical localization of B-43 in astrocytes and in some neurons which was observed in the previous study suggest that B-43 may be involved in the regulation of serine proteinases present in the brain or extravasated from the blood.

  13. Purification and characterization of elastase-specific inhibitor. Sequence homology with mucus proteinase inhibitor.

    Science.gov (United States)

    Sallenave, J M; Ryle, A P

    1991-01-01

    Elastase-specific inhibitor (ESI) was purified from sputum of patients with chronic bronchitis and compared with mucus proteinase inhibitor (MPI, BrI) isolated, without the use of affinity chromatography on an enzyme, from non-purulent sputum of a patient with bronchial carcinoma. The N-terminal sequence of 27 residues of the latter was determined and showed serine as the only N-terminus. The partial N-terminal amino-acid sequence of ESI shows some homology with MPI, especially around the reactive site of MPI for human neutrophil elastase. This region could therefore be the reactive site of ESI. The thermodynamic and kinetic constants of the reactions of ESI with human neutrophil elastase and with porcine pancreatic elastase show that ESI is a fast-acting inhibitor. PMID:2039600

  14. Expression of human α1-proteinase inhibitor in Aspergillus niger

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    Punt Peter J

    2007-10-01

    Full Text Available Abstract Background Human α1-proteinase inhibitor (α1-PI, also known as antitrypsin, is the most abundant serine protease inhibitor (serpin in plasma. Its deficiency is associated with development of progressive, ultimately fatal emphysema. Currently in the United States, α1-PI is available for replacement therapy as an FDA licensed plasma-derived (pd product. However, the plasma source itself is limited; moreover, even with efficient viral inactivation steps used in manufacture of plasma products, the risk of contamination from emerging viruses may still exist. Therefore, recombinant α1-PI (r-α1-PI could provide an attractive alternative. Although r-α1-PI has been produced in several hosts, protein stability in vitro and rapid clearance from the circulation have been major issues, primarily due to absent or altered glycosylation. Results We have explored the possibility of expressing the gene for human α1-PI in the filamentous fungus Aspergillus niger (A. niger, a system reported to be capable of providing more "mammalian-like" glycosylation patterns to secretable proteins than commonly used yeast hosts. Our expression strategy was based on fusion of α1-PI with a strongly expressed, secreted leader protein (glucoamylase G2, separated by dibasic processing site (N-V-I-S-K-R that provides in vivo cleavage. SDS-PAGE, Western blot, ELISA, and α1-PI activity assays enabled us to select the transformant(s secreting a biologically active glycosylated r-α1-PI with yields of up to 12 mg/L. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS analysis further confirmed that molecular mass of the r-α1-PI was similar to that of the pd-α1-PI. In vitro stability of the r-α1-PI from A. niger was tested in comparison with pd-α1-PI reference and non-glycosylated human r-α1-PI from E. coli. Conclusion We examined the suitability of the filamentous fungus A. niger for the expression of the human gene for α1-PI, a medium size

  15. Coffee cysteine proteinases and related inhibitors with high expression during grain maturation and germination

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    Lepelley Maud

    2012-03-01

    Full Text Available Abstract Background Cysteine proteinases perform multiple functions in seeds, including participation in remodelling polypeptides and recycling amino acids during maturation and germination. Currently, few details exist concerning these genes and proteins in coffee. Furthermore, there is limited information on the cysteine proteinase inhibitors which influence the activities of these proteinases. Results Two cysteine proteinase (CP and four cysteine proteinase inhibitor (CPI gene sequences have been identified in coffee with significant expression during the maturation and germination of coffee grain. Detailed expression analysis of the cysteine proteinase genes CcCP1 and CcCP4 in Robusta using quantitative RT-PCR showed that these transcripts accumulate primarily during grain maturation and germination/post germination. The corresponding proteins were expressed in E. coli and purified, but only one, CcCP4, which has a KDDL/KDEL C-terminal sequence, was found to be active after a short acid treatment. QRT-PCR expression analysis of the four cysteine proteinase inhibitor genes in Robusta showed that CcCPI-1 is primarily expressed in developing and germinating grain and CcCPI-4 is very highly expressed during the late post germination period, as well as in mature, but not immature leaves. Transcripts corresponding to CcCPI-2 and CcCPI-3 were detected in most tissues examined at relatively similar, but generally low levels. Conclusions Several cysteine proteinase and cysteine proteinase inhibitor genes with strong, relatively specific expression during coffee grain maturation and germination are presented. The temporal expression of the CcCP1 gene suggests it is involved in modifying proteins during late grain maturation and germination. The expression pattern of CcCP4, and its close identity with KDEL containing CP proteins, implies this proteinase may play a role in protein and/or cell remodelling during late grain germination, and that it is

  16. Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi

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    Leah Theresa Sigle

    2013-09-01

    Full Text Available Sandflies (Diptera: Psychodidae are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2. Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania.

  17. Pest protection conferred by a Beta vulgaris serine proteinase inhibitor gene.

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    Ann C Smigocki

    Full Text Available Proteinase inhibitors provide a means of engineering plant resistance to insect pests. A Beta vulgaris serine proteinase inhibitor gene (BvSTI was fused to the constitutive CaMV35S promoter for over-expression in Nicotiana benthamiana plants to study its effect on lepidopteran insect pests. Independently derived BvSTI transgenic tobacco T2 homozygous progeny were shown to have relatively high BvSTI gene transcript levels. BvSTI-specific polyclonal antibodies cross-reacted with the expected 30 kDA recombinant BvSTI protein on Western blots. In gel trypsin inhibitor activity assays revealed a major clear zone that corresponded to the BvSTI proteinase inhibitor that was not detected in the untransformed control plants. BvSTI-transgenic plants were bioassayed for resistance to five lepidopteran insect pests. Spodoptera frugiperda, S. exigua and Manduca sexta larvae fed BvSTI leaves had significant reductions in larval weights as compared to larvae fed on untransformed leaves. In contrast, larval weights increased relative to the controls when Heliothis virescens and Agrotis ipsilon larvae were fed on BvSTI leaves. As the larvae entered the pupal stage, pupal sizes reflected the overall larval weights. Some developmental abnormalities of the pupae and emerging moths were noted. These findings suggest that the sugar beet BvSTI gene may prove useful for effective control of several different lepidopteran insect pests in genetically modified tobacco and other plants. The sugar beet serine proteinase inhibitor may be more effective for insect control because sugar beet is cropped in restricted geographical areas thus limiting the exposure of the insects to sugar beet proteinase inhibitors and build up of non-sensitive midgut proteases.

  18. Insect and wound induced GUS gene expression from a Beta vulgaris proteinase inhibitor gene promoter

    Science.gov (United States)

    Inducible gene promoters that are specifically activated by pathogen invasion or insect pest attack are needed for effective expression of resistance genes to control plant diseases. In the present study, a promoter from a serine proteinase inhibitor gene (BvSTI) shown to be up-regulated in resist...

  19. Successful treatment of murine muscular dystrophy with the proteinase inhibitor leupeptin.

    OpenAIRE

    Sher, J H; Stracher, A.; Shafiq, S A; Hardy-Stashin, J

    1981-01-01

    Mice with genetic muscular dystrophy were treated with intraperitoneal injections of the proteinase inhibitor leupeptin, beginning before the onset of weakness. A significant number of the treated animals failed to develop histological evidence of dystrophy, compared with controls. Leupeptin treatment prevented (or delayed) the onset of muscular dystrophy in this experiment.

  20. Solution structure of the squash aspartic acid proteinase inhibitor (SQAPI) and mutational analysis of pepsin inhibition.

    Science.gov (United States)

    Headey, Stephen J; Macaskill, Ursula K; Wright, Michele A; Claridge, Jolyon K; Edwards, Patrick J B; Farley, Peter C; Christeller, John T; Laing, William A; Pascal, Steven M

    2010-08-27

    The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NMR and show that SQAPI shares the same fold as a plant cystatin. The structure is characterized by a four-strand anti-parallel beta-sheet gripping an alpha-helix in an analogous manner to fingers of a hand gripping a tennis racquet. Truncation and site-specific mutagenesis revealed that the unstructured N terminus and the loop connecting beta-strands 1 and 2 are important for pepsin inhibition, but the loop connecting strands 3 and 4 is not. Using ambiguous restraints based on the mutagenesis results, SQAPI was then docked computationally to pepsin. The resulting model places the N-terminal strand of SQAPI in the S' side of the substrate binding cleft, whereas the first SQAPI loop binds on the S side of the cleft. The backbone of SQAPI does not interact with the pepsin catalytic Asp(32)-Asp(215) diad, thus avoiding cleavage. The data show that SQAPI does share homologous structural elements with cystatin and appears to retain a similar protease inhibitory mechanism despite its different target. This strongly supports our hypothesis that SQAPI evolved from an ancestral cystatin.

  1. Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI) in Potyviruses Resistance

    OpenAIRE

    Chia-Wei Lin; Mei-Hsiu Su; Yu-Tsung Lin; Chien-Hung Chung; Hsin-Mei Ku

    2015-01-01

    Proteinase inhibitors are ubiquitous proteins that block the active center or interact allosterically with proteinases and are involved in plant physiological processes and defense responses to biotic and abiotic stresses. The CmSPI gene identified from Cucumis metuliferus encodes a serine type PI (8 kDa) that belongs to potato I type family. To evaluate the effect of silencing CmSPI gene on Papaya ringspot virus resistance, RNA interference (RNAi) with an inter-space hairpin RNA (ihpRNA) con...

  2. A sycamore cell wall polysaccharide and a chemically related tomato leaf polysaccharide possess similar proteinase inhibitor-inducing activities.

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    Ryan, C A; Bishop, P; Pearce, G

    1981-09-01

    A large pectic polysaccharide, called rhamnogalacturonan I, that is solubilized by a fungal endo-alpha-1,4-polygalacturonase from the purified walls of suspension-cultured sycamore cells possesses proteinase inhibitor-inducing activity similar to that of the proteinase inhibitor-inducing factor, a pectic-like oligosaccharide fraction isolated from tomato leaves. This suggests that the proteinase inhibitor-inducing activity resides in particular polysaccharide fragments which can be released when plant cell walls are exposed to appropriate enzyme degradation as a result of either wounding or pest attack.

  3. Three low molecular weight cysteine proteinase inhibitors of human seminal fluid: purification and enzyme kinetic properties.

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    Yadav, Vikash Kumar; Chhikara, Nirmal; Gill, Kamaldeep; Dey, Sharmistha; Singh, Sarman; Yadav, Savita

    2013-08-01

    The cystatins form a superfamily of structurally related proteins with highly conserved structural folds. They are all potent, reversible, competitive inhibitors of cysteine proteinases (CPs). Proteins from this group present differences in proteinase inhibition despite their high level of structural similarities. In this study, three cysteine proteinase inhibitors (CPIs) of low molecular weight were isolated from human seminal fluid (HSF) by affinity chromatography on carboxymethyl (CM)-papain-Sepharose column, purified using various chromatographic procedures and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Matrix-assisted laser desorption-ionization-time-of flight-mass spectrometry (MALDI-TOF-MS) identified these proteins as cystatin 9, cystatin SN, and SAP-1 (an N-terminal truncated form of cystatin S). All three CPIs suppressed the activity of papain potentially and showed remarkable heat stability. Interestingly SAP-1 also inhibits the activity of trypsin, chymotrypsin, pepsin, and PSA (prostate specific antigen) and acts as a cross-class protease inhibitor in in vitro studies. Using Surface Plasmon Resonance, we have also observed that SAP-1 shows a significant binding with all these proteases. These studies suggest that SAP-1 is a cross-class inhibitor that may regulate activity of various classes of proteases within the reproductive systems. To our knowledge, this is the first report about purification of CPIs from HSF; the identification of such proteins could provide better insights into the physiological processes and offer intimation for further research. PMID:23619703

  4. Primary structure of a cysteine proteinase inhibitor from the fruit of avocado (Persea americana Mill).

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    Kimura, M; Ikeda, T; Fukumoto, D; Yamasaki, N; Yonekura, M

    1995-12-01

    The complete amino acid sequence of a proteinaceous cysteine proteinase inhibitor from the fruit of avocado (avocado cystatin) is presented. The protein consists of 100 amino acid residues and has a molecular mass of 11,300 Da. Comparison of this sequence with sequences of plant cysteine proteinase inhibitors (phytocystatins), including oryzacystatins I and II from rice seeds, cowpea cystatin, and corn cystatin, showed that the avocado cystatin molecule has 60% and 54% residues identical with the two forms of the rice seed proteins, oryzacystatins I and II, respectively, and 64% and 63% with the cowpea and corn proteins, respectively. The totally conserved sequence, Gln-Val-Val-Ala-Gly, among several of the animal cystatins as well as phytocystatins, is at positions 47-51 in the avocado cystatin molecule.

  5. In situ localization of proteinase inhibitor mRNA in rice plant challenged by brown planthopper

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Proteinase inhibitor (PI) mRNA was localized by in situ hybridization in tissue sections of root, stem and leaf of the resistant rice (B5) plant fed by brown planthopper nymphs. In the rice material without BPH feeding, PI gene was expressed in the root, stem and leaf, while the abundance of PI mRNA was low. In the rice material fed by BPH, PI gene was expressed substantially in the parenchyma of rice stem and leaf, but weakly in the root. The results indicated that the PI gene was up-regulated in the rice plant challenged by brown planthopper. For the first time, we reported the expression changes of proteinase inhibitor gene in plant which was infested by a piercing/sucking insect.

  6. Inhibition of tryptase and chymase induced nucleated cell infiltration by proteinase inhibitors

    Institute of Scientific and Technical Information of China (English)

    Shao-heng HE; Han-qiu CHEN; Jian ZHENG

    2004-01-01

    AIM: To investigate the ability of proteinase inhibitors to modulate nucleated cell infiltration into the peritoneum of mice induced by tryptase and chymase. METHODS: Human lung tryptase and skin chymase were purified by a similar procedure involving high salt extraction, heparin agarose affinity chromatography followed by S-200 Sephacryl gel filtration chromatography. The actions of proteinase inhibitors on tryptase and chymase induced nucleated cell accumulation were examined with a mouse peritoneum model. RESULTS: A selective chymase inhibitor Z-Ile-GluPro-Phe-CO2Me (ZIGPPF) was able to inhibit approximately 90% neutrophil, 73% eosinophil, 87% lymphocyte and 60% macrophage accumulation induced by chymase at 16 h following injection. Soy bean trypsin inhibitor (SBTI), chymostatin, and α1-antitrypsin showed slightly less potency than ZIGPPF in inhibition of the actions of chymase. While all tryptase inhibitors tested were able to inhibit neutrophil, eosinophil, and macrophage accumulation provoked by tryptase at 16 h following injection, only leupeptin, APC366, and aprotinin were capable of inhibiting tryptase induced lymphocyte accumulation. The inhibitiors of tryptase tested were also able to inhibit tryptase induced neutrophil and eosinophil accumulation at 6 h following injection. When being injected alone, all inhibitors of chymase and tryptase at the concentrations tested by themselves had no significant effect on the accumulation of nucleated cells in the peritoneum of mice at both 6 h and 16 h. CONCLUSION: Proteinase inhibitors significantly inhibited tryptase and chymase-induced nucleated cell accumulation in vivo, and therefore they are likely to be developed as a novel class of anti-inflammatory drugs.

  7. Enzymatic response of the eucalypt defoliator Thyrinteina arnobia (Stoll) (Lepidoptera: Geometridae) to a bis-benzamidine proteinase Inhibitor. i.

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    Marinho-Prado, Jeanne Scardini; Lourenção, A L; Guedes, R N C; Pallini, A; Oliveira, J A; Oliveira, M G A

    2012-10-01

    Ingestion of proteinase inhibitors leads to hyperproduction of digestive proteinases, limiting the bioavailability of essential amino acids for protein synthesis, which affects insect growth and development. However, the effects of proteinase inhibitors on digestive enzymes can lead to an adaptive response by the insect. In here, we assessed the biochemical response of midgut proteinases from the eucalypt defoliator Thyrinteina arnobia (Stoll) to different concentrations of berenil, a bis-benzamidine proteinase inhibitor, on eucalyptus. Eucalyptus leaves were immersed in berenil solutions at different concentrations and fed to larvae of T. arnobia. Mortality was assessed daily. The proteolytic activity in the midgut of T. arnobia was assessed after feeding on plants sprayed with aqueous solutions of berenil, fed to fifth instars of T. arnobia for 48 h before midgut removal for enzymatic assays. Larvae of T. arnobia were able to overcome the effects of the lowest berenil concentrations by increasing their trypsin-like activity, but not as berenil concentration increased, despite the fact that the highest berenil concentration resulted in overproduction of trypsin-like proteinases. Berenil also prevented the increase of the cysteine proteinases activity in response to trypsin inhibition. PMID:23950094

  8. Isolation, characterization and cDNA sequencing of a Kazal family proteinase inhibitor from seminal plasma of turkey (Meleagris gallopavo).

    Science.gov (United States)

    Słowińska, Mariola; Olczak, Mariusz; Wojtczak, Mariola; Glogowski, Jan; Jankowski, Jan; Watorek, Wiesław; Amarowicz, Ryszard; Ciereszko, Andrzej

    2008-06-01

    The turkey reproductive tract and seminal plasma contain a serine proteinase inhibitor that seems to be unique for the reproductive tract. Our experimental objective was to isolate, characterize and cDNA sequence the Kazal family proteinase inhibitor from turkey seminal plasma and testis. Seminal plasma contains two forms of a Kazal family inhibitor: virgin (Ia) represented by an inhibitor of moderate electrophoretic migration rate (present also in the testis) and modified (Ib, a split peptide bond) represented by an inhibitor with a fast migration rate. The inhibitor from the seminal plasma was purified by affinity, ion-exchange and reverse phase chromatography. The testis inhibitor was purified by affinity and ion-exchange chromatography. N-terminal Edman sequencing of the two seminal plasma inhibitors and testis inhibitor were identical. This sequence was used to construct primers and obtain a cDNA sequence from the testis. Analysis of a cDNA sequence indicated that turkey proteinase inhibitor belongs to Kazal family inhibitors (pancreatic secretory trypsin inhibitors, mammalian acrosin inhibitors) and caltrin. The turkey seminal plasma Kazal inhibitor belongs to low molecular mass inhibitors and is characterized by a high value of the equilibrium association constant for inhibitor/trypsin complexes.

  9. A trypsin-like proteinase in the midgut of Ectomyelois ceratoniae Zeller (Lepidoptera: Pyralidae): purification, characterization, and host plant inhibitors.

    Science.gov (United States)

    Ranjbar, Mina; Zibaee, Arash; Sendi, Jalal Jalali

    2014-01-01

    A trypsin-like proteinase was purified and characterized in the midgut of Ectomyelois ceratoniae. A purification process that used Sepharyl G-100 and DEAE-cellulose fast flow chromatographies revealed a proteinase with specific activity of 66.7 μmol/min/mg protein, recovery of 27.04 and purification fold of 23.35. Molecular weight of the purified protein was found to be 35.8 kDa. Optimal pH and temperature were obtained 9 and 20°C for the purified trypsin proteinase, respectively. The purified enzyme was significantly inhibited by PMSF, TLCK, and SBTI as specific inhibitors of trypsins in which TLCK showed the highest inhibitory effect. Trypsin proteinase inhibitors were extracted from four varieties of pomegranate including Brait, Torsh-Sabz, May-Khosh, and Shirin by ion exchange chromatography. It was found that fractions 17-20 of Brait; fractions 18 and 21-26 of Torsh-Sabz; fractions 1-7, 11-17, and 19-21 of May-Khosh and fraction 8 for Shirin showed presence of trypsin inhibitor in these host. Comparison of their inhibitory effects on the purified trypsin proteinase of E. ceratoniae demonstrated that fractions from May-khosh variety had the highest effect on the enzyme among other extracted fractions. Characterization of serine proteinases of insects mainly trypsins is one of the promising methods to decrease population and damages via extracting their inhibitors and providing resistant varieties.

  10. Secretory leukocyte proteinase inhibitor is preferentially increased in patients with acute respiratory distress syndrome.

    Science.gov (United States)

    Sallenave, J M; Donnelly, S C; Grant, I S; Robertson, C; Gauldie, J; Haslett, C

    1999-05-01

    Inappropriate release of proteases from inflammatory and stromal cells can lead to destruction of the lung parenchyma. Antiproteinases such as alpha-1-proteinase inhibitor (alpha1-Pi), secretory leukocyte proteinase inhibitor (SLPI) and elastase-specific inhibitor (elafin) control excess production of human neutrophil elastase. In the present study, the concentrations of alpha1-Pi, SLPI and elafin found in bronchoalveolar lavage (BAL) fluid from control subjects, patients at risk of developing acute respiratory distress syndrome (ARDS) and patients with established ARDS were determined. Levels of all three inhibitors were raised in patients compared with normal subjects. SLPI was increased in the group of patients who were at risk of ARDS and went on to develop the condition, compared with the "at-risk" group who did not progress to ARDS (p=0.0083). Alpha1-Pi and elafin levels were similar in these two populations. In patients with established ARDS, both alpha1-Pi and SLPI levels were significantly increased, compared to patients at risk of ARDS who did (p=0.0089) or did not (p=0.0003) progress to ARDS. The finding of increased antiproteinases shortly before the development of acute respiratory distress syndrome provide further evidence for enhanced inflammation prior to clinical disease. PMID:10414400

  11. Differential gene expression for suicide-substrate serine proteinase inhibitors (serpins) in vegetative and grain tissues of barley

    DEFF Research Database (Denmark)

    Roberts, T.H.; Marttila, S.; Rasmussen, S.K.;

    2003-01-01

    Proteins of the serpin superfamily (similar to43 kDa) from mature cereal grains are in vitro suicide-substrate inhibitors of specific mammalian serine proteinases of the chymotrypsin family. However, unlike the 'standard-mechanism' serine proteinase inhibitors (kDa), the biological functions...... of plant serpins are unknown. Expression studies of genes encoding members of three subfamilies of serpins (BSZx, BSZ4 and BSZ7) in developing grain and vegetative tissues of barley (Hordeum vulgare L.) showed that transcripts encoding BSZx, which inhibits distinct proteinases at overlapping reactive...... their irreversible inhibitory mechanism in the inhibition of exogenous proteinases capable of breaking down seed storage proteins, and in the defence of specific cell types in vegetative tissues....

  12. Purification, crystallization and preliminary crystallographic studies of a Kunitz-type proteinase inhibitor from tamarind (Tamarindus indica) seeds.

    Science.gov (United States)

    Patil, Dipak N; Chaudhry, Anshul; Sharma, Ashwani K; Tomar, Shailly; Kumar, Pravindra

    2009-07-01

    A Kunitz-type proteinase inhibitor has been purified from tamarind (Tamarindus indica) seeds. SDS-PAGE analysis of a purified sample showed a homogeneous band corresponding to a molecular weight of 21 kDa. The protein was identified as a Kunitz-type proteinase inhibitor based on N-terminal amino-acid sequence analysis. It was crystallized by the vapour-diffusion method using PEG 6000. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 37.2, b = 77.1, c = 129.1 A. Diffraction data were collected to a resolution of 2.7 A. Preliminary crystallographic analysis indicated the presence of one proteinase inhibitor molecule in the asymmetric unit, with a solvent content of 44%.

  13. Secretion of mucus proteinase inhibitor and elafin by Clara cell and type II pneumocyte cell lines.

    Science.gov (United States)

    Sallenave, J M; Silva, A; Marsden, M E; Ryle, A P

    1993-02-01

    The regulation of proteinases secreted by neutrophils is very important for the prevention of tissue injury. We recently described the isolation of elafin from bronchial secretions, a new elastase-specific inhibitor that is also found in the skin of patients with psoriasis. In this study, we investigated the secretion of elafin and mucus proteinase inhibitor (MPI), another inhibitor showing sequence similarity with elafin, in two lung carcinoma cell lines, NCI-H322 and A549, which have features of Clara cells and type II alveolar cells, respectively. The results presented show that the two inhibitors are produced when the cells are cultured either in serum-free or in serum-containing media. MPI was detected immunologically as a unique molecule of M(r) 14 kD, in accordance with previous studies. Conversely, one or two elafin-immunoreactive species were detected depending on the cell line: a 12- to 14-kD species was observed in the A549 cell line, regardless of the culture conditions, whereas in the NCI-H322 cell line we detected a 6-kD species in serum-containing (10% fetal calf serum) conditions and a 12- to 14-kD species in serum-free conditions. The 12- to 14-kD molecule probably represents an active precursor of elafin. Whether the cleavage of the 12- to 14-kD precursor giving rise to the elafin molecule is of any physiologic significance is not known. In showing for the first time that MPI and elafin (and its precursor) are secreted by the A549 cell line, this report implicates the type II alveolar cell in the defense of the peripheral lung against the neutrophil elastase secreted during inflammation. PMID:8427705

  14. Hepatocyte growth factor activator is a potential target proteinase for Kazal-type inhibitor in turkey (Meleagris gallopavo) seminal plasma.

    Science.gov (United States)

    Słowińska, Mariola; Bukowska, Joanna; Hejmej, Anna; Bilińska, Barbara; Kozłowski, Krzysztof; Jankowski, Jan; Ciereszko, Andrzej

    2015-08-01

    A peculiar characteristic of turkey seminal plasma is the increased activity of serine proteinases. It is of interest if the single-domain Kazal-type inhibitor controls the activity of turkey seminal plasma proteinases. Pure preparations of the Kazal-type inhibitor and anti-Kazal-type inhibitor monospecific immunoglobulin Gs were used as ligands in affinity chromatography for proteinase isolation from turkey seminal plasma. Gene expression and the immunohistochemical detection of the single-domain Kazal-type inhibitor in the reproductive tract of turkey toms are described. The hepatocyte growth factor activator (HGFA) was identified in the binding fraction in affinity chromatography. Hepatocyte growth factor activator activity was inhibited by the Kazal-type inhibitor in a dose-dependent manner. This protease was a primary physiological target for the single-domain Kazal-type inhibitor. Numerous proteoforms of HGFA were present in turkey seminal plasma, and phosphorylation was the primary posttranslational modification of HGFA. In addition to HGFA, acrosin was a target proteinase for the single-domain Kazal-type inhibitor. In seminal plasma, acrosin was present only in complexes with the Kazal-type inhibitor and was not present as a free enzyme. The single-domain Kazal-type inhibitor was specific for the reproductive tract. The germ cell-specific expression of Kazal-type inhibitors in the testis indicated an important function in spermatogenesis; secretion by the epithelial cells of the epididymis and the ductus deferens indicated that the Kazal-type inhibitor was an important factor involved in the changes in sperm membranes during maturation and in the maintenance of the microenvironment in which sperm maturation occurred and sperm was stored. The role of HGFA in these processes remains to be established.

  15. Effects of pH on the association between the inhibitor cystatin and the proteinase chymopapain.

    Science.gov (United States)

    Reyes-Espinosa, Francisco; Arroyo-Reyna, Alfonso; Garcia-Gutierrez, Ponciano; Serratos, Iris N; Zubillaga, Rafael A

    2014-01-01

    Cysteine proteinases are involved in many aspects of physiological regulation. In humans, some cathepsins have shown another function in addition to their role as lysosomal proteases in intracellular protein degradation; they have been implicated in the pathogenesis of several heart and blood vessel diseases and in cancer development. In this work, we present a fluorometric and computational study of the binding of one representative plant cysteine proteinase, chymopapain, to one of the most studied inhibitors of these proteinases: chicken cystatin. The binding equilibrium constant, Kb, was determined in the pH range between 3.5 and 10.0, revealing a maximum in the affinity at pH 9.0. We constructed an atomic model for the chymopapain-cystatin dimer by docking the individual 3D protein structures; subsequently, the model was refined using a 100 ns NPT molecular dynamics simulation in explicit water. Upon scrutiny of this model, we identified 14 ionizing residues at the interface of the complex using a cutoff distance of 5.0 Å. Using the pKa values predicted with PROPKA and a modified proton-linkage model, we performed a regression analysis on our data to obtain the composite pKavalues for three isoacidic residues. We also calculated the electrostatic component of the binding energy (ΔGb,elec) at different pH values using an implicit solvent model and APBS software. The pH profile of this calculated energy compares well with the experimentally obtained binding energy, ΔGb. We propose that the residues that form an interchain ionic pair, Lys139A from chymopapain and Glu19B from cystatin, as well as Tyr61A and Tyr67A from chymopapain are the main residues responsible for the observed pH dependence in the chymopapain- cystatin affinity.

  16. Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI) in Potyviruses Resistance.

    Science.gov (United States)

    Lin, Chia-Wei; Su, Mei-Hsiu; Lin, Yu-Tsung; Chung, Chien-Hung; Ku, Hsin-Mei

    2015-07-01

    Proteinase inhibitors are ubiquitous proteins that block the active center or interact allosterically with proteinases and are involved in plant physiological processes and defense responses to biotic and abiotic stresses. The CmSPI gene identified from Cucumis metuliferus encodes a serine type PI (8 kDa) that belongs to potato I type family. To evaluate the effect of silencing CmSPI gene on Papaya ringspot virus resistance, RNA interference (RNAi) with an inter-space hairpin RNA (ihpRNA) construct was introduced into a PRSV-resistant C. metuliferus line. CmSPI was down-regulated in CmSPI RNAi transgenic lines in which synchronously PRSV symptoms were evident at 21 day post inoculation. Alternatively, heterogeneous expression of CmSPI in Nicotiana benthamiana was also conducted and showed that CmSPI can provide resistance to Potato virus Y, another member of Potyvirus, in transgenic N. benthamiana lines. This study demonstrated that CmSPI plays an important role in resistant function against potyviruses in C. metuliferus and N. benthamiana. PMID:26184285

  17. Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI in Potyviruses Resistance

    Directory of Open Access Journals (Sweden)

    Chia-Wei Lin

    2015-07-01

    Full Text Available Proteinase inhibitors are ubiquitous proteins that block the active center or interact allosterically with proteinases and are involved in plant physiological processes and defense responses to biotic and abiotic stresses. The CmSPI gene identified from Cucumis metuliferus encodes a serine type PI (8 kDa that belongs to potato I type family. To evaluate the effect of silencing CmSPI gene on Papaya ringspot virus resistance, RNA interference (RNAi with an inter-space hairpin RNA (ihpRNA construct was introduced into a PRSV-resistant C. metuliferus line. CmSPI was down-regulated in CmSPI RNAi transgenic lines in which synchronously PRSV symptoms were evident at 21 day post inoculation. Alternatively, heterogeneous expression of CmSPI in Nicotiana benthamiana was also conducted and showed that CmSPI can provide resistance to Potato virus Y, another member of Potyvirus, in transgenic N. benthamiana lines. This study demonstrated that CmSPI plays an important role in resistant function against potyviruses in C. metuliferus and N. benthamiana.

  18. The role of secretory leukocyte proteinase inhibitor and elafin (elastase-specific inhibitor/skin-derived antileukoprotease) as alarm antiproteinases in inflammatory lung disease

    OpenAIRE

    Sallenave Jean-Michel

    2000-01-01

    Abstract Secretory leukocyte proteinase inhibitor and elafin are two low-molecular-mass elastase inhibitors that are mainly synthesized locally at mucosal sites. It is thought that their physicochemical properties allow them to efficiently inhibit target enzymes, such as neutrophil elastase, released into the interstitium. Historically, in the lung, these inhibitors were first purified from secretions of patients with chronic obstructive pulmonary disease and cystic fibrosis. This suggested t...

  19. Transgenic tobacco plants harboring tomato proteinase inhibitor II gene and their insect resistance

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The plant expression vectors pBCT2 and pBT2 were constructed with the cDNA sequence (tin2) and genomic DNA sequence (tin2i) of tomato proteinase inhibitor II gene respectively. Then the two expression vectors were transferred into tobacco via the Agrobacterium tumefaciens strain LBA4404, and transgenic tobacco plants were generated. Molecular analysis and trypsin activity assay showed that both cDNA and genomic DNA were expressed properly in the transgenic plants. Insecticidal activities in these transgenic plants indicated that transgenic tobacco plants carrying tin2i sequence were more resistant to 2-instar larvae of Heliothis armigera Hubner than those carrying tin2 sequence. Therefore the intron of tin2i sequence might be a contributor to insecticidal activity of the transgenic tobacco.

  20. Antifeedant effects of proteinase inhibitors on feeding behaviors of adult western corn rootworm (Diabrotica virgifera virgifera).

    Science.gov (United States)

    Kim, Jae Hak; Mullin, Christopher A

    2003-04-01

    Low-molecular-weight peptidyl proteinase inhibitors (PIs) including leupeptin, calpain inhibitor I, and calpeptin were found to be potent antifeedants for adult western corn rootworm (WCR) against the phagostimulation of cucurbitacin B (Cuc B) or a corn pollen extract (CPE). Leupeptin was the strongest (ED50 = 0.36 and 0.55 nmol/disk for Cuc B and CPE, respectively) among PIs tested with an antifeedant potency much stronger than the steroid progesterone (ED50 = 2.29 and 5.05 nmol/disk for Cuc B and CPE, respectively), but slightly less than the reference alkaloid, strychnine (ED50 = 0.17 and 0.37 nmol/disk for Cuc B and CPE, respectively). All active PIs contain a di- or tripeptidyl aldehyde moiety, indicating that PIs exert their antifeedant effects by covalent interaction with putative sulfhydryl (SH) groups on taste receptors as do these PIs with cysteine proteinases. However, opposite inhibition potency against Cuc B versus CPE by two thiol-group reducing agents, DTT and L-cysteine, and the results with other cysteine-modifying reagents obscure the net functional role of SH groups at WCR taste chemoreceptors. Surprisingly, the model phagostimulant for diabroticites, Cuc B, was more easily counteracted by these feeding deterrents than the stimulants present in CPE. Three-dimensional structure-antifeedant relationships for the PIs suggest that a novel taste chemoreception mechanism exists for these peptidyl aldehydes or that they fit partially into a strychnine binding pocket on protein chemoreceptors. Favorable economic benefit may be achieved if PIs are discovered to be useful in adult WCR control, since both pre- and postingestive sites would be targeted. PMID:12775144

  1. Serine leucocyte proteinase inhibitor-treated monocyte inhibits human CD4(+) lymphocyte proliferation.

    Science.gov (United States)

    Guerrieri, Diego; Tateosian, Nancy L; Maffía, Paulo C; Reiteri, Romina M; Amiano, Nicolás O; Costa, María J; Villalonga, Ximena; Sanchez, Mercedes L; Estein, Silvia M; Garcia, Verónica E; Sallenave, Jean-Michel; Chuluyan, Héctor E

    2011-08-01

    Serine leucocyte proteinase inhibitor (SLPI) is the main serine proteinase inhibitor produced by epithelial cells and has been shown to be a pleiotropic molecule with anti-inflammatory and microbicidal activities. However, the role of SLPI on the adaptive immune response is not well established. Therefore, we evaluated the effect of SLPI on lymphocyte proliferation and cytokine production. Human peripheral blood mononuclear cells (PBMC) were treated with mitogens plus SLPI and proliferation was assessed by [(3) H]thymidine uptake. The SLPI decreased the lymphocyte proliferation induced by interleukin-2 (IL-2) or OKT3 monoclonal antibodies in a dose-dependent manner. Inhibition was not observed when depleting monocytes from the PBMC and it was restored by adding monocytes and SLPI. SLPI-treated monocyte slightly decreased MHC II and increased CD18 expression, and secreted greater amounts of IL-4, IL-6 and IL-10 in the cell culture supernatants. SLPI-treated monocyte culture supernatant inhibited the CD4(+) lymphocyte proliferation but did not affect the proliferation of CD8(+) cells. Moreover, IL-2 increased T-bet expression and the presence of SLPI significantly decreased it. Finally, SLPI-treated monocyte culture supernatant dramatically decreased interferon-γ but increased IL-4, IL-6 and IL-10 in the presence of IL-2-treated T cells. Our results demonstrate that SLPI target monocytes, which in turn inhibit CD4 lymphocyte proliferation and T helper type 1 cytokine secretion. Overall, these results suggest that SLPI is an alarm protein that modulates not only the innate immune response but also the adaptive immune response. PMID:21574992

  2. Serine leucocyte proteinase inhibitor-treated monocyte inhibits human CD4+ lymphocyte proliferation

    Science.gov (United States)

    Guerrieri, Diego; Tateosian, Nancy L; Maffía, Paulo C; Reiteri, Romina M; Amiano, Nicolás O; Costa, María J; Villalonga, Ximena; Sanchez, Mercedes L; Estein, Silvia M; Garcia, Verónica E; Sallenave, Jean-Michel; Chuluyan, Héctor E

    2011-01-01

    Serine leucocyte proteinase inhibitor (SLPI) is the main serine proteinase inhibitor produced by epithelial cells and has been shown to be a pleiotropic molecule with anti-inflammatory and microbicidal activities. However, the role of SLPI on the adaptive immune response is not well established. Therefore, we evaluated the effect of SLPI on lymphocyte proliferation and cytokine production. Human peripheral blood mononuclear cells (PBMC) were treated with mitogens plus SLPI and proliferation was assessed by [3H]thymidine uptake. The SLPI decreased the lymphocyte proliferation induced by interleukin-2 (IL-2) or OKT3 monoclonal antibodies in a dose-dependent manner. Inhibition was not observed when depleting monocytes from the PBMC and it was restored by adding monocytes and SLPI. SLPI-treated monocyte slightly decreased MHC II and increased CD18 expression, and secreted greater amounts of IL-4, IL-6 and IL-10 in the cell culture supernatants. SLPI-treated monocyte culture supernatant inhibited the CD4+ lymphocyte proliferation but did not affect the proliferation of CD8+ cells. Moreover, IL-2 increased T-bet expression and the presence of SLPI significantly decreased it. Finally, SLPI-treated monocyte culture supernatant dramatically decreased interferon-γ but increased IL-4, IL-6 and IL-10 in the presence of IL-2-treated T cells. Our results demonstrate that SLPI target monocytes, which in turn inhibit CD4 lymphocyte proliferation and T helper type 1 cytokine secretion. Overall, these results suggest that SLPI is an alarm protein that modulates not only the innate immune response but also the adaptive immune response. PMID:21574992

  3. Synthesis of the proteinase inhibitor LEKTI domain 6 by the fragment condensation method and regioselective disulfide bond formation.

    Science.gov (United States)

    Vasileiou, Zoe; Barlos, Kostas K; Gatos, Dimitrios; Adermann, Knut; Deraison, Celine; Barlos, Kleomenis

    2010-01-01

    Proteinase inhibitors are of high pharmaceutical interest and are drug candidates for a variety of indications. Specific kallikrein inhibitors are important for their antitumor activity and their potential application to the treatment of skin diseases. In this study we describe the synthesis of domain 6 of the kallikrein inhibitor Lympho-Epithilial Kazal-Type Inhibitor (LEKTI) by the fragment condensation method and site-directed cystine bridge formation. To obtain the linear LEKTI precursor, the condensation was best performed in solution, coupling the protected fragment 1-22 to 23-68. This method yielded LEKTI domain 6 of high purity and equipotent to the recombinantly produced peptide. PMID:20069636

  4. A serine proteinase inhibitor isolated from Tamarindus indica seeds and its effects on the release of human neutrophil elastase.

    Science.gov (United States)

    Fook, J M S L L; Macedo, L L P; Moura, G E D D; Teixeira, F M; Oliveira, A S; Queiroz, A F S; Sales, M P

    2005-05-01

    Proteinaceous inhibitors with high inhibitory activities against human neutrophil elastase (HNE) were found in seeds of the Tamarind tree (Tamarindus indica). A serine proteinase inhibitor denoted PG50 was purified using ammonium sulphate and acetone precipitation followed by Sephacryl S-300 and Sephadex G-50 gel filtration chromatographies. Inhibitor PG50 showed a Mr of 14.9 K on Sephadex G-50 calibrated column and a Mr of 11.6 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PG50 had selective activity while cysteine proteinases (papain and bromelain) and serine proteinases (porcine pancreatic elastase and bovine chymotrypsin) were not inhibited, it was strongly effective against serine proteinases such as bovine trypsin and isolated human neutrophil elastase. The IC50 value was determined to be 55.96 microg.mL-1. PG50 showed neither cytotoxic nor haemolytic activity on human blood cells. After pre-incubation of PG50 with cytochalasin B, the exocytosis of elastase was initiated using PAF and fMLP. PG50 exhibited different inhibition on elastase release by PAF, at 44.6% and on release by fMLP, at 28.4%. These results showed that PG50 preferentially affected elastase release by PAF stimuli and this may indicate selective inhibition on PAF receptors.

  5. Solution structure of PMP-C: a new fold in the group of small serine proteinase inhibitors.

    Science.gov (United States)

    Mer, G; Hietter, H; Kellenberger, C; Renatus, M; Luu, B; Lefèvre, J F

    1996-04-26

    The solution structure and the disulfide pairings of a 36-residue proteinase inhibitor isolated from the insect Locusta migratoria have been determined using NMR spectroscopy and simulated annealing calculations. The peptide, termed PMP-C, was previously shown to inhibit bovine alpha-chymotrypsin as well as human leukocyte elastase, and was also found to block high-voltage-activated Ca2+ currents in rat sensory neurones. PMP-C has a prolate ellipsoid shape and adopts a tertiary fold hitherto unobserved in the large group of small "canonical" proteinase inhibitors. The over-all fold consists mainly of three strands arranged in a right-handed twisted, antiparallel, beta-sheet that demarcates a cavity, together with a linear amino-terminal segment oriented almost perpendicular to the three strands of the beta-sheet. Inside the cavity a phenyl ring constitutes the centre of a hydrophobic core. The proteinase binding loop is located in the carboxy-terminal part of the molecule, between two cysteine residues involved in disulfide bridges. Its conformation resembles that found in other small canonical proteinase inhibitors. A comparison of PMP-C structure with the recently published solution structure of the related peptide PMP-D2 shows that the most significant differences are complementary changes involved in the stabilization of similar folds. This comparison led us to review the structure of PMP-D2 and to identify two salt bridges in PMP-D2.

  6. Isolation, cloning and structural characterisation of boophilin, a multifunctional Kunitz-type proteinase inhibitor from the cattle tick.

    Directory of Open Access Journals (Sweden)

    Sandra Macedo-Ribeiro

    Full Text Available Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine alpha-thrombin.boophilin complex, refined at 2.35 A resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S(1 pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9 degrees and is displaced by 6 A, while the C-terminal domain rotates almost 6 degrees accompanied by a 3 A displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P(1 residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin.boophilin.trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo.

  7. Potato type I and II proteinase inhibitors: modulating plant physiology and host resistance.

    Science.gov (United States)

    Turra, David; Lorito, Matteo

    2011-08-01

    Serine protease inhibitors (PIs) are a large and complex group of plant proteins. Members of the potato type I (Pin1) and II (Pin2) proteinase inhibitor families are among the first and most extensively characterized plant PIs. Many insects and phytopathogenic microorganisms use intracellular and extracellular serine proteases playing important roles in pathogenesis. Plants, however, are able to fight these pathogens through the activation of an intricate defence system that leads to the accumulation of various PIs, including Pin1 and Pin2. Several transgenic plants over-expressing members of the Pin1 and Pin2 families have been obtained in the last twenty years and their enhanced defensive capabilities demonstrated against insects, fungi and bacteria. Furthermore, Pin1 and Pin2 genetically engineered plants showed altered regulation of different plant physiological processes (e.g., dehydratation response, programmed cell death, plant growth, trichome density and branching), supporting an endogenous role in various plant species in addition to the well established defensive one. This review summarizes the current knowledge about Pin1 and Pin2 structure, the role of these proteins in plant defence and physiology, and their potential exploitation in biotechnology. PMID:21418020

  8. Changes of balance between proteinase and their inhibitors in blood of pigs with high-velocity missile wounds

    Institute of Scientific and Technical Information of China (English)

    周元国; 朱佩芳; 周继红; 李晓炎

    2003-01-01

    Objective: To study the effect of imbalance between lysosomal enzymes and their inhibitors in blood on disturbance of the local and whole body after trauma. Methods: The dynamic changes of lysosomal enzymes and proteinase inhibitors were studied in 12 pigs with femoral comminuted fractures in both hind limbs caused by high velocity missiles. Four normal pigs served as controls. Results: After injury, the activity of Cathepsin D in arterial plasma increased gradually and reached the highest level at 8 hours, acid phosphatase in serum began to increase at 12 hours and the value of serum elastase did not change significantly. The level of α1-antitrypsin, a proteinase inhibitor in plasma, decreased significantly in the early stage after injury [73.5%±6.4% and 81.0%±5.1% of the baseline value (1.67 μmol*ml-1*min-1± 0.29 μmol*ml-1*min-1) at l and 2 hours after injury, respectively, P<0.05], then increased gradually and was higher than the baseline value at 12 hours after injury. Conclusions: Imbalance between lysosomal enzymes and proteinase inhibitors occurs soon after injury, which might result in continuous tissue damage and play an important role in the disturbance of general reaction after injury.

  9. Rhabdovirus-induced apoptosis in a fish cell line is inhibited by a human endogenous acid cysteine proteinase inhibitor.

    Science.gov (United States)

    Björklund, H V; Johansson, T R; Rinne, A

    1997-07-01

    To determine the mechanisms of cell death in rhabdovirus-infected cells, we studied the infection of the epithelial papilloma of carp cell line with spring viremia of carp virus. Studies using electron microscopy, confocal microscopy, and agarose gel electrophoresis revealed changes in cell morphology and DNA fragmentation indicative of apoptosis. The virus-induced apoptosis was inhibited in cells treated with a human endogenous acid cysteine proteinase inhibitor. PMID:9188644

  10. Rhabdovirus-induced apoptosis in a fish cell line is inhibited by a human endogenous acid cysteine proteinase inhibitor.

    OpenAIRE

    Björklund, H V; Johansson, T R; Rinne, A

    1997-01-01

    To determine the mechanisms of cell death in rhabdovirus-infected cells, we studied the infection of the epithelial papilloma of carp cell line with spring viremia of carp virus. Studies using electron microscopy, confocal microscopy, and agarose gel electrophoresis revealed changes in cell morphology and DNA fragmentation indicative of apoptosis. The virus-induced apoptosis was inhibited in cells treated with a human endogenous acid cysteine proteinase inhibitor.

  11. Biospecific haemosorbents based on proteinase inhibitor. II. Efficiency of biospecific antiproteinase haemosorbent 'Ovosorb' in complex treatment of experimental generalized purulent peritonitis and acute destructive pancreatitis in dogs.

    Science.gov (United States)

    Platé, N A; Kirkovsky, V V; Antiperovich, O F; Nicolaichik, V V; Valueva, T A; Sinilo, S B; Moin, V M; Lobacheva, G A

    1994-03-01

    The biospecific antiproteinase haemosorbent (BAH) 'Ovosorb' containing, in the bulk of polyacryamide gel, the ovomucoid from whites of duck eggs, was used for a complex treatment of the experimental generalized purulent peritonitis and acute destructive pancreatitis in dogs. The efficiency of BAH was manifested in the significant reduction of lethality of the experimental animals, a more rapid liquidation of proteinasaemia, normalization in plasma of alpha 1-proteinase inhibitor and protein metabolism. Thus, by eliminating proteinases from circulation, Ovosorb contributes to the cessation of imbalance in the proteinase-inhibitor system and is efficient in the therapy of pathological states related to this imbalance. PMID:8031989

  12. Molecular interactions between an insect predator and its herbivore prey on transgenic potato expressing a cysteine proteinase inhibitor from rice.

    Science.gov (United States)

    Bouchard, Edith; Michaud, Dominique; Cloutier, Conrad

    2003-09-01

    Transgenic plants expressing resistance to herbivorous insects may represent a safe and sustainable pest control alternative if they do not interfere with the natural enemies of target pests. Here we examined interactions between oryzacystatin I (OCI), a proteinase inhibitor from rice genetically engineered into potato (Solanum tuberosum cv. Kennebec, line K52) to increase resistance to insect herbivory, and the insect predator Perillus bioculatus. This stinkbug is a relatively specialized predator of caterpillars and leaf-beetle larvae, and may also include plant sap in its predominantly carnivorous diet. One of its preferred prey is Colorado potato beetle (Leptinotarsa decemlineata), a major target of insect resistance development for potato field crops. Gelatin/sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that a major fraction of proteinase (gelatinase) activity in P. bioculatus extracts is OCI-sensitive. Among five gelatinolytic bands detected, the slowest-moving one (proteinase I) was inhibited strongly by purified OCI expressed in Escherichia coli or by OCI-transgenic potato extracts, while three other proteinases were partly sensitive to these treatments. There was also evidence of slight inhibition of proteinase I by untransformed potato foliage, suggesting the presence of a natural inhibitor related to OCI at low level in potato foliage. Interestingly, only about 50% of the maximum potential activity of proteinase I was recovered in extracts of P. bioculatus feeding on L. decemlineata larval prey on a diet of OCI-potato foliage, indicating that the predator was sensitive to OCI in the midgut of its prey. However, P. bioculatus on OCI-prey survived, grew and developed normally, indicating ability to compensate prey-mediated exposure to the OCI inhibitor. Confinement of P. bioculatus to potato foliage provided no evidence that potato plant-derived nutrition is a viable alternative to predation, restriction to potato foliage

  13. Regulation of secretory leukocyte proteinase inhibitor (SLPI) and elastase-specific inhibitor (ESI/elafin) in human airway epithelial cells by cytokines and neutrophilic enzymes.

    Science.gov (United States)

    Sallenave, J M; Shulmann, J; Crossley, J; Jordana, M; Gauldie, J

    1994-12-01

    The regulation of the activity of potentially harmful proteinases secreted by neutrophils during inflammation is important for the prevention of excessive tissue injury. Secretory leukocyte proteinase inhibitor (SLPI), also called antileukoprotease (ALP) or mucus proteinase inhibitor (MPI), is a serine proteinase inhibitor that has been found in a variety of mucous secretions and that is secreted by bronchial epithelial cells. We recently reported the presence of SLPI and of an elastase-specific inhibitor (ESI), also called elafin, in the supernatants of two cell lines, NCI-H322 and A549, which have features of Clara cells and type II alveolar cells, respectively. We showed in addition that epithelial cell lines produce the elastase-specific inhibitor as a 12 to 16 kD precursor of the elafin molecule (6 kD) called pre-elafin. In the present study, we show that NCI-H322 cells produced higher amounts of both inhibitors than A549 cells and that basal production of SLPI in both cell lines is higher than the production of elafin/pre-elafin. In addition, we show that interleukin-1 beta and tumor necrosis factor induce significant SLPI expression and are major inducers of elafin/pre-elafin expression. Moreover, induction is greater in A549 cells than in NCI-H322 cells. The implications of these findings for the peripheral airways are twofold: (1) alveolar epithelial cells may respond to cytokines secreted during the onset of inflammation by increasing their antiprotease shield; (2) elafin/pre-elafin seems to be a true local "acute phase reactant" whereas SLPI, in comparison, may be less responsive to local inflammatory mediators. PMID:7946401

  14. Opposite Effects on Spodoptera littoralis Larvae of High Expression Level of a Trypsin Proteinase Inhibitor in Transgenic Plants1

    Science.gov (United States)

    De Leo, Francesca; Bonadé-Bottino, Michel A.; Ceci, Luigi R.; Gallerani, Raffaele; Jouanin, Lise

    1998-01-01

    This work illustrates potential adverse effects linked with the expression of proteinase inhibitor (PI) in plants used as a strategy to enhance pest resistance. Tobacco (Nicotiana tabacum L. cv Xanthi) and Arabidopsis [Heynh.] ecotype Wassilewskija) transgenic plants expressing the mustard trypsin PI 2 (MTI-2) at different levels were obtained. First-instar larvae of the Egyptian cotton worm (Spodoptera littoralis Boisd.) were fed on detached leaves of these plants. The high level of MTI-2 expression in leaves had deleterious effects on larvae, causing mortality and decreasing mean larval weight, and was correlated with a decrease in the leaf surface eaten. However, larvae fed leaves from plants expressing MTI-2 at the low expression level did not show increased mortality, but a net gain in weight and a faster development compared with control larvae. The low MTI-2 expression level also resulted in increased leaf damage. These observations are correlated with the differential expression of digestive proteinases in the larval gut; overexpression of existing proteinases on low-MTI-2-expression level plants and induction of new proteinases on high-MTI-2-expression level plants. These results emphasize the critical need for the development of a PI-based defense strategy for plants obtaining the appropriate PI-expression level relative to the pest's sensitivity threshold to that PI. PMID:9808744

  15. Structural and functional characterization of proteinase inhibitors from seeds of Cajanus cajan (cv. ICP 7118).

    Science.gov (United States)

    Swathi, Marri; Lokya, Vadthya; Swaroop, Vanka; Mallikarjuna, Nalini; Kannan, Monica; Dutta-Gupta, Aparna; Padmasree, Kollipara

    2014-10-01

    Proteinase inhibitors (C11PI) from mature dry seeds of Cajanus cajan (cv. ICP 7118) were purified by chromatography which resulted in 87-fold purification and 7.9% yield. SDS-PAGE, matrix assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF) mass spectrum and two-dimensional (2-D) gel electrophoresis together resolved that C11PI possessed molecular mass of 8385.682 Da and existed as isoinhibitors. However, several of these isoinhibitors exhibited self association tendency to form small oligomers. All the isoinhibitors resolved in Native-PAGE and 2-D gel electrophoresis showed inhibitory activity against bovine pancreatic trypsin and chymotrypsin as well as Achaea janata midgut trypsin-like proteases (AjPs), a devastating pest of castor plant. Partial sequences of isoinhibitor (pI 6.0) obtained from MALDI-TOF/TOF analysis and N-terminal sequencing showed 100% homology to Bowman-Birk Inhibitors (BBIs) of leguminous plants. C11PI showed non-competitive inhibition against trypsin and chymotrypsin. A marginal loss (<15%) in C11PI activity against trypsin at 80 (°)C and basic pH (12.0) was associated with concurrent changes in its far-UV CD spectra. Further, in vitro assays demonstrated that C11PI possessed significant inhibitory potential (IC50 of 78 ng) against AjPs. On the other hand, in vivo leaf coating assays demonstrated that C11PI caused significant mortality rate with concomitant reduction in body weight of both larvae and pupae, prolonged the duration of transition from larva to pupa along with formation of abnormal larval-pupal and pupal-adult intermediates. Being smaller peptides, it is possible to express C11PI in castor to protect them against its devastating pest A. janata. PMID:25093261

  16. The M358R variant of α(1)-proteinase inhibitor inhibits coagulation factor VIIa.

    Science.gov (United States)

    Sheffield, William P; Bhakta, Varsha

    2016-02-12

    The naturally occurring M358R mutation of the plasma serpin α1-proteinase inhibitor (API) changes both its cleavable reactive centre bond to Arg-Ser and the efficacy with which it inhibits different proteases, reducing the rate of inhibition of neutrophil elastase, and enhancing that of thrombin, factor XIa, and kallikrein, by several orders of magnitude. Although another plasma serpin with an Arg-Ser reactive centre, antithrombin (AT), has been shown to inhibit factor VIIa (FVIIa), no published data are available with respect to FVIIa inhibition by API M358R. Recombinant bacterially-expressed API M358R and plasma-derived AT were therefore compared using gel-based and kinetic assays of FVIIa integrity and activity. Under pseudo-first order conditions of excess serpin over protease, both AT and API M358R formed denaturation-resistant inhibitory complexes with FVIIa in reactions accelerated by TF; AT, but not API M358R, also required heparin for maximal activity. The second order rate constant for heparin-independent API M358R-mediated FVIIa inhibition was determined to be 7.8 ± 0.8 × 10(2) M(-1)sec(-1). We conclude that API M358R inhibits FVIIa by forming inhibitory complexes of the serpin type more rapidly than AT in the absence of heparin. The likely 20-fold excess of API M358R over AT in patient plasma during inflammation raises the possibility that it could contribute to the hemorrhagic tendencies manifested by rare individuals expressing this mutant serpin. PMID:26797521

  17. The squash aspartic proteinase inhibitor SQAPI is widely present in the cucurbitales, comprises a small multigene family, and is a member of the phytocystatin family.

    Science.gov (United States)

    Christeller, John T; Farley, Peter C; Marshall, Richelle K; Anandan, Ananda; Wright, Michele M; Newcomb, Richard D; Laing, William A

    2006-12-01

    The squash (Cucurbita maxima) phloem exudate-expressed aspartic proteinase inhibitor (SQAPI) is a novel aspartic acid proteinase inhibitor, constituting a fifth family of aspartic proteinase inhibitors. However, a comparison of the SQAPI sequence to the phytocystatin (a cysteine proteinase inhibitor) family sequences showed approximately 30% identity. Modeling SQAPI onto the structure of oryzacystatin gave an excellent fit; regions identified as proteinase binding loops in cystatin coincided with regions of SQAPI identified as hypervariable, and tryptophan fluorescence changes were also consistent with a cystatin structure. We show that SQAPI exists as a small gene family. Characterization of mRNA and clone walking of genomic DNA (gDNA) produced 10 different but highly homologous SQAPI genes from Cucurbita maxima and the small family size was confirmed by Southern blotting, where evidence for at least five loci was obtained. Using primers designed from squash sequences, PCR of gDNA showed the presence of SQAPI genes in other members of the Cucurbitaceae and in representative members of Coriariaceae, Corynocarpaceae, and Begoniaceae. Thus, at least four of seven families of the order Cucurbitales possess member species with SQAPI genes, covering approximately 99% of the species in this order. A phylogenetic analysis of these Cucurbitales SQAPI genes indicated not only that SQAPI was present in the Cucurbitales ancestor but also that gene duplication has occurred during evolution of the order. Phytocystatins are widespread throughout the plant kingdom, suggesting that SQAPI has evolved recently from a phytocystatin ancestor. This appears to be the first instance of a cystatin being recruited as a proteinase inhibitor of another proteinase family.

  18. Ixodes scapularis tick serine proteinase inhibitor (serpin gene family; annotation and transcriptional analysis

    Directory of Open Access Journals (Sweden)

    Chalaire Katelyn C

    2009-05-01

    Full Text Available Abstract Background Serine proteinase inhibitors (Serpins are a large superfamily of structurally related, but functionally diverse proteins that control essential proteolytic pathways in most branches of life. Given their importance in the biology of many organisms, the concept that ticks might utilize serpins to evade host defenses and immunizing against or disrupting their functions as targets for tick control is an appealing option. Results A sequence homology search strategy has allowed us to identify at least 45 tick serpin genes in the Ixodes scapularis genome that are structurally segregated into 32 intronless and 13 intron-containing genes. Nine of the intron-containing serpins occur in a cluster of 11 genes that span 170 kb of DNA sequence. Based on consensus amino acid residues in the reactive center loop (RCL and signal peptide scanning, 93% are putatively inhibitory while 82% are putatively extracellular. Among the 11 different amino acid residues that are predicted at the P1 sites, 16 sequences possess basic amino acid (R/K residues. Temporal and spatial expression analyses revealed that 40 of the 45 serpins are differentially expressed in salivary glands (SG and/or midguts (MG of unfed and partially fed ticks. Ten of the 38 serpin genes were expressed from six to 24 hrs of feeding while six and fives genes each are predominantly or exclusively expressed in either MG and SG respectively. Conclusion Given the diversity among tick species, sizes of tick serpin families are likely to be variable. However this study provides insight on the potential sizes of serpin protein families in ticks. Ticks must overcome inflammation, complement activation and blood coagulation to complete feeding. Since these pathways are regulated by serpins that have basic residues at their P1 sites, we speculate that I. scapularis may utilize some of the serpins reported in this study to manipulate host defense. We have discussed our data in the context of

  19. In vivo and in vitro effect of Acacia nilotica seed proteinase inhibitors on Helicoverpa armigera (Hübner) larvae

    Indian Academy of Sciences (India)

    S Ramesh Babu; B Subrahmanyam; Srinivasan; I M Santha

    2012-06-01

    Acacia nilotica proteinase inhibitor (AnPI) was isolated by ammonium sulphate precipitation followed by chromatography on DEAE-Sephadex A-25 and resulted in a purification of 10.68-fold with a 19.5% yield. Electrophoretic analysis of purified AnPI protein resolved into a single band with molecular weight of approximately 18.6+1.00 kDa. AnPI had high stability at different pH values (2.0 to 10.0) except at pH 5.0 and are thermolabile beyond 80°C for 10 min. AnPI exhibited effective against total proteolytic activity and trypsin-like activity, but did not show any inhibitory effect on chymotrypsin activity of midgut of Helicoverpa armigera. The inhibition kinetics studies against H. armigera gut trypsin are of non-competitive type. AnPI had low affinity for H. armigera gut trypsin when compared to SBTI. The partially purified and purified PI proteins-incorporated test diets showed significant reduction in mean larval and pupal weight of H. armigera. The results provide important clues in designing strategies by using the proteinase inhibitors (PIs) from the A. nilotica that can be expressed in genetically engineered plants to confer resistance to H. armigera.

  20. Purification and characterization of a Bowman-Birk proteinase inhibitor from the seeds of black gram (Vigna mungo).

    Science.gov (United States)

    Prasad, E R; Dutta-Gupta, A; Padmasree, K

    2010-03-01

    A proteinase inhibitor (BgPI) was purified from black gram, Vigna mungo (cv. TAU-1) seeds by using ammonium sulfate fractionation, followed by ion-exchange, affinity and gel-filtration chromatography. BgPI showed a single band in SDS-PAGE under non-reducing condition with an apparent molecular mass of approximately 8kDa correlating to the peak 8041.5Da in matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrum. BgPI existed in different isoinhibitor forms with pI values ranging from 4.3 to 6.0. The internal sequence "SIPPQCHCADIR" of a peak 1453.7 m/z, obtained from MALDI-TOF-TOF showed 100% similarity with Bowman-Birk inhibitor (BBI) family. BgPI exhibited non-competitive-type inhibitory activity against both bovine pancreatic trypsin (K(i) of 309.8nM) and chymotrypsin (K(i) of 10.7muM), however, with a molar ratio of 1:2 with trypsin. BgPI was stable up to a temperature of 80 degrees C and active over a wide pH range between 2 and 12. The temperature-induced conformational changes in secondary structure are reversed when BgPI was cooled from 90 to 25 degrees C. Further, upon reduction with dithiothreitol, BgPI lost both its inhibitory activity as well as secondary structural conformation. Lysine residue(s) present in the reactive site of BgPI play an important role in inhibiting the bovine trypsin activity. The present study provides detailed biochemical characteristic features of a BBI type serine proteinase inhibitor isolated from V. mungo.

  1. Is a cysteine proteinase inhibitor involved in the regulation of petal wilting in senescing carnation (Dianthus caryophyllus L.) flowers?

    Science.gov (United States)

    Sugawara, Hiroaki; Shibuya, Kenichi; Yoshioka, Toshihito; Hashiba, Teruyoshi; Satoh, Shigeru

    2002-03-01

    Senescence of carnation petals is accompanied by autocatalytic ethylene production and wilting of the petals; the former is caused by the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase genes and the latter is related to the expression of a cysteine proteinase (CPase) gene. CPase is probably responsible for the degradation of proteins, leading to the decomposition of cell components and resultant cell death during the senescence of petals. The carnation plant also has a gene for the CPase inhibitor (DC-CPIn) that is expressed abundantly in petals at the full opening stage of flowers. In the present study, DC-CPIn cDNA was cloned and expressed in E. coli. The recombinant DC-CPIn protein completely inhibited the activities of a proteinase (CPase) extracted from carnation petals and papain. Northern blot analysis showed that the mRNA for CPase (DC-CP1) accumulated in large amounts, whereas that for DC-CPIn disappeared, corresponding to the onset of petal wilting in flowers undergoing natural senescence and exogenous ethylene-induced senescence. Based on these findings, a role of DC-CPIn in the regulation of petal wilting is suggested; DC-CPIn acts as a suppressor of petal wilting, which probably functions to fine-tune petal wilting in contrast to coarse tuning, the up-regulation of CPase activity by gene expression.

  2. The urinary excretion of epidermal growth factor in the rat is reduced by aprotinin, a proteinase inhibitor

    DEFF Research Database (Denmark)

    Jørgensen, P E; Raaberg, Lasse; Poulsen, Steen Seier;

    1990-01-01

    in vivo is processed by an aprotinin inhibitable proteinase. EGF is produced in the kidneys as a precursor with a molecular weight of approximately 130 kDa. In rat urine, nanomolar amounts of 6 kDa EGF are excreted per 24 h together with small amounts of high molecular weight forms of EGF. During i......The present study on the rat shows that i.v. administration of the proteinase inhibitor aprotinin reduces the urinary output of immunoreactive epidermal growth factor (EGF) while the amount of immunoreactive EGF in the kidneys is increased. This indicates that the EGF-precursor in the rat kidney.......v. administration of aprotinin the median urinary output of immunoreactive EGF is reduced to 15% of the excretion of control rats (23 pmol/2 h versus 157 pmol/2 h, P less than 0.001). Especially the excretion of 6 kDa EGF is reduced (median excretion 12 pmol/2 h versus 134 pmol/2 h, P less than 0.001). The amount...

  3. Matrix metalloproteinases-2, -9 and tissue inhibitor of metallo-proteinase-1 in lung cancer invasion and metastasis

    Institute of Scientific and Technical Information of China (English)

    MING Shu-hong; SUN Tie-ying; XIAO Wei; XU Xiao-mao

    2005-01-01

    @@ Lung cancer is a major cause of death from malignant disease due to its high incidence, malignant behavior and lack of major advancements in treatment strategies. The ability to invade tissues and establish colonies at remote sites is a defining characteristic of malignant neoplasms. Matrix metalloproteinases (MMPs) are zinc proteinases that degrade compounds of extracellular matrix (ECM). These enzymes have been implicated in tumour invasion and metastasis through degrading many extracellular matrix proteins especially MMP-2 and MMP-9, which are regarded as markers of tumour invasion and metastasis.1 The purpose of this study is to examine the role of MMP-9, MMP-2, tissue inhibitor of metalloproteinase-1 (TIMP-1) and MMP-9/TIMP-1 in tumour invasion and metastasis as well as the relationships between the mRNA expression of MMP-9 in white blood cells and MMP-9 levels in the plasma.

  4. Negative Effects of a Nonhost Proteinase Inhibitor of ~19.8 kDa from Madhuca indica Seeds on Developmental Physiology of Helicoverpa armigera (Hübner)

    OpenAIRE

    Farrukh Jamal; Dushyant Singh; Pandey, Prabhash K.

    2014-01-01

    An affinity purified trypsin inhibitor from the seed flour extracts of Madhuca indica (MiTI) on denaturing polyacrylamide gel electrophoresis showed that MiTI consisted of a single polypeptide chain with molecular mass of ~19.8 kDa. MiTI inhibited the total proteolytic and trypsin-like activities of the midgut proteinases of Helicoverpa armigera larvae by 87.51% and 76.12%, respectively, at concentration of 5 µg/mL with an IC50 of 1.75 µg/mL against trypsin like midgut proteinases. The enzyme...

  5. Basis for the Specificity and Activation of the Serpin Protein Z-dependent Proteinase Inhibitor (ZPI) as an Inhibitor of Membrane-associated Factor Xa

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Xin; Dementiev, Alexey; Olson, Steven T.; Gettins, Peter G.W. (UIC)

    2012-12-13

    The serpin ZPI is a protein Z (PZ)-dependent specific inhibitor of membrane-associated factor Xa (fXa) despite having an unfavorable P1 Tyr. PZ accelerates the inhibition reaction {approx}2000-fold in the presence of phospholipid and Ca{sup 2+}. To elucidate the role of PZ, we determined the x-ray structure of Gla-domainless PZ (PZ{sub {Delta}GD}) complexed with protein Z-dependent proteinase inhibitor (ZPI). The PZ pseudocatalytic domain bound ZPI at a novel site through ionic and polar interactions. Mutation of four ZPI contact residues eliminated PZ binding and membrane-dependent PZ acceleration of fXa inhibition. Modeling of the ternary Michaelis complex implicated ZPI residues Glu-313 and Glu-383 in fXa binding. Mutagenesis established that only Glu-313 is important, contributing {approx}5-10-fold to rate acceleration of fXa and fXIa inhibition. Limited conformational change in ZPI resulted from PZ binding, which contributed only {approx}2-fold to rate enhancement. Instead, template bridging from membrane association, together with previously demonstrated interaction of the fXa and ZPI Gla domains, resulted in an additional {approx}1000-fold rate enhancement. To understand why ZPI has P1 tyrosine, we examined a P1 Arg variant. This reacted at a diffusion-limited rate with fXa, even without PZ, and predominantly as substrate, reflecting both rapid acylation and deacylation. P1 tyrosine thus ensures that reaction with fXa or most other arginine-specific proteinases is insignificant unless PZ binds and localizes ZPI and fXa on the membrane, where the combined effects of Gla-Gla interaction, template bridging, and interaction of fXa with Glu-313 overcome the unfavorability of P1 Tyr and ensure a high rate of reaction as an inhibitor.

  6. A group-specific inhibitor of lysosomal cysteine proteinases selectively inhibits both proteolytic degradation and presentation of the antigen dinitrophenyl-poly-L-lysine by guinea pig accessory cells to T cells

    DEFF Research Database (Denmark)

    Buus, S; Werdelin, O

    1986-01-01

    A limited intralysosomal proteolytic degradation is probably a key event in the accessory cell processing of large protein antigens before their presentation to T cells. With the aid of highly specific inhibitors of proteinases, we have examined the role of proteolysis in the presentation of anti...... inhibitor. Another inhibitor, pepstatin A, which selectively blocks aspartic proteinases, did not block the presentation of dinitrophenyl-poly-L-lysine. The results identify cysteine proteinases, probably lysosomal, as one of the groups of enzymes involved in antigen processing....

  7. Synthesis and structure-activity relationships of a series of penicillin-derived HIV proteinase inhibitors containing a stereochemically unique peptide isostere.

    Science.gov (United States)

    Holmes, D S; Bethell, R C; Cammack, N; Clemens, I R; Kitchin, J; McMeekin, P; Mo, C L; Orr, D C; Patel, B; Paternoster, I L

    1993-10-15

    A series of HIV-1 proteinase inhibitors was synthesized based upon a single penicillin derived thiazolidine moiety. Reaction of the C-4 carboxyl group with (R)-phenylalaninol gave amide 10 which was a moderately potent inhibitor of HIV-1 proteinase (IC50 = 0.15 microM). Further modifications based on molecular modeling studies led to compound 48 which contained a stereochemically unique statine-based isostere. This was a potent competitive inhibitor (Ki = 0.25 nM) with antiviral activity against HIV-1 in vitro (5 microM). Neither modification to the benzyl group in an attempt to improve interaction with the S2' pocket, nor introduction of a hydrogen bond donating group to interact with residue Gly48' resulted in improved inhibitory or antiviral activity. PMID:8230099

  8. Cystatin M/E expression is restricted to differentiated epidermal keratinocytes and sweat glands: a new skin-specific proteinase inhibitor that is a target for cross-linking by transglutaminase.

    NARCIS (Netherlands)

    Zeeuwen, P.L.J.M.; Vlijmen-Willems, I.M.J.J. van; Jansen, B.J.H.; Sotiropoulou, G.; Curfs, J.H.A.J.; Meis, J.F.G.M.; Janssen, J.J.M.; Ruissen, F. van; Schalkwijk, J.

    2001-01-01

    Using serial analysis of gene expression on cultured human keratinocytes we found high expression levels of genes putatively involved in host protection and defense, such as proteinase inhibitors and antimicrobial proteins. One of these expressed genes was the recently discovered cysteine proteinase

  9. The role of secretory leukocyte proteinase inhibitor and elafin (elastase-specific inhibitor/skin-derived antileukoprotease as alarm antiproteinases in inflammatory lung disease

    Directory of Open Access Journals (Sweden)

    Sallenave Jean-Michel

    2000-08-01

    Full Text Available Abstract Secretory leukocyte proteinase inhibitor and elafin are two low-molecular-mass elastase inhibitors that are mainly synthesized locally at mucosal sites. It is thought that their physicochemical properties allow them to efficiently inhibit target enzymes, such as neutrophil elastase, released into the interstitium. Historically, in the lung, these inhibitors were first purified from secretions of patients with chronic obstructive pulmonary disease and cystic fibrosis. This suggested that they might be important in controlling excessive neutrophil elastase release in these pathologies. They are upregulated by 'alarm signals' such as bacterial lipopolysaccharides, and cytokines such as interleukin-1 and tumor necrosis factor and have been shown to be active against Gram-positive and Gram-negative bacteria, so that they have joined the growing list of antimicrobial 'defensin-like' peptides produced by the lung. Their site of synthesis and presumed functions make them very attractive candidates as potential therapeutic agents under conditions in which the excessive release of elastase by neutrophils might be detrimental. Because of its natural tropism for the lung, the use of adenovirus-mediated gene transfer is extremely promising in such applications.

  10. The role of secretory leukocyte proteinase inhibitor and elafin (elastase-specific inhibitor/skin-derived antileukoprotease) as alarm antiproteinases in inflammatory lung disease.

    Science.gov (United States)

    Sallenave, J M

    2000-01-01

    Secretory leukocyte proteinase inhibitor and elafin are two low-molecular-mass elastase inhibitors that are mainly synthesized locally at mucosal sites. It is thought that their physicochemical properties allow them to efficiently inhibit target enzymes, such as neutrophil elastase, released into the interstitium. Historically, in the lung, these inhibitors were first purified from secretions of patients with chronic obstructive pulmonary disease and cystic fibrosis. This suggested that they might be important in controlling excessive neutrophil elastase release in these pathologies. They are upregulated by 'alarm signals' such as bacterial lipopolysaccharides, and cytokines such as interleukin-1 and tumor necrosis factor and have been shown to be active against Gram-positive and Gram-negative bacteria, so that they have joined the growing list of antimicrobial 'defensin-like' peptides produced by the lung. Their site of synthesis and presumed functions make them very attractive candidates as potential therapeutic agents under conditions in which the excessive release of elastase by neutrophils might be detrimental. Because of its natural tropism for the lung, the use of adenovirus-mediated gene transfer is extremely promising in such applications. PMID:11667971

  11. Novel alleles among soybean Bowman-Birk proteinase inhibitor gene families

    Institute of Scientific and Technical Information of China (English)

    WANG YuePing; CHEN XiongTing; QIU LiJuan

    2008-01-01

    Trypsin inhibitors have been found in various animals, plants and microorganisms. There were two types of trypsin inhibitors in soybean including Bowman-Birk protease inhibitors (BBI) and Kunitz in-hibitors (KTI). The different BBI genes from wild soybean (G.soja) and cultivated soybean (G max) formed a multigene family. We constructed a cDNA library of cultivar 'SuiNong 14' seed at the R7 growth stage using the SMART Kit. Seventeen contigs or singletons were highly homologous to soy-bean protease inhibitors. Contigs of 5, 35, 8 and 9 were highly homologous to BBI family members BBI-A1, BBI-A2, BBI-C and BBI-D, respectively. Sequence analyses showed there were novel allelic varia-tions among the 4 BBI members in SuiNong 14. Based on the comparison of soybean seed cDNA li-braries from different developmental stages, it was apparent that the expression of trypsin inhibitors increased during seed development in soybean. Phylogenetic analysis of BBI gene sequences among dicotyledonous and monocotyledonous plants demonstrated that these genes shared a common pro-genitor.

  12. Expression, purification and characterization of recombinant human serine proteinase inhibitor Kazal-type 6 (SPINK6) in Pichia pastoris.

    Science.gov (United States)

    Lu, Hairong; Huang, Jinjiang; Li, Guodong; Ge, Kuikui; Wu, Hongyu; Huang, Qingshan

    2012-03-01

    Human serine proteinase inhibitor Kazal-type 6 (SPINK6) belongs to the medically important SPINK family. Malfunctions of SPINK members are linked to many diseases, including pancreatitis, skin barrier defects, and cancer. SPINK6 has been shown to selectively inhibit Kallikrein-related peptidases (KLKs) in human skin. As a SPINK protein, it contains a typical Kazal domain, which requires three intramolecular disulfide bonds for correct folding and activity. Preparation of functional protein is a prerequisite for studying this important human factor. Here, we report the successful generation of tagless SPINK6 using a yeast expression system. The recombinant protein was secreted and purified by cation exchange and size-exclusion chromatography. The protein identity was confirmed by MALDI-TOF MS and N-terminal sequencing. Pichia pastoris-derived recombinant human SPINK6 (rhSPINK6) showed higher inhibitory activity against Kallikrein-related peptidase 14 (KLK14) (K(i)=0.16 nM) than previously reported Escherichia coli-derived rhSPINK6 (K(i)=0.5 nM). This protein also exhibited moderate inhibition of bovine trypsin (K(i)=33 nM), while previous E. coli-derived rhSPINK6 did not. The results indicate that P. pastoris is a better system to generate active rhSPINK6, warranting further studies on this medically important SPINK family candidate.

  13. The Plant-Derived Bauhinia bauhinioides Kallikrein Proteinase Inhibitor (rBbKI) Attenuates Elastase-Induced Emphysema in Mice.

    Science.gov (United States)

    Martins-Olivera, Bruno Tadeu; Almeida-Reis, Rafael; Theodoro-Júnior, Osmar Aparecido; Oliva, Leandro Vilela; Neto Dos Santos Nunes, Natalia; Olivo, Clarice Rosa; Vilela de Brito, Marlon; Prado, Carla Máximo; Leick, Edna Aparecida; Martins, Mílton de Arruda; Oliva, Maria Luiza Vilela; Righetti, Renato Fraga; Tibério, Iolanda de Fátima Lopes Calvo

    2016-01-01

    Background. Elastase mediates important oxidative actions during the development of chronic obstructive pulmonary disease (COPD). However, few resources for the inhibition of elastase have been investigated. Our study evaluated the ability of the recombinant plant derived Bauhinia bauhinioides Kallikrein proteinase Inhibitor (rBbKI) to modulate elastase-induced pulmonary inflammation. Methods. C57Bl/6 mice were given intratracheal elastase (ELA group) or saline (SAL group) and were treated intraperitoneally with rBbKI (ELA-rBbKI and SAL-rBbKI groups). At day 28, the following analyses were performed: (I) lung mechanics, (II) exhaled nitric oxide (ENO), (III) bronchoalveolar lavage fluid (BALF), and (IV) lung immunohistochemical staining. Results. In addition to decreasing mechanical alterations and alveolar septum disruption, rBbKI reduced the number of cells in the BALF and decreased the cellular expression of TNF-α, MMP-9, MMP-12, TIMP-1, eNOS, and iNOS in airways and alveolar walls compared with the ELA group. rBbKI decreased the volume proportion of 8-iso-PGF2α, collagen, and elastic fibers in the airways and alveolar walls compared with the ELA group. A reduction in the number of MUC-5-positive cells in the airway walls was also observed. Conclusion. rBbKI reduced elastase-induced pulmonary inflammation and extracellular matrix remodeling. rBbKI may be a potential pharmacological tool for COPD treatment.

  14. α1Proteinase inhibitor regulates CD4+ lymphocyte levels and is rate limiting in HIV-1 disease.

    Directory of Open Access Journals (Sweden)

    Cynthia L Bristow

    Full Text Available BACKGROUND: The regulation of adult stem cell migration through human hematopoietic tissue involves the chemokine CXCL12 (SDF-1 and its receptor CXCR4 (CD184. In addition, human leukocyte elastase (HLE plays a key role. When HLE is located on the cell surface (HLE(CS, it acts not as a proteinase, but as a receptor for α(1proteinase inhibitor (α(1PI, α(1antitrypsin, SerpinA1. Binding of α(1PI to HLE(CS forms a motogenic complex. We previously demonstrated that α(1PI deficiency attends HIV-1 disease and that α(1PI augmentation produces increased numbers of immunocompetent circulating CD4(+ lymphocytes. Herein we investigated the mechanism underlying the α(1PI deficiency that attends HIV-1 infection. METHODS AND FINDINGS: Active α(1PI in HIV-1 subjects (median 17 µM, n = 35 was significantly below normal (median 36 µM, p220 CD4 cells/µl, CD4(+ lymphocytes were correlated solely with active α(1PI (r(2 = 0.93, p<0.0001, n = 26. The monoclonal anti-HIV-1 gp120 antibody 3F5 present in HIV-1 patient blood is shown to bind and inactivate human α(1PI. Chimpanzee α(1PI differs from human α(1PI by a single amino acid within the 3F5-binding epitope. Unlike human α(1PI, chimpanzee α(1PI did not bind 3F5 or become depleted following HIV-1 challenge, consistent with the normal CD4(+ lymphocyte levels and benign syndrome of HIV-1 infected chimpanzees. The presence of IgG-α(1PI immune complexes correlated with decreased CD4(+ lymphocytes in HIV-1 subjects. CONCLUSIONS: This report identifies an autoimmune component of HIV-1 disease that can be overcome therapeutically. Importantly, results identify an achievable vaccine modification with the novel objective to protect against AIDS as opposed to the current objective to protect against HIV-1 infection.

  15. Occurrence of Two Distinct Types of Tissue Inhibitors of Metallo-proteinases-2 in Fugu rubripes

    Institute of Scientific and Technical Information of China (English)

    Yoshihiro Yokoyama; Hiroshi Tsukamoto; Tohru Suzuki; Shohshi Mizuta; Reiji Yoshinaka

    2005-01-01

    In this study, genes of two distinct tissue inhibitors of metalloproteinases-2 (TIMP-2) from Japanese puffer fish Fugu rubripes, Fugu TIMP-2a and TIMP-2b, were cloned. The open reading frames of Fugu TIMP-2a and TIMP-2b cDNAs are composed of 660 and 657 nucleotides and 220 and 219 amino acids, respectively. Both Fugu TIMP-2s contain 12 cysteine residues, whichmight form six disulfide bonds as in other animals TIMP-2s. Reverse-transcribed polymerase chain reaction analysis showed the mRNAs of Fugu TIMP-2a and TIMP-2b to be expressed in some tissues examined with different expression patterns. These findings suggest that the two distinct Fugu TIMP-2s might perform different functions in Fugu tissues.

  16. Engineering D-helix of antithrombin in alpha-1-proteinase inhibitor confers antiinflammatory properties on the chimeric serpin.

    Science.gov (United States)

    Yang, L; Dinarvand, P; Qureshi, S H; Rezaie, A R

    2014-07-01

    Antithrombin (AT) is a heparin-binding serpin in plasma which regulates the proteolytic activity of procoagulant proteases of the clotting cascade. In addition to being an anticoagulant, AT also exhibits antiinflammatory activities when it binds to cell surface heparan sulfate proteoglycans (HSPGs) on the endothelium via its basic residues of D-helix to elicit intracellular signalling responses. By contrast to AT, α1-proteinase inhibitor (α1-PI) is a non-heparin-binding serpin that exhibits very slow reactivity with coagulation proteases and possesses no HSPG-dependent antiinflammatory properties. To determine whether the antiinflammatory signaling specificity of AT can be transferred to α1-PI, we replaced the D-helix of human α1-PI with the corresponding sequence of human AT and expressed the chimeric serpin α1-PI/D-helix) in a bacterial expression system. High molecular weight heparin bound to α1-PI/D-helix and accelerated the inhibition of thrombin by the serpin mutant by a template mechanism reminiscent of the cofactor effect of heparin on inhibition of thrombin by AT. Like AT, α1-PI/D-helix exhibited antiinflammatory properties in both cellular and animal models. Thus, α1-PI/D-helix inhibited the barrier-disruptive effect of proinflammatory cytokines and inhibited the activation of nuclear factor-κB transcription factor in lipopolysaccharide-stimulated endothelial cells by a concentration-dependent manner. Furthermore, the chimeric serpin reduced lipopolysaccharide-mediated lethality, elicited a vascular protective effect and inhibited infiltration of activated leukocytes to the peritoneal cavity of mice in an HMGB1-mediated inflammatory model. These results suggest that grafting the D-helix of AT to α1-PI confers antiinflammatory properties on the serpin and that the chimeric serpin may have therapeutic utility for treating inflammatory disorders. PMID:24522239

  17. The response to epidermal growth factor of human maxillary tumor cells in terms of tumor growth, invasion and expression of proteinase inhibitors.

    Science.gov (United States)

    Mizoguchi, H; Komiyama, S; Matsui, K; Hamanaka, R; Ono, M; Kiue, A; Kobayashi, M; Shimizu, N; Welgus, H G; Kuwano, M

    1991-11-11

    Three cancer cell lines, IMC-2, IMC-3 and IMC-4, were established from a single tumor of a patient with maxillary cancer. We examined responses to epidermal growth factor (EGF) of these 3 cell lines with regard to cell growth and tumor invasion. The growth rate of IMC-2 in nude mice was markedly faster than that of the IMC-3 and IMC-4 cell lines. Assay for invasion through fibrin gels showed significantly enhanced invasive capacity of IMC-2 cells in response to EGF, but no change for IMC-3 and IMC-4 cells. We examined response to EGF of IMC-2 cells with regard to expression of a growth-related oncogene (c-fos), proteinases and their inhibitors. Expression of c-fos was transiently increased in IMC-2 cells at rates comparable to those seen in the 2 other lines in the presence of EGF. There was no apparent effect of EGF on the expression of urokinase-type plasminogen activator and 72-kDa type-IV collagenase in IMC-2 cells. In contrast, EGF specifically enhanced the expression of plasminogen activator inhibitor-I (PAI-I) and tissue inhibitor of metalloproteinases-I (TIMP-I) in IMC-2 cells. Our data suggest that proteinase inhibitors or other related factors may play an important role in tumor growth and invasion in response to EGF.

  18. Bmcystatin, a cysteine proteinase inhibitor characterized from the tick Boophilus microplus

    International Nuclear Information System (INIS)

    The bovine tick Rhipicephalus (Boophilus) microplus is a blood-sucking animal, which is responsible for Babesia spp and Anaplasma marginale transmission for cattle. From a B. microplus fat body cDNA library, 465 selected clones were sequenced randomly and resulted in 60 Contigs. An open reading frame (ORF) contains 98 amino acids named Bmcystatin, due to 70% amino acid identity to a classical type 1 cystatin from Ixodes scapularis tick (GenBank Accession No. DQ066227). The Bmcystatin amino acid sequence analysis showed two cysteine residues, theoretical pI of 5.92 and Mr of 11kDa. Bmcystatin gene was cloned in pET 26b vector and the protein expressed using bacteria Escherichia coli BL21 SI. Recombinant Bmcystatin (rBmcystatin) purified by affinity chromatography on Ni-NTA-agarose column and ionic exchange chromatography on HiTrap Q column presented molecular mass of 11kDa, by SDS-PAGE and the N-terminal amino acid sequenced revealed unprocessed N-terminal containing part of pelB signal sequence. Purified rBmcystatin showed to be a C1 cysteine peptidase inhibitor with Ki value of 0.1 and 0.6nM for human cathepsin L and VTDCE (vitellin degrading cysteine endopeptidase), respectively. The rBmcystatin expression analyzed by semi-quantitative RT-PCR confirmed the amplification of a specific DNA sequence (294bp) in the fat body and ovary cDNA preparation. On the other hand, a protein band was detected in the fat body, ovary, and the salivary gland extracts using anti-Bmcystatin antibody by Western blot. The present results suggest a possible role of Bmcystatin in the ovary, even though the gene was cloned from the fat body, which could be another site of this protein synthesis

  19. Negative Effects of a Nonhost Proteinase Inhibitor of ~19.8 kDa from Madhuca indica Seeds on Developmental Physiology of Helicoverpa armigera (Hübner

    Directory of Open Access Journals (Sweden)

    Farrukh Jamal

    2014-01-01

    Full Text Available An affinity purified trypsin inhibitor from the seed flour extracts of Madhuca indica (MiTI on denaturing polyacrylamide gel electrophoresis showed that MiTI consisted of a single polypeptide chain with molecular mass of ~19.8 kDa. MiTI inhibited the total proteolytic and trypsin-like activities of the midgut proteinases of Helicoverpa armigera larvae by 87.51% and 76.12%, respectively, at concentration of 5 µg/mL with an IC50 of 1.75 µg/mL against trypsin like midgut proteinases. The enzyme kinetic studies demonstrated that MiTI is a competitive inhibitor with a Ki value of 4.1×10−10 M for Helicoverpa trypsin like midgut proteinases. In vivo experiments with different concentrations of MiTI in artificial diet (0.5, 1.0, and 1.5% w/w showed an effective downfall in the larval body weight and an increase in larval mortality. The concentration of MiTI in the artificial diet to cause 50% mortality (LD50 of larvae was 1.5% w/w and that to cause reduction in mass of larvae by 50% (ED50 was 1.0% w/w. Nutritional indices observations suggest the toxic and adverse effects of MiTI on the growth and development of H. armigera larvae. The results suggest a strong bioinsecticidal potential of affinity purified MiTI which can be exploited in insect pest management of crop plants.

  20. Negative effects of a nonhost proteinase inhibitor of ~19.8 kDa from Madhuca indica seeds on developmental physiology of Helicoverpa armigera (Hübner).

    Science.gov (United States)

    Jamal, Farrukh; Singh, Dushyant; Pandey, Prabhash K

    2014-01-01

    An affinity purified trypsin inhibitor from the seed flour extracts of Madhuca indica (MiTI) on denaturing polyacrylamide gel electrophoresis showed that MiTI consisted of a single polypeptide chain with molecular mass of ~19.8 kDa. MiTI inhibited the total proteolytic and trypsin-like activities of the midgut proteinases of Helicoverpa armigera larvae by 87.51% and 76.12%, respectively, at concentration of 5 µg/mL with an IC50 of 1.75 µg/mL against trypsin like midgut proteinases. The enzyme kinetic studies demonstrated that MiTI is a competitive inhibitor with a K i value of 4.1 × 10(-10) M for Helicoverpa trypsin like midgut proteinases. In vivo experiments with different concentrations of MiTI in artificial diet (0.5, 1.0, and 1.5% w/w) showed an effective downfall in the larval body weight and an increase in larval mortality. The concentration of MiTI in the artificial diet to cause 50% mortality (LD50) of larvae was 1.5% w/w and that to cause reduction in mass of larvae by 50% (ED50) was 1.0% w/w. Nutritional indices observations suggest the toxic and adverse effects of MiTI on the growth and development of H. armigera larvae. The results suggest a strong bioinsecticidal potential of affinity purified MiTI which can be exploited in insect pest management of crop plants. PMID:25298962

  1. Purification and Partial Characterization of Trypsin-Specific Proteinase Inhibitors from Pigeonpea Wild Relative Cajanus platycarpus L. (Fabaceae) Active against Gut Proteases of Lepidopteran Pest Helicoverpa armigera.

    Science.gov (United States)

    Swathi, Marri; Mishra, Prashant K; Lokya, Vadthya; Swaroop, Vanka; Mallikarjuna, Nalini; Dutta-Gupta, Aparna; Padmasree, Kollipara

    2016-01-01

    Proteinase inhibitors (PIs) are natural defense proteins of plants found to be active against gut proteases of various insects. A pigeonpea wild relative Cajanus platycarpus was identified as a source of resistance against Helicoverpa armigera, a most devastating pest of several crops including pigeonpea. In the light of earlier studies, trypsin-specific PIs (CpPI 63) were purified from mature dry seeds of C. platycarpus (ICPW-63) and characterized their biochemical properties in contributing to H. armigera resistance. CpPI 63 possessed significant H. armigera gut trypsin-like proteinase inhibitor (HGPI) activity than trypsin inhibitor (TI) activity. Analysis of CpPI 63 using two-dimensional (2-D) electrophoresis and matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that it contained several isoinhibitors and small oligomers with masses ranging between 6 and 58 kDa. The gelatin activity staining studies suggest that these isoinhibitors and oligomers possessed strong inhibitory activity against H. armigera gut trypsin-like proteases (HGPs). The N-terminal sequence of the isoinhibitors (pI 6.6 and pI 5.6) of CpPI 63 exhibited 80% homology with several Kunitz trypsin inhibitors (KTIs) as well as miraculin-like proteins (MLPs). Further, modification of lysine residue(s) lead to 80% loss in both TI and HGPI activities of CpPI 63. In contrast, the TI and HGPI activities of CpPI 63 were stable over a wide range of temperature and pH conditions. The reported results provide a biochemical basis for pod borer resistance in C. platycarpus. PMID:27656149

  2. Perspectives of digestive pest control with proteinase inhibitors that mainly affect the trypsin-like activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae

    Directory of Open Access Journals (Sweden)

    M.E. Pereira

    2005-11-01

    Full Text Available The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4ºC and the supernatants were used in enzymatic assays at 30ºC, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis was 1.14 ± 0.15 absorbance variation min-1 mg protein-1, at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis was 0.217 ± 0.02 mmol p-nitroaniline min-1 mg protein-1. The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50ºC, a temperature that reduces enzyme stability to 80 and 60% of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.

  3. Cytotoxic Compounds from Zanthoxylum Americanum

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Four pyranocoumarins: dipetaline, alloxanthoxyletin, xanthoxyletin, and xanthyletin, and two lignans: sesamin and asarinin were isolated from the northern prickly ash, Zanthoxylum americanum. To varying degrees, all six compounds inhibited the incorporation of tritiated thymidine into human leukemia (HL-60) cells and the inhibitory effect was dependent on the structures of the isolated compounds.

  4. Cysteine proteinases and cystatins

    Directory of Open Access Journals (Sweden)

    Adeliana S. Oliveira

    2003-01-01

    Full Text Available This review describeds the definition, localization, functions and examples of cysteine proteinases and their protein inhibitors in vertebrate, non-vertebrate animals and plants. These inhibitors are related with defense mechanisms of plant against pests. It also describes the factors involved in the specific cysteine proteinase-cystatin interaction and high degree of affinity and large specificity in this interaction which are not only represented by the compatibility between amino acid residues of the active site involved in catalysis, but also of all amino acid residues that participante in the enzyme-inhibitor interaction.Nesta revisão foram descritas definições, localizações, funções e exemplos de proteinases cisteínicas e suas proteinas inibidoras em animais vertebrados e invertebrados e plantas. Tratamos principalmente com aqueles inibidores que são relatados com o mecanismo de defesa da planta contra pestes. Em adição, comentamos sobre recentes trabalhos que contribuíram para uma melhor compreenção dos fatores envolvidos na interação específica proteinase cisteínica-cistatina. Por outro lado, chamamos atenção para o alto grau de afinidade e grande especificidade na interação que não são apenas representadas pela compatibilidade entre os residuos de aminoácidos do sítio ativo envolvidos na catalise, mas também de todos os resíduos de aminoácidos que participam da interação enzima-inibidor.

  5. Cloning of a Potato Proteinase Inhibitor Gene PINII-2x from Diploid Potato (Solanum phurejia L.) and Transgenic Investigation of Its Potential to Confer Insect Resistance in Rice

    Institute of Scientific and Technical Information of China (English)

    Qing-Yun Bu; Liang Wu; Shi-Hu Yang; Jian-Min Wan

    2006-01-01

    Both cDNA and a genomic DNA fragment encoding a new potato proteinase inhibitor Ⅱ were isolated from a diploid potato IVP101 (Solanum phurejia L.) and named PINⅡ-2x. Nucleotide sequencing confirmed that the DNA fragment of PINⅡ-2xwas 580 bp, including a 115-bp intron and two exons. The deduced PINⅡ-2x protein contained an intact signal peptide and two active sites. The PINⅡ-2x gene and its deduced PINⅡ-2x protein had 88% and 93% homology with another tetraploid potato proteinase inhibitor Ⅱ, respectively. Northern blotting analysis indicated that the mRNA of PINⅡ-2x gene was wound induced in potato leaves. Binary vector pNAR301 and pNAR302 were constructed for rice transformation, in which the PINⅡ-2x cDNA was driven,respectively, by rice actin I promoter (Actl) and maize ubiquitin promoter (Ubil). Via an Agrobacteriummediated method, these two constructs were transferred into japonica rice cv. Xiushui 63. PCR and Southern blotting analysis for transgenic rice revealed the integration of the PINⅡ-2x gene. Northern blotting analysis also confirmed transcripts of the PINⅡ-2x gene in transgenic rice plants. Insect bioassays using stripe stem borer (Chilo suppressalis Walker) demonstrated that the average weight and body length of larvae in transgenic plants were only nearly 50% and 61% of those of larvae in control plants, respectively.These results indicate that the PINⅡ-2x gene should be an effective insect-resistance gene and could be valuable for application in crop breeding for insect resistance.

  6. Plasmin: indigenous milk proteinase

    Directory of Open Access Journals (Sweden)

    Samir Kalit

    2002-06-01

    Full Text Available The most important characteristic of plasmin, as significant indigenous milk proteinase, its concentration, concentration measuring procedure and activity of plasmin are described. The most important factors, which have an influence on concentration and plasmin activity in milk, are stage of lactation and mastitis (high somatic cell count – SCC. In high SCC milk indigenous proteinase activity increased, especially in plasmin and plasminogen system.Specific hydrolytic activity of plasmin during primary proteolysis of some casein fractions is described. ß-CN is most susceptible fraction, but αs1-CN and αs2-Cn are less susceptible to degradation by plasmin. Almost all fractions of κ-CN are resistant to degradation by plasmin. Activation of plasminogen to plasmin is very complex biochemical process influenced by activators and inhibitors in milk, and can be increased in high SCC milk. There are many various types of inhibitors in milk serum and ßlactoglobulin is the most important after its thermal denaturation. Addition of aprotinin and soybean tripsin inhibitors in milk inhibits plasmin activity. Most important characteristic of plasmin is its thermostability onpasteurisation and even sterilisation. Mechanism of thermal inactivation of plasmin with developing covalent disulphide interaction between molecule of plasmin and serum proteins (mostly ß-laktoglobulin is described. Thermosensitive inhibitors of plasminogen activators and inhibitors of plasmin are inactivated by short pasteurisation and therefore increase plasmin activity,while higher temperature and longer treatment time inactivate plasmin activity.

  7. Oncostatin M, but not interleukin-6 or leukemia inhibitory factor, stimulates expression of alpha1-proteinase inhibitor in A549 human alveolar epithelial cells.

    Science.gov (United States)

    Sallenave, J M; Tremblay, G M; Gauldie, J; Richards, C D

    1997-06-01

    Alpha-1 proteinase inhibitor (A1-Pi) is the main serine proteinase inhibitor found in human plasma and is a potent elastase inhibitor in various tissues, including lung. A1-Pi is expressed and induced in liver during inflammatory responses but can also be produced by epithelial cells. Since hepatocyte A1-Pi production is stimulated by interleukin-6 (IL-6) and other gp130-cytokines, such as leukemia inhibitory factor (LIF) and oncostatin M (OM), we investigated the role of these cytokines in regulating A1-Pi in lung epithelial cells. We show that OM, a monocyte and T cell product, can specifically and potently induce A1-Pi production in lung-derived A549 alveolar (epithelial) cells, as well as in liver-derived HepG2 cells. Both A1-Pi protein (as detected by ELISA and Western blots) and mRNA levels were enhanced 20-fold to 30-fold in A549 cells. OM was also able to stimulate the expression of tissue inhibitor of metalloproteinase-1 in these cells. Interestingly, other members of the IL-6 family (IL-6 and LIF) had little or no effect on A549 cells, and proinflammatory cytokines, such as IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) also had no stimulatory effect on A1-Pi synthesis in A549 cells. Costimulation with IL-1 beta resulted in a decrease in A1-Pi production from OM-stimulated A549 cells. However, IL-6 production was synergistically enhanced. OM was also able to stimulate A1-Pi production from a bronchial epithelial primary cell line, whereas an intestinal epithelial cell line HT29 responded to IL-6 but not OM. These results suggest that lung levels A1-Pi could be derived not only from liver and inflammatory cells but also from epithelial cells, which can be upregulated on stimulation by OM. This may have implications for regulation of local activity of human neutrophil elastase (HNE) in such diseases as emphysema and cystic fibrosis. PMID:9198001

  8. Using a Caesalpinia echinata Lam. protease inhibitor as a tool for studying the roles of neutrophil elastase, cathepsin G and proteinase 3 in pulmonary edema.

    Science.gov (United States)

    Cruz-Silva, Ilana; Neuhof, Christiane; Gozzo, Andrezza Justino; Nunes, Viviane Abreu; Hirata, Izaura Yoshico; Sampaio, Misako Uemura; Figueiredo-Ribeiro, Rita de Cássia; Neuhof, Heinz; Araújo, Mariana da Silva

    2013-12-01

    Acute lung injury (ALI) is characterized by neutrophil infiltration and the release of proteases, mainly elastase (NE), cathepsin G (Cat G) and proteinase 3 (PR3), which can be controlled by specific endogenous inhibitors. However, inhibitors of these proteases have been isolated from different sources, including plants. For this study, CeEI, or Caesalpinia echinata elastase inhibitor, was purified from C. echinata (Brazil-wood) seeds after acetone fractionation, followed by ion exchange and reversed phase chromatographic steps. Characterization with SDS-PAGE, stability assays, amino acid sequencing and alignment with other protein sequences confirmed that CeEI is a member of the soybean Kunitz trypsin inhibitor family. Like other members of this family, CeEI is a 20 kDa monomeric protein; it is stable within a large pH and temperature range, with four cysteine residues forming two disulfide bridges, conserved amino acid residues and leucine-isoleucine residues in the reactive site. CeEI was able to inhibit NE and Cat G at a nanomolar range (with K(i)s of 1.9 and 3.6 nM, respectively) and inhibited PR3 within a micromolar range (K(i) 3.7 μM), leading to hydrolysis of specific synthetic substrates. In a lung edema model, CeEI reduced the lung weight and pulmonary artery pressure until 180 min after the injection of zymosan-activated polymorphonuclear neutrophils. In experiments performed in the presence of a Cat G and PR3, but not an NE inhibitor, lung edema was reduced only until 150 min and pulmonary artery pressure was similar to that of the control. These results confirm that NE action is crucial to edema establishment and progression. Additionally, CeEI appears to be a useful tool for studying the physiology of pulmonary edema and provides a template for molecular engineering and drug design for ALI therapy.

  9. Triangular gold nanoparticles conjugated with peptide ligands: a new class of inhibitor for Candida albicans secreted aspartyl proteinase.

    Science.gov (United States)

    Jebali, Ali; Hajjar, Farzaneh Haji Esmaeil; Hekmatimoghaddam, Seyedhossein; Kazemi, Bahram; De La Fuente, Jesus M; Rashidi, Mohsen

    2014-08-15

    The aim of this study was to find the peptide ligands to inhibit Candida albicans secreted aspartyl proteinase 2 (Sap2). First, a ligand library, containing 300 different peptides, was constructed, and their interaction with Sap2 was separately calculated by molecular dynamic software. Second, 10 peptide ligands with the lowest intermolecular energy were selected. Then, triangular gold nanoparticles were synthesized, and separately conjugated with the peptide ligands. After synthesis, antifungal property and Sap inactivation of conjugated triangular gold nanoparticles, peptide ligands, and naked triangular gold nanoparticle were separately assessed, against thirty clinical isolates of C. albicans. In this study, we measured the uptake of conjugated and naked nanoparticles by atomic adsorption spectroscopy. This study showed that naked triangular gold nanoparticle and all conjugated triangular gold nanoparticles had high antifungal activity, but no peptide ligands had such activity. Of 300 peptide ligands, the peptide containing N-Cys-Lys-Lys-Arg-Met-Met-Lys-Ser-Met-Cys-C and its conjugate had the highest capability to inhibit Sap. Moreover, the uptake assay demonstrated that triangular gold nanoparticles conjugated with the peptide ligand had the highest uptake.

  10. RESEARCH ON PROTEINASE INHIBITORS OF BEANS PHASEOLUS VULGARIS TO MAKE PLANT PROTECTION PRODUCTS FROM PESTS AND DISEASES

    OpenAIRE

    Pavlovskaya, N.; Gagarina, I.; Dzumabaeva, B.; Dzangalina, E.

    2014-01-01

    An animal body and seed plants have a complex of proteolytic ferments which react in reserve protein breakdown to amino acids in food digestion and seed sprouting. At present a few hundreds of peptidohydrolases of different origin have been described. In regulation of proteolysis inhibitors of proteolytic ferments react. In a living organism they are presented by means of specific protein. Inhibitors have an ability to slow down or stop fermentation. They react in immunity apoptosis, protect ...

  11. Measurement of homonuclear three-bond J(HNH{alpha}) coupling constants in unlabeled peptides complexed with labeled proteins: Application to a decapeptide inhibitor bound to the proteinase domain of the NS3 protein of hepatitis C virus (HCV)

    Energy Technology Data Exchange (ETDEWEB)

    Cicero, Daniel O.; Barbato, Gaetano; Koch, Uwe; Ingallinella, Paolo; Bianchi, Elisabetta; Sambucini, Sonia; Neddermann, Petra; De Francesco, Raffaele; Pessi, Antonello; Bazzo, Renzo

    2001-05-15

    A new isotope-filtered experiment has been designed to measure homonuclear three-bond J(H{sup N}H{sup {alpha}}) coupling constants of unlabeled peptides complexed with labeled proteins. The new experiment is based on the 3D HNHA pulse scheme, and belongs to the 'quantitative J-correlation' type. It has been applied to a decapeptide inhibitor bound to the proteinase domain of the NS3 protein of human hepatitis C virus (HCV)

  12. Inhibitory effects of a Kunitz-type inhibitor from Pithecellobium dumosum (Benth) seeds against insect-pests' digestive proteinases.

    Science.gov (United States)

    Rufino, Fabiola P S; Pedroso, Vanessa M A; Araujo, Jonalson N; França, Anderson F J; Rabêlo, Luciana M A; Migliolo, Ludovico; Kiyota, Sumika; Santos, Elizeu A; Franco, Octavio L; Oliveira, Adeliana S

    2013-02-01

    Pithecellobium dumosum is a tree belonging to the Mimosoideae subfamily that presents various previously characterized Kunitz-type inhibitors. The present study provides a novel Kunitz-trypsin inhibitor isoform purified from P. dumosum seeds. Purification procedure was performed by TCA precipitation followed by a trypsin-Sepharose chromatography and a further reversed-phase HPLC. Purified inhibitor (PdKI-4) showed enhanced inhibitory activity against bovine trypsin and chymotrypsin. Furthermore, PdKI-4 showed remarkable inhibitory activity against serine proteases from the coleopterans Callosobruchus maculatus and Zabrotes subfasciatus, and the lepidopterans Alabama argillacea and Telchin licus. However, PdKI-4 was unable to inhibit porcine pancreatic elastase, pineapple bromelain and Carica papaya papain. SDS-PAGE showed that PdKI-4 consisted of a single polypeptide chain with molecular mass of 21 kDa. Kinetic studies demonstrated that PdKI-4 is probably a competitive inhibitor with a Ki value of 5.7 × 10(-10) M for bovine trypsin. PdKI-4 also showed higher stability over a wide range of temperature (37-100 °C) and pH (2-12). N-termini sequence was obtained by Edman degradation showing higher identity with other Mimosoideae subfamily Kunitz-type inhibitor members. In summary, data here reported indicate the biotechnological potential of PdKI-4 for development of products against insect-pests.

  13. Effect of quercetin on expression of matrix metallo-proteinases and tissue inhibitor of matalloproteinase-1 in cultured rat hepatic stellate cells

    Institute of Scientific and Technical Information of China (English)

    康鲁平; 齐荔红; 张俊平; 周斌

    2003-01-01

    Objective: To study the effects of quercetin (QU) on matrix metallo-proteinases (MMPs), the tissue inhibitor of matalloproteinase-1 (TIMP-1), procollagen I and 2 proteoglycans (decorin and biglycan) mRNA expression in cultured rat hepatic stellate cell line HSC-T6 cells.Methods: Cells were treated with different concentrations of QU (12.5, 25, 50 μmol/L) or drug solvent (0.1 % Me2SO) for 24 h.mRNA expression was determined by reverse transcription polymerase chain reaction (RT-PCR).Results: QU (12.5 - 50 μmol/L) enhanced collagenase (rat MMP-13) and membrane type1-MMP (MMP-14) mRNA expression, decreased procollagen I mRNA expression in a concentration-dependent manner, but did not affect gelatinase-A (MMP-2), TIMP-1, decorin and biglycan expression.Conclusion: QU may decrease matrix deposition and increase matrix degradation, which might be beneficial to liver fibrosis.

  14. Trichocystatin-2 (TC-2): an endogenous inhibitor of cysteine proteinases in Trichomonas vaginalis is associated with TvCP39.

    Science.gov (United States)

    Puente-Rivera, Jonathan; Ramón-Luing, Lucero de los Ángeles; Figueroa-Angulo, Elisa Elvira; Ortega-López, Jaime; Arroyo, Rossana

    2014-09-01

    The causal agent of trichomoniasis is a parasitic protist, Trichomonas vaginalis, which is rich in proteolytic activity, primarily carried out by cysteine proteases (CPs). Some CPs are known virulence factors. T. vaginalis also possesses three genes encoding endogenous cystatin-like CP inhibitors. The aim of this study was to identify and characterize one of these CP inhibitors. Using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), a cystatin-like peptidase inhibitor dubbed Trichocystatin-2 (TC-2) was identified in the T. vaginalis active degradome in association with TvCP39, a 39kDa CP involved in cytotoxicity. To characterize the TC-2 inhibitor, we cloned and expressed the tvicp-2 gene, purified the recombinant protein (TC-2r), and produced a specific polyclonal antibody (α-TC-2r). This antibody recognized a 10kDa protein band by western blotting. An indirect immunofluorescence assay (IFA) and cell fractionation assays using the α-TC-2r antibody showed that TC-2 was localized in the cytoplasm and lysosomes and that it colocalized with TvCP39. TC-2r showed inhibitory activity against papain, cathepsin-L, and TvCP39 in trichomonad extracts and live parasites but not legumain-like CPs. Live trichomonads treated with TC-2r showed reduced trichomonal cytotoxicity to HeLa cell monolayers in a TC-2r-concentration-dependent manner. In this study, we identified and characterized an endogenous cystatin-like inhibitor in T. vaginalis, TC-2, which is associated with TvCP39 and appears to regulate the cellular damage caused by T. vaginalis.

  15. The recombinant prepro region of TvCP4 is an inhibitor of cathepsin L-like cysteine proteinases of Trichomonas vaginalis that inhibits trichomonal haemolysis.

    Science.gov (United States)

    Cárdenas-Guerra, Rosa Elena; Ortega-López, Jaime; Flores-Pucheta, Claudia Ivonne; Benítez-Cardoza, Claudia Guadalupe; Arroyo, Rossana

    2015-02-01

    Trichomonas vaginalis expresses multiple proteinases, mainly of the cysteine type (CPs). A cathepsin L-like 34kDa CP, designated TvCP4, is synthesized as a 305-amino-acid precursor protein. TvCP4 contains the prepro fragment and the catalytic triad that is typical of the papain-like CP family of clan CA. The aim of this work was to determine the function of the recombinant TvCP4 prepro region (ppTvCP4r) as a specific inhibitor of CPs. We cloned, expressed, and purified the recombinant TvCP4 prepro region. The conformation of the purified and refolded ppTvCP4r polypeptide was verified by circular dichroism spectroscopy and fluorescence emission spectra. The inhibitory effect of ppTvCP4r was tested on protease-resistant extracts from T. vaginalis using fluorogenic substrates for cathepsin L and legumain CPs. In 1-D zymograms, the inhibitory effect of ppTvCP4r on trichomonad CP proteolytic activity was observed in the ∼97, 65, 39, and 30 kDa regions. By using 2-D zymograms and mass spectrometry, several of the CPs inhibited by ppTvCP4r were identified. A clear reduction in the proteolytic activity of several cathepsin L-like protein spots (TvCP2, TvCP4, TvCP4-like, and TvCP39) was observed compared with the control zymogram. Moreover, pretreatment of live parasites with ppTvCP4r inhibited trichomonal haemolysis in a concentration dependent manner. These results confirm that the recombinant ppTvCP4 is a specific inhibitor of the proteolytic activity of cathepsin L-like T. vaginalis CPs that is useful for inhibiting virulence properties depending on clan CA papain-like CPs.

  16. Effect of adding Matrix Metallo proteinase inhibitors on the degree of conversion of monomers to polymer an experimental bonding agent

    Directory of Open Access Journals (Sweden)

    Ghavam M.

    2009-11-01

    Full Text Available "nBackground and Aim: In spite of the achievements in the field of dental adhesives, we are facing challenges with dentine bonding resistance, strength and stability. According to recent studies the role of MMP inhibitors in association with bonding,s persistence and leakage reduction and restoration,s persistence is important. The aim of this study was to investigate the effect of doxycycline as a MMP inhibitor on the degree of conversion (DC of an experimental dental adhesive. "nMaterials and Methods: In this experimental study, a new dental adhesive blend was prepared by mixing doxycycline monohydrate (in concentrations of 0.0, 0.25, 0.5, and 1 wt.% with monomers. The monomers were composed of 12% Bis-GMA and 10% TMPTMA, 28% HEMA, and 50% Ethanol by weight for all groups. Comphorquinone and amines were chosen as photo initiator system. Degree of conversion of all adhesives was measured using FTIR spectroscopy. The results were analyzed using one-way ANOVA and Tukey post hoc tests. "nResults: The results showed that addition of 0.25, 0.5, and 1 weight percent doxycycline did not significantly reduce the DC of the adhesives compared to 0.0% control group (p>0.05%. "nConclusion: According to the results of this study, adding doxycycline to the adhesives did not adversely affect the DC.

  17. Global proteome changes in larvae of Callosobruchus maculatus Coleoptera:Chrysomelidae:Bruchinae) following ingestion of a cysteine proteinase inhibitor

    DEFF Research Database (Denmark)

    Nogueira, Fábio C S; Silva, Carlos P; Alexandre, Daniel;

    2012-01-01

    proteomic changes induced in the intestinal tract of larval C. maculatus challenged by the ingestion of cystatin, a cysteine peptidase inhibitor, was investigated by a nanoLC-MS/MS approach. The ingestion of cystatin caused a delay in the development of the larvae, but the mortality was not high, indicating....... Ingestion of cystatin led to significant changes in the proteome of both the midgut epithelia and midgut contents. We have observed that proteins related to plant cell wall degradation, particularly the key glycoside hydrolases of the families GH5 (endo-β-1,4-mannanase) and GH 28 (polygalacturonase) were...... overexpressed. Conversely, α-amylases were downexpressed, indicating that an increase in hemicelluloses digestion helps the larvae to cope with the challenge of cystatin ingestion. Furthermore, a number of proteins associated with transcription/translation and antistress reactions were among the cystatin...

  18. Serum and fecal canine α1-proteinase inhibitor concentrations reflect the severity of intestinal crypt abscesses and/or lacteal dilation in dogs.

    Science.gov (United States)

    Heilmann, Romy M; Parnell, Nolie K; Grützner, Niels; Mansell, Joanne; Berghoff, Nora; Schellenberg, Stefan; Reusch, Claudia E; Suchodolski, Jan S; Steiner, Jörg M

    2016-01-01

    Gastrointestinal (GI) protein loss, due to lymphangiectasia or chronic inflammation, can be challenging to diagnose. This study evaluated the diagnostic accuracy of serum and fecal canine α1-proteinase inhibitor (cα1PI) concentrations to detect crypt abscesses and/or lacteal dilation in dogs. Serum and fecal cα1PI concentrations were measured in 120 dogs undergoing GI tissue biopsies, and were compared between dogs with and without crypt abscesses/lacteal dilation. Sensitivity and specificity were calculated for dichotomous outcomes. Serial serum cα1PI concentrations were also evaluated in 12 healthy corticosteroid-treated dogs. Serum cα1PI and albumin concentrations were significantly lower in dogs with crypt abscesses and/or lacteal dilation than in those without (both P <0.001), and more severe lesions were associated with lower serum cα1PI concentrations, higher 3 days-mean fecal cα1PI concentrations, and lower serum/fecal cα1PI ratios. Serum and fecal cα1PI, and their ratios, distinguished dogs with moderate or severe GI crypt abscesses/lacteal dilation from dogs with only mild or none such lesions with moderate sensitivity (56-92%) and specificity (67-81%). Serum cα1PI concentrations increased during corticosteroid administration. We conclude that serum and fecal α1PI concentrations reflect the severity of intestinal crypt abscesses/lacteal dilation in dogs. Due to its specificity for the GI tract, measurement of fecal cα1PI appears to be superior to serum cα1PI for diagnosing GI protein loss in dogs. In addition, the serum/fecal cα1PI ratio has an improved accuracy in hypoalbuminemic dogs, but serum cα1PI concentrations should be carefully interpreted in corticosteroid-treated dogs. PMID:26631946

  19. A recombinant plasmid of composite cysteine proteinase inhibitor/glyceraldehyde-3-phosphate dehydrogenase gene of periodic Brugia malayi functions on DNA immunity in the host

    Directory of Open Access Journals (Sweden)

    Z Fang

    2016-01-01

    Full Text Available Objectives: Both cysteine proteinase inhibitors (CPIs and glyceraldehyde-3-phosphate dehydrogenase (GAPDH play important roles in the pathogenesis of parasites and their relationship with the hosts. We constructed a new eukaryotic recombinant expression plasmid pcDNA3.1(+-BmCPI/BmGAPDH of periodic Brugia malayi for investigation of the DNA vaccine-elicited immune responses. Materials and Methods: We cloned a gene encoding the CPIs and GAPDH from periodic B. malayi into vector pcDNA3.1. The composited plasmid or the control was injected into the tibialis anterior muscle of the hind leg in BALB/c mice, respectively. The target genes were detected by reverse transcription-polymerase chain reaction in muscle tissues. The stimulation index (SI of T-lymphocyte proliferation and the levels of interferon-gamma (INF-g and interleukin-4 ( IL-4 in serum were detected by thiazolyl blue tetrazolium blue and enzyme-linked immunosorbent assays. Results: The pcDNA3.1(+-BmCPI/BmGAPDH was amplified from muscle tissues of the mice after immunisation. The SI of the immunised group was significantly higher than that of the two control groups (P < 0.05. The levels of INF-g and IL-4 of pcDNA3.1(+-BmCPI/BmGAPDH group were both higher than those of the two control groups (P < 0.05. The level of INF-g of pcDNA3.1(+-BmCPI/BmGAPDH group was significantly higher than that of pcDNA3.1(+-BmCPI/CpG group (P < 0.05. Conclusions: We conclude that the recombinant plasmid pcDNA3.1(+-BmCPI/BmGAPDH could elicit specific humoural and cellular immune responses in mice.

  20. Phage display of the serpin alpha-1 proteinase inhibitor randomized at consecutive residues in the reactive centre loop and biopanned with or without thrombin.

    Directory of Open Access Journals (Sweden)

    Benjamin M Scott

    Full Text Available In spite of the power of phage display technology to identify variant proteins with novel properties in large libraries, it has only been previously applied to one member of the serpin superfamily. Here we describe phage display of human alpha-1 proteinase inhibitor (API in a T7 bacteriophage system. API M358R fused to the C-terminus of T7 capsid protein 10B was directly shown to form denaturation-resistant complexes with thrombin by electrophoresis and immunoblotting following exposure of intact phages to thrombin. We therefore developed a biopanning protocol in which thrombin-reactive phages were selected using biotinylated anti-thrombin antibodies and streptavidin-coated magnetic beads. A library consisting of displayed API randomized at residues 357 and 358 (P2-P1 yielded predominantly Pro-Arg at these positions after five rounds of thrombin selection; in contrast the same degree of mock selection yielded only non-functional variants. A more diverse library of API M358R randomized at residues 352-356 (P7-P3 was also probed, yielding numerous variants fitting a loose consensus of DLTVS as judged by sequencing of the inserts of plaque-purified phages. The thrombin-selected sequences were transferred en masse into bacterial expression plasmids, and lysates from individual colonies were screening for API-thrombin complexing. The most active candidates from this sixth round of screening contained DITMA and AAFVS at P7-P3 and inhibited thrombin 2.1-fold more rapidly than API M358R with no change in reaction stoichiometry. Deep sequencing using the Ion Torrent platform confirmed that over 800 sequences were significantly enriched in the thrombin-panned versus naïve phage display library, including some detected using the combined phage display/bacterial lysate screening approach. Our results show that API joins Plasminogen Activator Inhibitor-1 (PAI-1 as a serpin amenable to phage display and suggest the utility of this approach for the selection

  1. Morphological Variation among 23 Xiphinema americanum Populations.

    Science.gov (United States)

    Cho, M R; Robbins, R T

    1991-01-01

    Morphometrics of 23 United States populations of Xiphinema americanum sensu lato, sharing the characteristics of an offset lip region and conoid tail, were examined and analyzed statistically by canonical discriminant analysis (CDA). Specimens were collected from Arkansas, Georgia, Tennessee, Mississippi, Florida, Oklahoma, California, and North Dakota. Eleven measurements and body ratios obtained from female specimens were used in the analysis. Xiphinema americanum, X. bricolensis, X. californicum, X. citricolum, X, intermedium, X. tarjanense, and X. thornei, and one undescribed species were identified among the 23 populations. Three groups -- X. americanum-group, X. californicum-group, and X. intermedium-group (X. intermedium and X. tarjanense) -- were formed and four populations belonging to four different species were separated consistently from these groups in CDA scatterplots of the 23 populations. Composition of the groups was somewhat related to the geographical origins of the populations in the groups. A population from California had morphometrics intermediate between X. americanum and X. californicum. Separation between the X. americanum-group and X. californicum-group in the CDA scatterplots was not as distinct as that between them and the X. intermedium-group or between any of the three groups and the four single outlying populations.

  2. The Onchocerca volvulus cysteine proteinase inhibitor, Ov-CPI-2, is a target of protective antibody response that increases with age.

    Directory of Open Access Journals (Sweden)

    Fidelis Cho-Ngwa

    Full Text Available BACKGROUND: Despite considerable efforts, a suitable vaccine against Onchocerca volvulus infection has remained elusive. Herein, we report on the use of molecular tools to identify and characterize O. volvulus antigens that are possibly associated with the development of concomitant immunity in onchocerciasis. METHODOLOGY/PRINCIPAL FINDINGS: Third-stage larvae (L3 and molting L3 (mL3 O. volvulus stage-specific cDNA libraries were screened with a pool of sera from chronically infected patients who had likely developed such immunity. The 87 immunoreactive clones isolated were grouped into 20 distinct proteins of which 12 had already been cloned and/or characterized before and 4 had been proven to be protective in a small O. volvulus animal model. One of these, onchocystatin (Ov-CPI-2, a previously characterized O. volvulus cysteine proteinase inhibitor was, overall, the most abundant clone recognized by the immune sera in both the L3 and mL3 cDNA libraries. To further characterize its association with protective immunity, we measured the IgG subclass and IgE class specific responses to the antigen in putatively immune (PI and infected (INF individuals living in a hyperendemic area in Cameroon. It appeared that both groups had similar IgG3 and IgE responses to the antigen, but the INF had significantly higher IgG1 and IgG4 responses than the PI individuals (p<0.05. In the INF group, the IgG3 levels increased significantly with the age of the infected individuals (r = 0.241; p<0.01. The IgG1 responses in the INF were high regardless of age. Notably, culturing L3 in vitro in the presence of anti-Ov-CPI-2 monospecific human antibodies and naïve neutrophils resulted in almost complete inhibition of molting of L3 to L4 and to cytotoxicity to the larvae. CONCLUSIONS/SIGNIFICANCE: These results add to the knowledge of protective immunity in onchocerciasis and support the possible involvement of anti-Ov-CPI-2 IgG1 and/or IgG3 cytophilic antibodies in the

  3. Gamma globulin, Evan's blue, aprotinin A PLA2 inhibitor, tetracycline and antioxidants protect epithelial cells against damage induced by synergism among streptococcal hemolysins, oxidants and proteinases: relation to the prevention of post-streptococcal sequelae and septic shock.

    Science.gov (United States)

    Ginsburg, I; Sadovnic, M

    1998-11-01

    An in vitro model was employed to study the potential role of streptococcal extra-cellular products, rich in streptolysin O, in cellular injury as related to streptococcal infections and post-streptococcal sequelae. Extra-cellular products (EXPA) rich in streptolysin O were isolated from type 4, group A hemolytic streptococci grown in a chemostat, in a synthetic medium. EXPA induced moderate cytopathogenic changes in monkey kidney epithelial cells and in rat heart cells pre-labeled with 3H-arachidonate. However very strong toxic effects were induced when EXP was combined with oxidants (glucose oxides generated H2O2, AAPH-induced peroxyl radical (ROO.), NO generated by sodium nitroprusside) and proteinases (plasmin, trypsin). Cell killing was distinctly synergistic in nature. Cell damage induced by the multi-component cocktails was strongly inhibited either by micromolar amounts of gamma globulin, and Evan's blue which neutralized SLO activity, by tetracycline, trasylol (aprotinin), epsilon amino caproic acid and by soybean trypsin inhibitor, all proteinase inhibitors as well as by a non-penetrating PLA2 inhibitor A. The results suggest that fasciitis, myositis and sepsis resulting from infections with hemolytic streptococci might be caused by a coordinated 'cross-talk' among microbial, leukocyte and additional host-derived pro-inflammatory agents. Since attempts to prolong lives of septic patients by the exclusive administration of single antagonists invariably failed, it is proposed that the administration of 'cocktails' of putative inhibitors against major pro-inflammatory agonizes generated in inflammation and infection might protect against the deleterious effects caused by the biochemical and pharmacological cascades which are known to be activated in sepsis. PMID:9848686

  4. A novel Glycine soja cysteine proteinase inhibitor GsCPI14, interacting with the calcium/calmodulin-binding receptor-like kinase GsCBRLK, regulated plant tolerance to alkali stress.

    Science.gov (United States)

    Sun, Xiaoli; Yang, Shanshan; Sun, Mingzhe; Wang, Sunting; Ding, Xiaodong; Zhu, Dan; Ji, Wei; Cai, Hua; Zhao, Chaoyue; Wang, Xuedong; Zhu, Yanming

    2014-05-01

    It has been well demonstrated that cystatins regulated plant stress tolerance through inhibiting the cysteine proteinase activity under environmental stress. However, there was limited information about the role of cystatins in plant alkali stress response, especially in wild soybean. Here, in this study, we focused on the biological characterization of a novel Glycine soja cystatin protein GsCPI14, which interacted with the calcium/calmodulin-binding receptor-like kinase GsCBRLK and positively regulated plant alkali stress tolerance. The protein-protein interaction between GsCBRLK and GsCPI14 was confirmed by using split-ubiquitin based membrane yeast two-hybrid analysis and bimolecular fluorescence complementation assay. Expression of GsCPI14 was greatly induced by salt, ABA and alkali stress in G. soja, and GsCBRLK overexpression (OX) in Glycine max promoted the stress induction of GmCPI14 expression under stress conditions. Furthermore, we found that GsCPI14-eGFP fusion protein localized in the entire Arabidopsis protoplast and onion epidermal cell, and GsCPI14 showed ubiquitous expression in different tissues of G. soja. In addition, we gave evidence that the GST-GsCPI14 fusion protein inhibited the proteolytic activity of papain in vitro. At last, we demonstrated that OX of GsCPI14 in Arabidopsis promoted the seed germination under alkali stress, as evidenced by higher germination rates. GsCPI14 transgenic Arabidopsis seedlings also displayed better growth performance and physiological index under alkali stress. Taken together, results presented in this study demonstrated that the G. soja cysteine proteinase inhibitor GsCPI14 interacted with the calcium/calmodulin-binding receptor-like kinase GsCBRLK and regulated plant tolerance to alkali stress.

  5. Transmission of Nepoviruses by Xiphinema americanum-group Nematodes.

    Science.gov (United States)

    Brown, D J; Halbrendt, J M; Robbins, R T; Vrain, T C

    1993-09-01

    The transmission of North American nepoviruses by putative species belonging to the Xiphinema americanum-group is reviewed. Xiphinema americanum sensu stricto, X. californicum, and X. rivesi each transmit cherry rasp leaf (CRLV), tobacco ringspot (TobRSV), and tomato ringspot nepovirus (TomRSV), and X. bricolensis is a vector of TomRSV. The apparent lack of specificity in the transmission of North American nepoviruses by X. americanum-group species markedly contrasts with the specific associations between European nepoviruses and their vector nematode species. Two complementary projects are described examining the taxonomic identity of putative species in the X. americanum-group, their morphological and genetic relationships, their ontogeny, and their ability to transmit viruses.

  6. 杜梨CPI基因的克隆、序列分析及表达%Cloning, sequencing and expression of a cysteine proteinase inhibitor gene (PbCPI) from Pyrus betulaefolia Bunge

    Institute of Scientific and Technical Information of China (English)

    李慧; 丛郁; 常有宏; 蔺经; 盛宝龙

    2011-01-01

    植物半胱氨酸蛋白酶抑制剂(Cysteine proteinase inhibitor,CPI)在植物的抗逆基因工程中发挥着越来越重要的作用,分离和克隆植物CPI基因进而研究该基因的功能是植物抗逆基因工程研究的热点.为从分子水平上揭示CPI基因在杜梨防御机制中所起的作用,利用RACE和PCR方法,从杜梨种子中克隆CPI基因的cDNA和DNA序列,并采用跨内含子表达引物进行半定量RT-PCR来分析该基因在不同胁迫条件下的表达情况.结果表明:PbCPI基因cDNA长度为987 bp,开放阅读框包含738个核苷酸,编码1个由信号肽(26个氨基酸)和成熟肽(219个氨基酸)组成的多肽.该多肽预测的等电点为6.68,估计的相对分子质量为27 190.其对应基因组DNA序列由3个外显子(1 ~302 bp,401 ~772 bp,1615~1 897 bp)和2个内含子(303~400 bp,773~1 614 bp)组成.通过PSORT进行亚细胞定位分析发现PbCPI蛋白位于内质网上.PbCPI基因编码的多肽具有植物CPI产生抑制活性所必需的一级结构:2个甘氨酸残基( Gly46-Gly47)、假定的反应域QXVXG(Q90 -V91 -V92 -A93 -G94)和A/PW基序(p120-w121);并包含植物CPI家族高度保守的特征序列模式LARFAVQEHN、QVVAG和YQAKVWVKPW.进化树分析表明PbCP1和蔷薇科植物CPI蛋白位于分子进化树的同一发育分支上,并且与苹果MdCPI(AAO19652)蛋白具有较高的一致性(95.92%).杜梨叶片中PbCPI为诱导型表达,高温(30℃)、低温(4℃)、NaCl、机械损伤、MeJA或ABA处理4h后其表达量明显上调,即其对温度胁迫、盐碱、机械损伤和外源激素处理均存在转录响应,这表明该基因参与了杜梨对生物或非生物胁迫的防御机制.%Plant cysteine proteinase inhibitor (CPI) has played more and more important roles in the fields of plant genetic engineering for resistance to adverse environments. It is one of the hot issues to isolate and validate CPI gene functions in the stress-tolerance gene engineering at present

  7. Silencing Brassinosteroid Receptor BRI1 Impairs Herbivory-elicited Accumulation of Jasmonic Acid-isoleucine and Diterpene Glycosides, but not Jasmonic Acid and Trypsin Proteinase Inhibitors in Nicotiana attenuata

    Institute of Scientific and Technical Information of China (English)

    Da-Hai Yang; lan T.Baldwin; Jianqiang Wu

    2013-01-01

    The brassinosteroid (BR) receptor,BR insensitive 1 (BRI1),plays a critical role in plant development,but whether BRI1-mediated BR signaling is involved in plant defense responses to herbivores was largely unknown.Here,we examined the function of BRI1 in the resistance of Nicotiana attenuata (Solanaceae) to its specialist insect herbivore Manduca sexta.Jasmonic acid (JA) and JA-isoleucine conjugate (JA-Ile) are important hormones that mediate resistance to herbivores and we found that after wounding or simulated herbivory NaBRI1 had little effect on JA levels,but was important for the induction of JA-Ile.Further experiments revealed that decreased JAR (the enzyme for JA-Ile production) activity and availability of lie in NaBRI1-silenced plants were likely responsible for the low JA-Ile levels.Consistently,M.sexta larvae gained more weight on NaBRI1-silenced plants than on the control plants.Quantification of insect feeding-induced secondary metabolites revealed that silencing NaBRI1 resulted in decreased levels of carbon-rich defensive secondary metabolites (hydroxygeranyllinalool diterpene glycosides,chlorogenic acid,and rutin),but had little effect on the nitrogen-rich ones (nicotine and trypsin proteinase inhibitors).Thus,NaBRI1-mediated BR signaling is likely involved in plant defense responses to M.sexta,including maintaining JA-Ile levels and the accumulation of several carbon-rich defensive secondary metabolites.

  8. Controlled intracellular proteolysis during postpartal involution of the uterus: characterization and regulation of an alkaline proteinase.

    Science.gov (United States)

    Roth, M; Hoechst, M; Afting, E G

    1981-01-01

    The postpartal involution of the uterus is predominantly due to cellular hypotrophy. This implies an intracellular proteolytic system which must be carefully controlled pre and post partum. We have characterized and partially purified a proteinase with an alkaline pH-optimum of activity and a proteinase inhibitor protein which inhibits this proteinase very strongly. The alkaline proteinase copurifies with the actomyosin complex of the uterine myometrium and degrades the actomyosin complex with a concomitant loss of its myosin-ATPase activity. The alkaline proteinase is a very labile enzyme, markedly sensitive to SH-group modifying agents and has very high molecular weight at the present state of purification. This proteolytic enzyme could specifically be separated from the main components of the actomyosin complex by extraction with low ionic strength phosphate buffers. The proteinase inhibitor protein may control the activity of this alkaline proteinase during pregnancy and involution. The inhibitor protein raises 15-fold during pregnancy, possibly blocks important steps of intracellular proteolysis and permits organ growth. The dramatic fall of the inhibitor protein activity after parturition, which precedes the loss of weight, could release the proteolytic system, including the alkaline proteinase, and permits controlled intracellular degradation.

  9. Localization and possible role of membrane type metallo-proteinase and tissue inhibitors of metalloproteinase-1 in early stages of placentation

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Human placental tissues from the first and second trimesters of gestation have been investigated using riboprobe in situ hybridisation of mRNA sequences coding for membrane type metalloproteinase (MT-1-MMP) and tissue inhibitors of metalloproteinase-1 (TIMP-1). Results show that (i) both mRNAs express at a relatively high level in the chorion laeve trophoblast cells and the adjacent decidual cells of fetal membrane; (ii) the most abundant expression of the two mRNAs was found in the extravillous trophoblast between Rohrs and Nitabuch striae of basal plate, trophoblast shell and gland cells of the decidua; (iii) isolated or small groups of cytotrophoblast cells in the chorionic villi and in the cells lining arterioles in decidua and stem villi also expressed both MT-1-MMP and TIMP-1 at defferent extents. The data suggest that the coordinated expression of the MT-MMP and its inhibitor TIMP in defferent cells of the placental tissue may play an essential role in trophoblast invasion and angiogenesis related to placentation in the first two trimesters of gestation. They may also have an ability to effect separation of fetal from material tissue at a favorable junctional site during parturition.

  10. Multigene family for Bowman-Birk type proteinase inhibitors of wild soja and soybean: the presence of two BBI-A genes and pseudogenes.

    Science.gov (United States)

    Deshimaru, Masanobu; Yoshimi, Shingo; Shioi, Seijiro; Terada, Shigeyuki

    2004-06-01

    Genes for Bowman-Birk type protease inhibitors (BBIs) of wild soja (Glycine soja) and soybean (Glycine max) comprise a multigene family. The organization of the genes for wild soja BBIs (wBBIs) was elucidated by an analysis of their cDNAs and the corresponding genomic sequences, and compared with the counterparts in the soybean. The cDNAs encoding three types of wild soja BBIs (wBBI-A, -C, and -D) were cloned. Two subtypes of cDNAs for wBBI-A, designated wBBI-A1 and -A2, were further identified. Similar subtypes (sBBI-A1 and -A2) were also found in the soybean genome. cDNA sequences for wBBIs were highly homologous to those for the respective soybean homologs. Phylogenetic analysis of these cDNAs demonstrated the evolutional proximity between these two leguminae strains.

  11. Plasma levels of alpha1-antichymotrypsin and secretory leukocyte proteinase inhibitor in healthy and chronic obstructive pulmonary disease (COPD subjects with and without severe α1-antitrypsin deficiency

    Directory of Open Access Journals (Sweden)

    Sveger Tomas

    2007-01-01

    Full Text Available Abstract Background Individuals with severe Z α1-antitrypsin (AAT deficiency have a considerably increased risk of developing chronic obstructive lung disease (COPD. It has been hypothesized that compensatory increases in levels of other protease inhibitors mitigate the effects of this AAT deficiency. We analysed plasma levels of AAT, α1-antichymotrypsin (ACT and secretory leukocyte protease inhibitor (SLPI in healthy (asymptomatic and COPD subjects with and without AAT deficiency. Methods Studied groups included: 71 asymptomatic AAT-deficient subjects (ZZ, n = 48 and SZ, n = 23, age 31 ± 0.5 identified during Swedish neonatal screening for AAT deficiency between 1972 and 1974; age-matched controls (MM, n = 57, age 30.7 ± 0.6; older asymptomatic ZZ (n = 10; healthy MM (n = 20, age 53 ± 9.6; and COPD patients (ZZ, n = 10, age 47.4 ± 11 and MM, n = 10, age 59.4 ± 6.7. Plasma levels of SLPI, AAT and ACT were analysed using ELISA and immunoelectrophoresis. Results No significant difference was found in plasma ACT and SLPI levels between the healthy MM and the ZZ or SZ subjects in the studied groups. Independent of the genetic variant, subjects with COPD (n = 19 had elevated plasma levels of SLPI and ACT relative to controls (n = 153 (49.5 ± 7.2 vs 40.7 ± 9.1 ng/ml, p Conclusion Our findings show that plasma levels of ACT and SLPI are not elevated in subjects with genetic AAT deficiency compared MM controls and do not appear to compensate for the deficiency of plasma AAT.

  12. Inibidores de proteases de hospedeiros nativos e exóticos e sua ação em intestinos de lagartas de Thyrinteina leucoceraea Proteinase inhibitors of novel and native host plants and their action in midgut of Thyrinteina leucoceraea caterpillars

    Directory of Open Access Journals (Sweden)

    Jeanne Scardini Marinho

    2008-12-01

    hosts (also Myrtaceae in Brazil and introduced from Australia, suffer attacks by T. leucoceraea, which became a severe pest of this plant. Plants can defend themselves against herbivores using proteinase inhibitors which reduce insect development and lead them to death. Thus, based on studies on the development of T. leucoceraea caterpillars on these two hosts and plant defense, this work aimed to verify the production of proteinase inhibitors by guava and eucalyptus plants upon T. leucoceraea attack, and to observe the biochemical response of the midgut of the caterpillars to these inhibitors. Eucalyptus plants produced more proteinase inhibitors than guava plants. The good development of T. leucoceraea in eucalyptus plants despite the high concentration of proteinase inhibitors may be due to an increase of enzyme activity in the caterpillars' midgut. Our data suggest that T. leucoceraea developed an adaptation to the proteinase inhhibitor produced by eucalyptus plants, by increasing serine-proteinase and cys-proteinase activities.

  13. Metalloproteinase activity secreted by fibrogenic cells in the processing of prolysyl oxidase. Potential role of procollagen C-proteinase.

    Science.gov (United States)

    Panchenko, M V; Stetler-Stevenson, W G; Trubetskoy, O V; Gacheru, S N; Kagan, H M

    1996-03-22

    Lysyl oxidase is secreted from fibrogenic cells as a 50-kDa proenzyme that is proteolytically processed to the mature enzyme in the extracellular space. To characterize the secreted proteinase activity, a truncated, recombinant form of lysyl oxidase was prepared as a proteinase substrate containing the sequence of the propeptide cleavage region. The processing proteinase activity secreted by cultured fibrogenic cells resists inhibitors of serine or aspartyl proteinases as well as tissue inhibitor of matrix metalloproteinases-2 (MMP-2) but is completely inhibited by metal ion chelators. Known metalloproteinases were tested for their activity toward this substrate. Carboxyl-terminal procollagen proteinase (C-proteinase), MMP-2, and conditioned fibrogenic cell culture medium cleave the lysyl oxidase substrate to the size of the mature enzyme. The NH2-terminal sequence generated by arterial smooth muscle conditioned medium and the C-proteinase but not by MMP-2, i.e. Asp-Asp-Pro-Tyr, was identical to that previously identified in mature lysyl oxidase isolated from connective tissue. The C-proteinase activity against the model substrate was inhibited by a synthetic oligopeptide mimic of the cleavage sequence (Ac-Met-Val-Gly-Asp-Asp-Pro-Tyr-Asn-amide), whereas this peptide also inhibited the generation of lysyl oxidase activity in the medium of fetal rat lung fibroblasts in culture. In toto, these results identify a secreted metalloproteinase activity participating in the activation of prolysyl oxidase, identify inhibitors of the processing activity, and implicate procollagen C-proteinase in this role.

  14. Vector Capability of Xiphinema americanum sensu lato in California.

    Science.gov (United States)

    Griesbach, J A; Maggenti, A R

    1989-10-01

    Seven field populations of Xiphinema americanum sensu lato from California's major agronomic areas were tested for their ability to transmit two nepoviruses, including the prune brownline, peach yellow bud, and grapevine yellow vein strains of tomato ringspot virus and the bud blight strain of tobacco ringspot virus. Two field populations transmitted all isolates, one population transmitted all tomato ringspot virus isolates but failed to transmit bud blight strain of tobacco ringspot virus, and the remaining four populations failed to transmit any virus. Only one population, which transmitted all isolates, bad been associated with field spread of a nepovirus. As two California populations of Xiphinema americanum sensu lato were shown to have the ability to vector two different nepoviruses, a nematode taxonomy based on a parsimony of virus-vector relationship is not practical for these populations. Because two California populations of X. americanum were able to vector tobacco ringspot virus, commonly vectored by X. americanum in the eastern United States, these western populations cannot be differentiated from eastern populations by vector capability tests using tobacco ringspot virus.

  15. Inhibitors of lysosomal cysteine proteases

    Directory of Open Access Journals (Sweden)

    Lyanna O. L.

    2011-04-01

    Full Text Available The review is devoted to the inhibitors of cysteine proteinases which are believed to be very important in many biochemical processes of living organisms. They participate in the development and progression of numerous diseases that involve abnormal protein turnover. One of the main regulators of these proteinases is their specific inhibitors: cystatins. The aim of this review was to present current knowledge about endogenous inhibitors of lysosomal cysteine proteases and their synthetic analogs.

  16. Distribution of Xiphinema americanum and Related Species in North America.

    Science.gov (United States)

    Robbins, R T

    1993-09-01

    All species of the Xiphinema americanum-group and their synonyms are listed. The North American species reported are listed by state or province. Among these species, X. rivesi has the most widely reported distribution. Six species (X. diffusum, X. floridae, X. laevistriatum, X. luci, X. shell, and X. tarjanense) have been reported from only Florida. The reports of X. pachtaicum, X. sheri, and X. luci did not include morphometrics and need to be confirmed; X. brevicolle from California was identified before Lamberti and Bleve-Zacheo described 15 new species in 1979 and similarly needs to be confirmed. Because of the proliferation of species in this group, reports of X. americanum (sensu stricto) before 1979 are questionable. Extraction techniques for longidorids are discussed.

  17. Serological Strains of Tobacco Ringspot Virus Transmitted by Xiphinema americanum.

    Science.gov (United States)

    Rush, M C

    1970-07-01

    Five serological strains of tobacco ringspot virus isolated from naturally infected tobacco in North Carolina, and a strain isolated from watermelon in the Rio Grande Valley of Texas were transmitted from cucumber to cucumber by mass-screened and handpicked Xiphinerna americanum from North Carolina. The Eucharis mottle strain from Peru was not transmitted, indicating that a specific strain-vector relationship may exist between the geographically isolated strains from North and South America.

  18. Xiphinema americanum as Affected by Soil Organic Matter and Porosity.

    Science.gov (United States)

    Ponchillia, P E

    1972-07-01

    The effects of four soil types, soil porosity, particle size, and organic matter were tested on survival and migration of Xiphinema americanum. Survival and migration were significantly greater in silt loam than in clay loam and silty clay soils. Nematode numbers were significantly greater in softs planted with soybeans than in fallow softs. Nematode survival was greatest at the higher of two pore space levels in four softs. Migration of X. americanum through soft particle size fractions of 75-150, 150-250, 250-500, 500-700, and 700-1,000 mu was significantly greater in the middle three fractions, with the least occurring in the smallest fraction. Additions of muck to silt loam and loamy sand soils resulted in reductions in survival and migration of the nematode. The fulvic acid fraction of muck, extracted with sodium hydroxide, had a deleterious effect on nematode activity. I conclude that soils with small amounts of air-filled pore space, extremes in pore size, or high organic matter content are deleterious to the migration and survival of X. americanum, and that a naturally occurring toxin affecting this species may be present in native soft organic matter.

  19. Purification and characterization of major extracellular proteinases from Trichophyton rubrum.

    Science.gov (United States)

    Asahi, M; Lindquist, R; Fukuyama, K; Apodaca, G; Epstein, W L; McKerrow, J H

    1985-11-15

    Two extracellular proteinases that probably play a central role in the metabolism and pathogenesis of the most common dermatophyte of man, Trichophyton rubrum, were purified to homogeneity. Size-exclusion chromatography and Chromatofocusing were used to purify the major proteinases 42-fold from crude fungal culture filtrate. The major enzyme has pI 7.8 and subunit Mr 44 000, but forms a dimer of Mr approx. 90 000 in the absence of reducing agents. A second enzyme with pI 6.5 and subunit Mr 36 000, was also purified. It is very similar in substrate specificity to the major enzyme but has lower specific activity, and may be an autoproteolysis product. The major proteinase has pH optimum 8, a Ca2+-dependence maximum of 1 mM, and was inhibited by serine-proteinase inhibitors, especially tetrapeptidyl chloromethane derivatives with hydrophobic residues at the P-1 site. Kinetic studies also showed that tetrapeptides containing aromatic or hydrophobic residues at P-1 were the best substrates. A kcat./Km of 27 000 M-1 X S-1 was calculated for the peptide 3-carboxypropionyl-Ala-Ala-Pro-Phe-p-nitroanilide. The enzyme has significant activity against keratin, elastin and denatured type I collagen (Azocoll).

  20. Ribosomal and Mitochondrial DNA Analyses of Xiphinema americanum-Group Populations.

    Science.gov (United States)

    Lazarova, Stela S; Malloch, Gaynor; Oliveira, Claudio M G; Hübschen, Judith; Neilson, Roy

    2006-12-01

    The 18S ribosomal DNA (rDNA) and cytochrome oxidase I region of mitochondrial DNA (mtDNA) were sequenced for 24 Xiphinema americanum-group populations sourced from a number of geographically disparate locations. Sequences were subjected to phylogenetic analysis and compared. 18S rDNA strongly suggested that only X. pachtaicum, X. simile (two populations) and a X. americanum s.l. population from Portugal were different from the other 20 populations studied, whereas mtDNA indicated some heterogeneity between populations. Phylogenetically, based on mtDNA, an apparent dichotomy existed amongst X. americanum-group populations from North America and those from Asia, South America and Oceania. Analyses of 18S rDNA and mtDNA sequences underpin the classical taxonomic issues of the X. americanum-group and cast doubt on the degree of speciation within the X. americanum-group.

  1. Specificity of proteinase K at P2 to P3' sub-sites and its comparison to other serine proteases.

    Science.gov (United States)

    Qasim, Mohammad A

    2014-01-01

    Specificity of the commercially important serine protease, proteinase K, has been investigated by measuring free energies of association of proteinase K with turkey ovomucoid third domain inhibitor variants at contact positions P2, P1, P1', P2', and P3'. Correlations of these values were run with similar values that have been obtained for six other serine proteases. Among the six proteases, subtilisin Carlsberg shows a near perfect correlation (Pearson Product correlation coefficient = 0.93 to 0.99) with proteinase K at all of these positions. Proteinase K has only 35% sequence identity with subtilisin Carlsberg, yet, the two enzymes are nearly identical in their specificity at P2 to P3' positions. With other serine proteases such as bovine chymotrypsin, human leukocyte elastase, porcine pancreatic elastase, Streptomyces griseus protease A and B, proteinase K showed relatively poor or no correlation.

  2. Inhibitors

    Science.gov (United States)

    ... wrong place in the body. Immune Tolerance Induction (ITI) Therapy: The goal of ITI therapy is to stop the inhibitor reaction from ... body to accept clotting factor concentrate treatments. With ITI therapy, people receive large amounts of clotting factor ...

  3. Thiol-activated serine proteinases from nymphal hemolymph of the African migratory locust, Locusta migratoria migratorioides.

    Science.gov (United States)

    Hanzon, Jacob; Smirnoff, Patricia; Applebaum, Shalom W; Mattoo, Autar K; Birk, Yehudith

    2003-02-01

    Two unique serine proteinase isoenzymes (LmHP-1 and LmHP-2) were isolated from the hemolymph of African migratory locust (Locusta migratoria migratorioides) nymphs. Both have a molecular mass of about 23 kDa and are activated by thiol-reducing agents. PMSF abolishes enzymes activity only after thiol activation, while the cysteine proteinase inhibitors E-64, iodoacetamide, and heavy metals fail to inhibit the thiol-activated enzymes. The N-terminal sequence was determined for the more-abundant LmHP-2 isoenzyme. It exhibits partial homology to that of other insect serine proteinases and similar substrate specificity and inhibition by the synthetic and protein trypsin inhibitors pABA, TLCK, BBI, and STI. The locust trypsins LmHP-1 and LmHP-2 constitute a new category of serine proteases wherein the active site of the enzyme is exposed by thiol activation without cleavage of peptide bonds. PMID:12559979

  4. Estimativa da área foliar de plantas daninhas: Solanum americanum Mill Leaf area determination of weeds: Solanum americanum Mill

    Directory of Open Access Journals (Sweden)

    Gustavo R. Tofoli

    1998-12-01

    Full Text Available A maria pretinha (Solanum americanum Mill é uma planta daninha infestante de diversas culturas e além da competição pode causar outros problemas. Nos estudos envolvendo a biologia e o controle de plantas daninhas, a área foliar é uma das mais importantes características a serem avaliadas, mas tem sido pouco estudada porque sua determinação exige equipamentos sofisticados ou utiliza técnicas destrutivas. Visando obter equações que permitissem a estimativa da área foliar desta planta daninha utilizando características lineares do limbo foliar, facilmente mensuráveis em plantas no campo, foram estudadas correlações entre a área foliar real e as seguintes características das folhas: comprimento ao longo da nervura principal (C, largura máxima do limbo (L e o produto (C x L. Para tanto, foram mensuradas 200 folhas coletadas de plantas sujeitas às mais diversas condições ecológicas em que a espécie sobrevive, considerando-se todas as folhas das plantas desde que não apresentassem deformações oriundas de fatores, tais como, pragas, moléstias e granizo. Todas as equações, lineares simples, geométricas e exponenciais, permitiram boa estimativa da área foliar (Af da maria pretinha. Do ponto de vista prático, sugere-se optar pela equação linear simples envolvendo o produto (C x L, a qual apresentou o menor QM Resíduo. Assim, a estimativa da área foliar de S. americanum pode ser efetuada pela equação AF = 0,5632 x (C x L, com coeficiente de determinação (R2 de valor igual a 0,9516.Solanum americanum is a very aggressive weed that, besides competition, can cause many other problems. Despite being one of the most important parameters to be analyzed, only few studies have been carried out concerning the leaf area mainly because its determination demands sophisticated equipment or destructive techniques. Aiming to develop equations that allow to estimate the leaf area of this weed using linear measure of the leaf

  5. Redescription of Xiphinema americanum Cobb, 1913 with Comments on Its Morphometric Variations.

    Science.gov (United States)

    Lamberti, F; Golden, A M

    1984-04-01

    Xiphinema americanum is redescribed and illustrated from material collected from Arlington Cemetery, near Cobb's type locality (Falls Church, Virginia), Morphometric data showing variations within this species are given for three additional populations.

  6. The Botrytis cinerea aspartic proteinase family

    NARCIS (Netherlands)

    Have, ten A.; Espino, J.J.; Dekkers, E.; Sluyter, Van S.; Brito, N.; Kay, J.; González, C.; Kan, van J.A.L.

    2010-01-01

    The ascomycete plant pathogen Botrytis cinerea secretes aspartic proteinase (AP) activity. Functional analysis was carried out on five aspartic proteinase genes (Bcap1-5) reported previously. Single and double mutants lacking these five genes showed neither a reduced secreted proteolytic activity, n

  7. Seasonal Population Fluctuation of Xiphinema americanum and X. rivesi in New York and Pennsylvania Orchards.

    Science.gov (United States)

    Jaffee, B A; Harrison, M B; Shaffer, R L; Strang, M B

    1987-07-01

    The population fluctuation and composition of Xiphinema americanum (sensu stricto) and X. rivesi were studied in a New York apple orchard (only X. americanum present), a Pennsylvania apple orchard (both X. americanum and X. rivesi present), and a Pennsylvania peach orchard (X. americanum, X. rivesi, and X. californicum present). Few clear trends in population fluctuation or composition were observed. The adult female was the predominant stage in most sample periods, and the reproductive period was limited to late spring and early summer. Only a few of the females at any sample period were gravid. All stages were present throughout the year, and all stages overwintered. Eggs in soil were not monitored. In the Pennsylvania apple orchard, X. americanum and X. rivesi were easily separated by morphological characteristics; however, the two species did not display differences in population structure or composition. The predominance of adults, the relatively low reproductive rates, and the association of these species with stable habitats suggest that the life strategies of X. americanum and X. rivesi are K-selected as opposed to r-selected.

  8. The cysteine proteinases of the pineapple plant.

    Science.gov (United States)

    Rowan, A D; Buttle, D J; Barrett, A J

    1990-03-15

    The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct.

  9. Proteinase activity regulation by glycosaminoglycans

    Directory of Open Access Journals (Sweden)

    Tersariol I.L.S.

    2002-01-01

    Full Text Available There are few reports concerning the biological role and the mechanisms of interaction between proteinases and carbohydrates other than those involved in clotting. It has been shown that the interplay of enzymes and glycosaminoglycans is able to modulate the activity of different proteases and also to affect their structures. From the large number of proteases belonging to the well-known protease families and also the variety of carbohydrates described as widely distributed, only few events have been analyzed more deeply. The term "family" is used to describe a group of proteases in which every member shows an evolutionary relationship to at least one other protease. This relationship may be evident throughout the entire sequence, or at least in that part of the sequence responsible for catalytic activity. The majority of proteases belong to the serine, cysteine, aspartic or metalloprotease families. By considering the existing limited proteolysis process, in addition to the initial idea that the proteinases participate only in digestive processes, it is possible to conclude that the function of the enzymes is strictly limited to the cleavage of intended substrates since the destruction of functional proteins would result in normal tissue damage. In addition, the location as well as the eventual regulation of protease activity promoted by glycosaminoglycans can play an essential role in the development of several physiopathological conditions.

  10. Coronavirus 3CLpro proteinase cleavage sites: Possible relevance to SARS virus pathology

    Directory of Open Access Journals (Sweden)

    Blom Nikolaj

    2004-06-01

    Full Text Available Abstract Background Despite the passing of more than a year since the first outbreak of Severe Acute Respiratory Syndrome (SARS, efficient counter-measures are still few and many believe that reappearance of SARS, or a similar disease caused by a coronavirus, is not unlikely. For other virus families like the picornaviruses it is known that pathology is related to proteolytic cleavage of host proteins by viral proteinases. Furthermore, several studies indicate that virus proliferation can be arrested using specific proteinase inhibitors supporting the belief that proteinases are indeed important during infection. Prompted by this, we set out to analyse and predict cleavage by the coronavirus main proteinase using computational methods. Results We retrieved sequence data on seven fully sequenced coronaviruses and identified the main 3CL proteinase cleavage sites in polyproteins using alignments. A neural network was trained to recognise the cleavage sites in the genomes obtaining a sensitivity of 87.0% and a specificity of 99.0%. Several proteins known to be cleaved by other viruses were submitted to prediction as well as proteins suspected relevant in coronavirus pathology. Cleavage sites were predicted in proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR, transcription factors CREB-RP and OCT-1, and components of the ubiquitin pathway. Conclusions Our prediction method NetCorona predicts coronavirus cleavage sites with high specificity and several potential cleavage candidates were identified which might be important to elucidate coronavirus pathology. Furthermore, the method might assist in design of proteinase inhibitors for treatment of SARS and possible future diseases caused by coronaviruses. It is made available for public use at our website: http://www.cbs.dtu.dk/services/NetCorona/.

  11. Luminal proteinases from Plodia interpunctella and the hydrolysis of Bacillus thuringiensis CryIA(c) protoxin.

    Science.gov (United States)

    Oppert, B; Kramer, K J; Johnson, D; Upton, S J; Mcgaughey, W H

    1996-06-01

    The ability of proteinases in gut extracts of the Indianmeal moth, Plodia interpunctella, to hydrolyze Bacillus thuringiensis (Bt) protoxin, casein, and rho-nitroanilide substrates was investigated. A polyclonal antiserum to protoxin CryIA(c) was used in Western blots to demonstrate slower protoxin processing by gut enzymes from Bt subspecies entomocidus-resistant larvae than enzymes from susceptible or kurstaki-resistant strains. Enzymes from all three strains hydrolyzed N-alpha-benzoyl-L-arginine rho-nitroanilide, N-succinyl-ala-ala-pro-phenylalanine rho-nitroanilide, and N-succinyl-ala-ala-pro-leucine rho-nitroanilide. Zymograms and activity blots were used to estimate the apparent molecular masses, number of enzymes, and relative activities in each strain. Several serine proteinase inhibitors reduced gut enzyme activities, with two soybean trypsin inhibitors, two potato inhibitors, and chymostatin the most effective in preventing protoxin hydrolysis.

  12. Soil Property Influences on Xiphinema americanum Populations as Related to Maturity of Loess-Derived Soils.

    Science.gov (United States)

    Schmitt, D P

    1973-10-01

    Field populations of Xiphinerna americanum around roots of Syringa vulgaris 'President Lincoln' were larger in Marshall silty clay loam, a medially developed loess soil, than in Monona silt loam, a minimally developed loess soil. Most X. amerieanum occurred in the top 15 cm of soil, with few below 30 cm. Maximum numbers occurred in August of both years in the Marshall soil, and in August 1969 and June 1970 in the Monona soil. Population fluctuations during the growing season were coincident with changes in soil moisture content. Although the population fluctuation pattern was the same at each depth tested, the adult-to-juvenile ratio increased in one soil while it decreased in the other. Numbers of X. americanum decreased as root weights decreased within a soil profile, but they were not correlated with root weights over all soils and depths. More X. americanum were recovered from the Marshall than from the Monona soil, but fibrous root weights were greater in the Monona soil. Survival of X. americanum in soil columns in growth chamber experiments was better in the Marshall than in the Monona soil. Movement and survival were different in identically textured Monona A and B horizon soils. Factors related to the ion exchange sites may affect X. americanum.

  13. Morphological Comparisons Between Xiphinema rivesi Daimasso and X. americanum Cobb Populations from the Eastern United States.

    Science.gov (United States)

    Wojtowlcz, M R; Golden, A M; Forer, L B; Stouffer, R F

    1982-10-01

    Though in the past Xiphinema americanum has been the most commonly reported dagger nematode in the eastern United States, our studies revealed the presence in Pennsvlvania of a previously unrecognized and unreported species related to X. americanum, Morphometric data and photomicrographs establish the identity of this form as X. rivesi and show expected variations in populations of this species from various locations. Similar data and illustrations are given for X. americanum populations from Pennsylvania and other areas, showing variations and relationships. Xiphinema rivesi is widely distributed in the fruit producing area of south-central Pennsylvania and is also reported herein from raspberry in Vermont and apple in Maryland and New York. This species is frequently found in fruit growing areas of Pennsylvania associated with tomato ringspot virus-induced diseases and is also found associated with corn, bluegrass sod, and alfalfa.

  14. Solanum americanum: reservoir for Potato virus Y and Cucumber mosaic virus in sweet pepper crops

    Directory of Open Access Journals (Sweden)

    Monika Fecury Moura

    2014-03-01

    Full Text Available Weeds can act as important reservoirs for viruses. Solanum americanum (Black nightshade is a common weed in Brazil and samples showing mosaic were collected from sweet pepper crops to verify the presence of viruses. One sample showed mixed infection between Cucumber mosaic virus (CMV and Potato virus Y (PVY and one sample showed simple infection by PVY. Both virus species were transmitted by plant extract and caused mosaic in tomato (Solanum lycopersicum cv. Santa Clara, sweet pepper (Capsicum annuum cv. Magda, Nicotiana benthamiana and N. tabaccum TNN, and local lesions on Chenopodium quinoa, C. murale and C. amaranticolor. The coat protein sequences for CMV and PVY found in S. americanum are phylogenetically more related to isolates from tomato. We conclude that S. americanum can act as a reservoir for different viruses during and between sweet pepper crop seasons.

  15. Autoactivation of proteinase A initiates activation of yeast vacuolar zymogens

    DEFF Research Database (Denmark)

    van den Hazel, H B; Kielland-Brandt, Morten; Winther, Jakob R.

    1992-01-01

    The Saccharomyces cerevisiae PEP4 gene encodes proteinase A, an aspartyl protease. pep4 mutants are defective in the activation of many vacuolar hydrolases, including proteinase B. We have expressed a pep4 mutation which directs the accumulation of pro-proteinase A with a defective active site. C...... of the mutant zymogen, owing to a strong, proteinase-B-dependent, phenotypic lag. In a proteinase-B-negative strain, processing of pro-proteinase A led to an active form of a higher molecular mass than the normal mature form....

  16. 肺间质纤维化大鼠肺组织基质金属蛋白酶及其组织抑制因子含量变化%Changes of lung tissue matrix metallo proteinase and its tissue inhibitor in pulmonary fibrosis rats

    Institute of Scientific and Technical Information of China (English)

    黄日红; 吴泰华; 张中和

    2001-01-01

    观察肺纤维化形成过程中基质金属蛋白酶(Matrix Metallo proteinas 简称MMPs)及其组织抑制因子(Tissue inhibitors of Metallo proteinases 简称TIMPs)含量的变化,探讨其在肺纤维化发病中的作用.将W istar大鼠60只,随机均分为对照组及模型组,气管内注入博莱霉素A5 5mg/kg,制备肺间质纤维化动物模型,观察注药后1、3、7、14及28d肺脏病理变化,利用酶谱法及免疫印记法分析肺组织MMP-2、MMP-9,TIMP-1的含量变化.结果显示各模型组pro-MMP-2、MMP -2、TIMP-1蛋白含量均较对照组增加,尤其7、14及28d组MMP-2较前明显增多.而MMP- 9变化不很明显.提示在肺纤维化形成过程中, pro-MMP-2、MMP-2 及TIMP-1都有所增高,MMP/TIMP比例失衡是最终导致肺间质纤维化形成的重要因素.

  17. Effect of Three Plant Species on Population Densities of Xiphinema americanum and X. rivesi.

    Science.gov (United States)

    Georgi, L L

    1988-07-01

    A taxonomic revision of the Xiphinema americanum species complex has necessitated a reexamination of the host range of species in the complex before recommendations can be made with confidence on the likelihood that specific crops will be damaged. Toward this end, populations of X. americanum and X. rivesi collected from apple orchards in eastern and western New York state were evaluated after 3 months in pots planted with cucumber, apple, or dandelion seedlings. Eastern and western New York populations of both nematode species declined on cucumber but increased to similar final densities on apple and dandelion.

  18. Diversity of Xiphinema americanum-group Species and Hierarchical Cluster Analysis of Morphometrics.

    Science.gov (United States)

    Lamberti, F; Ciancio, A

    1993-09-01

    Of the 39 species composing the Xiphinema americanum group, 14 were described originally from North America and two others have been reported from this region. Many species are very similar morphologically and can be distinguished only by a difficult comparison of various combinations of some morphometric characters. Study of morphometrics of 49 populations, including the type populations of the 39 species attributed to this group, by principal component analysis and hierarchical cluster analysis placed the populations into five subgroups, proposed here as the X. brevicolle subgroup (seven species), the X. americanum subgroup (17 species), the X. taylori subgroup (two species), the X. pachtaicum subgroup (eight species), and the X. lambertii subgroup (five species).

  19. Restriction Fragment Length Polymorphism Separates Species of the Xiphinema americanum Group.

    Science.gov (United States)

    Vrain, T C

    1993-09-01

    The Xiphinema americanum group of species is responsible for vectoring several important virus diseases to perennial crops. Variability of transmission of viruses by different species, and difficulties in separating species by morphometric measurements alone, make it essential to reassess the taxonomic position of several species in the group. The measurement of DNA sequence variability is a sensitive assay that can re-evaluate the separation of species and populations from each other. This study describes how an RFLP approach, in which the restriction sites in transcribed spacers of ribosomal repeats were detected, confirmed the separation of 16 populations of these species into X. americanum, X. rivesi, X. pacificum, and X. bricolensis.

  20. Recombinant expression and bioactivity assay of Kazal-type serine proteinase inhibitor(Fc-Kazal) from Fenneropenaeus chinensis%中国明对虾Kazal型丝氨酸蛋白酶抑制剂基因(Fc-Kazal)的重组表达及活性分析

    Institute of Scientific and Technical Information of China (English)

    黄明; 刘逸尘; 张亦陈; 孙妍; 孙金生

    2011-01-01

    Kazal型丝氨酸蛋白酶抑制剂可以通过精确调控丝氨酸蛋白酶的活力,在生物体的防御应答等众多生物过程中发挥重要作用.以前期克隆的中国明对虾Kaza1型丝氨酸蛋白酶抑制剂基因(Fc-Kazal,GenBank注册号为DQ318856)为基础,对其功能结构域进行序列比对和进化分析;组织表达分析结果表明,该基因在血细胞、鳃和淋巴器官等组织中高水平表达,而在眼柄、神经和肌肉中无表达;利用原核表达系统对该基因成熟肽区域成功进行了重组表达,纯化后的目的蛋白最终得率为0.4 g/L培养液;活性分析结果显示,复性后的rFc-Kazal对鳗弧菌、金黄色葡萄球菌、杀鲑气单胞菌、苏云金芽孢杆菌有明显的抑菌作用.%Chinese shrimp (Fenneropenaeus chinensis) is one of the most important aquaculture animals in China. The studies on the innate immune responses of shrimp, especially on immune defense against the main crustacean pathogens, will provide more knowledge of shrimp immunity to prevent infectious diseases. Arthropod innate defence responses[e. G. Prophenoloxidase(proPO) activation and Toll pathway initiation] and many other biological processes are mediated by serine proteinase( SP) cascades. If the activity of SPs is out of control, it will be fatal to organisms. Serine protease inhibitors ( SPI) play a critical role in precise regulation of SP activity, and also directly participate in the selection and digestion of pathogen. One of the well known SPI is the Kazal-type SPI which are usually multi-domain proteins containing more than one Kazal domain. Each domain contains 50-60 amino acids with six cysteine residues forming a 1 -5,2 -4, 3-6 disulphide bridges resulting in a characteristic three-dimensional structure. The inhibitory specificity of a Kazal domain varies with a different reactive PI amino acid, which is the second amino acid after the second Cys. However,the knowledge about the Kazal-type SPI in

  1. Ethylene-regulated expression of a carnation cysteine proteinase during flower petal senescence.

    Science.gov (United States)

    Jones, M L; Larsen, P B; Woodson, W R

    1995-06-01

    The senescence of carnation (Dianthus caryophyllus L.) flower petals is regulated by the phytohormone ethylene and is associated with considerable catabolic activity including the loss of protein. In this paper we present the molecular cloning of a cysteine proteinase and show that its expression is regulated by ethylene and associated with petal senescence. A 1600 bp cDNA was amplified by polymerase chain reaction using a 5'-specific primer and 3'-nonspecific primer designed to amplify a 1-aminocyclopropane-1-carboxylate synthase cDNA from reverse-transcribed stylar RNA. The nucleotide sequence of the cloned product (pDCCP1) was found to share significant homology to several cysteine proteinases rather than ACC synthase. A single open reading frame of 428 amino acids was shown to share significant homology with other plant cysteine proteinases including greater than 70% identity with a cysteine proteinase from Arabidopsis thaliana. Amino acids in the active site of cysteine proteinases were conserved in the pDCCP1 peptide. RNA gel blot analysis revealed that the expression of pDCCP1 increased substantially with the onset of ethylene production and senescence of petals. Increased pDCCP1 expression was also associated with ethylene production in other senescing floral organs including ovaries and styles. The pDCCP1 transcript accumulated in petals treated with exogenous ethylene within 3 h and treatment of flowers with 2,5-norbornadiene, an inhibitor of ethylene action, prevented the increase in pDCCP1 expression in petals. The temporal and spatial patterns of pDCCP1 expression suggests a role for cysteine proteinase in the loss of protein during floral senescence.

  2. Parasitism of Xiphinema rivesi and X. americanum by Zoosporic Fungi.

    Science.gov (United States)

    Jafee, B A

    1986-01-01

    Living Xiphinema americanum (Xa) and X. rivesi (Xr) extracted from soil samples and stored for 1-5 days at 4 or 20 C contained aseptate fungal hyphae. The fungi directly penetrated the nematode's cuticle from spores encysted near the head. Penetration through the stoma, vulva, or anus was rare. Catenaria anguillulae (Cat), Lagenidium caudatura (Lag), Aphanomyces sp. (Aph), and Leptolegnia sp. (Lep) were isolated into pure culture from infected nematodes. The pathogenicity of these zoosporic fungi was determined by incubating mixed freshly extracted Xa and Xr in 2% soil extract (pH = 6.7, conductivity = 48 mumhos, 20 +/- 2 C) containing zoospores obtained from single-spore isolates. After 4 days, Cat, Lag, Aph, and Lep had infected 78, 18, 13, and 22%, respectively, of the nematodes. Both Xa and Xr were infected by every fungus; however, the relative susceptibility of Xa and Xr to these fungi was not determined. All noninoculated control nematodes remained uninfected and alive. In a second experiment, parasitism of Xa and Xr by Aph and Lep was increased when nematodes were incubated in 2% soil extract for 4 days before exposure to zoospores. In a third experiment, parasitism of Xa and Xr by Cat was greater in diluted saturation soil extract (conductivity = 100-400 mumhos) than in undiluted saturation extract (conductivity = 780 mumhos). Cat produced small zoospores (4-mum-d), bulbous infection hyphae, and assimilative hyphae of varying diameters in nematodes, whereas Lag, Aph, and Lep produced large zoospores (8-mum-d) and tubular, uniform infection and assimilative hyphae in nematodes.

  3. The Overmyer mastodon (Mammut americanum) from Fulton County, Indiana

    Science.gov (United States)

    Woodman, N.; Branstrator, J.W.

    2008-01-01

    In June 1978 the partial skeleton of an American mastodon, Mammut americanum, was salvaged from a drainage ditch in Fulton County, north-central Indiana. The remains were recovered mostly from ca. 170-260 cm below the current land surface in marl overlain by peat and peaty marl. The stratigraphy of the site indicates that the remains were deposited in a small, open-water pond that subsequently filled. The skeleton, which is 41-48% complete, is that of a mature female, ca. 30-34 y old at death based on dental eruption and wear. Postcranial bone measurements indicate that this individual was relatively large for a female. Radiocarbon dating of wood from under the pelvis of the mastodon provided a maximum date of 12,575 ? 260 14C y BP [15,550-13,850 cal y BP] for the animal, which is up to 2575 14C y before the species is believed to have become extinct. Pollen samples from the site corroborate the interpretation that the regional climate was cooler and more humid than at present and supported a mixed spruce-deciduous parkland assemblage. The relatively small size of the molars of this and other mastodons from Indiana supports the hypothesis that late-glacial mastodons - just prior to their extinction - were smaller in size relative to earlier, full-glacial conspecifics. The relationship between molar size and body size is not clear, however, and there may be geographical factors as well as a temporal influence to size variation in these animals.

  4. Plasmodium falciparum proteinases: cloning of the putative gene coding for the merozoite proteinase for erythrocyte invasion (MPEI and determination of hydrolysis sites of spectrin by Pf37 proteinase

    Directory of Open Access Journals (Sweden)

    I. Florent

    1994-01-01

    Full Text Available Numerous proteinase activities have been shown to be essential for the survival of Plasmodium falciparum. One approach to antimalarial chemotherapy, would be to block specifically one or several of these activities, by using compounds structurally analogous to the substrates of these proteinases. Such a strategy requires a detailed knowledge of the active site of the proteinase, in order to identify the best substrate for the proteinase. Aiming at developing such a strategy, two proteinases previously identified in our laboratory, were chosen for further characterization of their molecular structure and properties: the merozoite proteinase for erythrocytic invasion (MPEI, involved in the erythrocyte invasion by the merozoites, and the Pf37 proteinase, which hydrolyses human spectrin in vitro.

  5. Molecular basis of Colorado potato beetle adaptation to potato plant defence at the level of digestive cysteine proteinases

    NARCIS (Netherlands)

    Gruden, K.; Kuipers, A.G.J.; Guncar, G.; Slapar, N.; Strukelj, B.; Jongsma, M.A.

    2004-01-01

    Potato synthesises high levels of proteinase inhibitors in response to insect attack. This can adversely affect protein digestion in the insects, leading to reduced growth, delayed development and lowered fecundity. Colorado potato beetle overcomes this defence mechanism by changing the composition

  6. Rickettsia parkeri Transmission to Amblyomma americanum by Cofeeding with Amblyomma maculatum (Acari: Ixodidae) and Potential for Spillover.

    Science.gov (United States)

    Wright, Chelsea L; Sonenshine, Daniel E; Gaff, Holly D; Hynes, Wayne L

    2015-09-01

    Amblyomma americanum (L.) is a human-biting ixodid tick distributed throughout much of the southeastern United States. Rickettsia parkeri is a member of the spotted fever group rickettsiae and causes a febrile illness in humans commonly referred to as "Tidewater spotted fever" or "R. parkeri rickettsiosis." Although the Gulf Coast tick, Amblyomma maculatum Koch, is the primary vector of R. parkeri, a small proportion of A. americanum have also been shown to harbor R. parkeri. The purpose of this investigation was to determine whether R. parkeri is spilling over into A. americanum in eastern Virginia and also to determine through laboratory experiments, whether A. americanum can acquire R. parkeri by cofeeding alongside infected ticks. Of 317 wild-caught, flat adult A. americanum tested from 29 counties and independent cities in coastal Virginia, a single female A. americanum was positive for R. parkeri, suggesting that R. parkeri is spilling over into this species, but at very low rates (Rickettsia amblyommii, however, were less likely to acquire R. parkeri, suggesting that infection with R. amblyommii may prevent R. parkeri from establishing infection in A. americanum.

  7. Prokaryotic expression and purification of Trichinella pseudospiralis serine proteinase inhibitor%伪旋毛虫丝氨酸蛋白酶抑制剂基因的原核表达及其纯化

    Institute of Scientific and Technical Information of China (English)

    王艳凤; 白雪; 刘晓雷; 王楠; 丁静; 刘明远

    2014-01-01

    目的 原核表达并纯化伪旋毛虫丝氨酸蛋白酶抑制剂(Trichinella pseudospiralis serine protease inhibitor,Tpserpin)基因,并鉴定其抗原性.方法 从伪旋毛虫肌幼虫提取总RNA,RT-PCR扩增Tp-serpin基因,插入原核表达载体pET-28a(+)中,构建重组表达质粒pET-28a-Tp-serpin,转化大肠埃希菌(E coli)Rosetta gami(DE3),IPTG诱导表达,表达产物经SDS-PAGE分析后,用Ni-NTA Agarose亲和层析纯化,纯化产物经SDS-PAGE分析纯度,Western blot 鉴定反应原性.结果 重组表达质粒pET-28a-Tp-serpin经PCR、双酶切及测序鉴定证明构建正确,与GenBank中登录的Tp-serpin基因相似性达99%.表达的重组Tp-serpin蛋白相对分子量约43 000,表达量约占菌体总蛋白的40%,主要以可溶性形式存在;纯化后的重组Tp-serpin蛋白纯度达95%以上,可被伪旋毛虫感染60 d的猪血清特异性识别,具有较好的反应原性.结论 成功构建了重组表达质粒pET-28a-Tp-serpin,并在E.coli Rosetta gami(DE3)中表达了重组蛋白,为旋毛虫病的血清学诊断候选抗原的研制及开发提供了科学依据,也为阐明serpin在伪旋毛虫入侵时期调节宿主免疫反应的作用奠定了基础.

  8. Proteinase K improves quantitative acylation studies.

    Science.gov (United States)

    Fränzel, Benjamin; Fischer, Frank; Steegborn, Clemens; Wolters, Dirk Andreas

    2015-01-01

    Acetylation is a common PTM of proteins but is still challenging to analyze. Only few acetylome studies have been performed to tackle this issue. Yet, the detection of acetylated proteins in complex cell lysates remains to be improved. Here, we present a proteomic approach with proteinase K as a suitable protease to identify acetylated peptides quantitatively. We first optimized the digestion conditions using an artificial system of purified bovine histones to find the optimal protease. Subsequently, the capability of proteinase K was demonstrated in complex HEK293 cell lysates. Finally, SILAC in combination with MudPIT was used to show that quantification with proteinase K is possible. In this study, we identified a sheer number of 557 unique acetylated peptides originating from 633 acetylation sites.

  9. Efficacy of granular deltamethrin against Ixodes scapularis and Amblyomma americanum (Acari: Ixodidade) nymphs.

    Science.gov (United States)

    Schulze, T L; Jordan, R A; Hung, R W; Taylor, R C; Markowski, D; Chomsky, M S

    2001-03-01

    A single barrier application of granular deltamethrin to the woodland edges of a forested residential community in late spring significantly reduced the abundance of Ixodes scapularis Say nymphs. The application also suppressed the population of Amblyomma americanum (L.) nymphs, which recently became established in the study area. The efficacy of deltamethrin is compared with other commonly used acaricides.

  10. Epidemiology of Xiphinema americanum and Tomato ringspot virus on Red Raspberry, Rubus idaeus.

    Science.gov (United States)

    The population dynamics and the ability of Xiphinema americanum to transmit Tomato ringspot virus (ToRSV) are poorly understood in the raspberry pathosystem. Soil samples were collected monthly from 1999 through 2002 in a ToRSV infected 'Willamette' red raspberry field in Clark County, WA, USA. Pop...

  11. An acarologic survey and Amblyomma americanum distribution map with implications for tularemia risk in Missouri

    Science.gov (United States)

    Brown, H.E.; Yates, K.F.; Dietrich, G.; MacMillan, K.; Graham, C.B.; Reese, S.M.; Helterbrand, Wm. S.; Nicholson, W.L.; Blount, K.; Mead, P.S.; Patrick, S.L.; Eisen, R.J.

    2011-01-01

    In the United States, tickborne diseases occur focally. Missouri represents a major focus of several tickborne diseases that includes spotted fever rickettsiosis, tularemia, and ehrlichiosis. Our study sought to determine the potential risk of human exposure to human-biting vector ticks in this area. We collected ticks in 79 sites in southern Missouri during June 7-10, 2009, which yielded 1,047 adult and 3,585 nymphal Amblyomma americanum, 5 adult Amblyomma maculatum, 19 adult Dermacentor variabilis, and 5 nymphal Ixodes brunneus. Logistic regression analysis showed that areas posing an elevated risk of exposure to A. americanum nymphs or adults were more likely to be classified as forested than grassland, and the probability of being classified as elevated risk increased with increasing relative humidity during the month of June (30-year average). Overall accuracy of each of the two models was greater than 70% and showed that 20% and 30% of the state were classified as elevated risk for human exposure to nymphs and adults, respectively. We also found a significant positive association between heightened acarologic risk and counties reporting tularemia cases. Our study provides an updated distribution map for A. americanum in Missouri and suggests a wide-spread risk of human exposure to A. americanum and their associated pathogens in this region. Copyright ?? 2011 by The American Society of Tropical Medicine and Hygiene.

  12. Neuronal projections from the Haller's organ and palp sensilla to the synganglion of Amblyomma americanum

    Science.gov (United States)

    The present study was conducted to elucidate the neuronal pathways between peripheral olfactory and taste sensilla and the synganglion in an Ixodidae tick species. The tarsus of the front legs (olfactory nerves) and the fourth palpal segment (gustatory nerves) of unfed Amblyomma americanum males and...

  13. Proteinase-antiproteinase balance in tracheal aspirates from neonates.

    Science.gov (United States)

    Sluis, K B; Darlow, B A; Vissers, M C; Winterbourn, C C

    1994-02-01

    We wanted to identify the inhibitors of neutrophil elastase, quantify their activities in the upper airways of neonates, and relate these to the presence of active elastase and the likelihood of elastolytic injury occurring due to inhibitory capacity being overwhelmed. Activities of neutrophil elastase and its inhibitors were measured in tracheal aspirates from 17 infants, 10 of whom subsequently developed bronchopulmonary dysplasia. All aspirates contained immunologically detectable alpha 1-proteinase inhibitor (alpha 1-PI), but their inhibitory capacity against neutrophil elastase ranged from being undetectable to being in excess of the amount of alpha 1-PI detected immunologically. When the alpha 1-PI was removed from each of the aspirates, using a specific antibody, from 0-50% of the original activity remained, indicating the presence of another elastase inhibitor. Its properties were consistent with it being the low molecular mass, secretory leucoproteinase inhibitor (SLPI), also known as bronchial antileucoproteinase. The alpha 1-PI was from 0-100% active. Most of the inactive inhibitor was shown by western blotting to be complexed with elastase, with a small amount of cleaved material. There was no evidence of major oxidative inactivation. Free elastase was detected in only three of the aspirates; these had little or no detectable elastase inhibitory capacity, and most of their alpha 1-PI was complexed. Elastase load, comprising the sum of free and complexed elastase, correlated closely with myeloperoxidase activity, a recognized marker of inflammatory activity. Active SLPI levels showed a positive correlation with gestational age (r = 0.66). We conclude that most neutrophil elastase in the upper airways of ventilated infants is complexed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7909297

  14. Proteinase genes of cheese starter cultures

    NARCIS (Netherlands)

    Kok, Jan

    1991-01-01

    The proteolytic enzymes of lactococci are of eminent importance for milk fermentations. By the combined action of proteinases and peptidases milk protein is degraded to peptides and amino acids which are required for cell growth and contribute to the organoleptic properties of the foods. The importa

  15. Evaluation of DEET and eight essential oils for repellency against nymphs of the lone star tick, Amblyomma americanum (Acari: Ixodidae)

    Science.gov (United States)

    Eight commercially available essential oils (oregano, clove, thyme, vetiver, sandalwood, cinnamon, cedarwood, and peppermint) were evaluated for repellency against host-seeking nymphs of the lone star tick, Amblyomma americanum. Concentration- repellency response was established using the vertical ...

  16. Proteinases in Naegleria Fowleri (strain NF3), a pathogenic amoeba: a preliminary study.

    Science.gov (United States)

    Mat Amin, Nakisah

    2004-12-01

    Naegleria fowleri is a free-living amoeba, known as a causative agent for a fatal disease of the central nervous system (CNS) in man such as Primary amoebic meningoencephalitis (PAM). Factors contributing to its pathogenicity and its distribution in the environment have been investigated by previous researchers. In case of its pathogenicity, several enzymes such as phospolipase A and sphingomyelinase, have been proposed to probably act as aggressors in promoting PAM but no study so far have been conducted to investigate the presence of proteinase enzyme in this amoeba although a 56kDa cystein proteinase enzyme has been identified in Entamoeba histolytica as an important contributing factor in the amoeba's virulence. In this preliminary study, a pathogenic amoeba, Naegleria fowleri (strain NF3) was examined for the presence of proteinases. Samples of enzymes in this amoeba were analysed by electrophoresis using SDS-PAGE-gelatin gels. The results showed that this amoeba possesses at least two high molecular weight proteinases on gelatin gels; their apparent molecular weights are approximately 128 kDa and approximately 170 kDa. Band of approximately 128 kDa enzyme is membrane-associated and its activity is higher at alkaline pH compared with lower pH; at lower pH, its activity is greatly stimulated by DTT. The approximately 170 kDa band enzyme appears to be inactivated at pH 8.0, at lower ph its activity is higher and DTT-dependance. The activity of this enzyme is partially inhibited by inhibitor E-64 but markedly inhibited to antipain suggesting it belongs to the cysteine proteinase group.

  17. A chestnut seed cystatin differentially effective against cysteine proteinases from closely related pests.

    Science.gov (United States)

    Pernas, M; Sánchez-Monge, R; Gómez, L; Salcedo, G

    1998-12-01

    Cystatin CsC, a cysteine proteinase inhibitor from chestnut (Castanea sativa) seeds, has been purified and characterized. Its full-length cDNA clone was isolated from an immature chestnut cotyledon library. The inhibitor was expressed in Escherichia coli and purified from bacterial extracts. Identity of both seed and recombinant cystatin was confirmed by matrix-assisted laser desorption/ionization mass spectrometry analysis, two-dimensional electrophoresis and N-terminal sequencing. CsC has a molecular mass of 11,275 Da and pI of 6.9. Its amino acid sequence includes all three motifs that are thought to be essential for inhibitory activity, and shows significant identity to other phytocystatins, especially that of cowpea (70%). Recombinant CsC inhibited papain (Ki 29 nM), ficin (Ki 65 nM), chymopapain (Ki 366 nM), and cathepsin B (Ki 473 nM). By contrast with most cystatins, it was also effective towards trypsin (Ki 3489 nM). CsC is active against digestive proteinases from the insect Tribolium castaneum and the mite Dermatophagoides farinae, two important agricultural pests. Its effects on the cysteine proteinase activity of two closely related mite species revealed the high specificity of the chestnut cystatin.

  18. Roles of the Picornaviral 3C Proteinase in the Viral Life Cycle and Host Cells

    Directory of Open Access Journals (Sweden)

    Di Sun

    2016-03-01

    Full Text Available The Picornaviridae family comprises a large group of non-enveloped viruses that have a major impact on human and veterinary health. The viral genome contains one open reading frame encoding a single polyprotein that can be processed by viral proteinases. The crucial 3C proteinases (3Cpros of picornaviruses share similar spatial structures and it is becoming apparent that 3Cpro plays a significant role in the viral life cycle and virus host interaction. Importantly, the proteinase and RNA-binding activity of 3Cpro are involved in viral polyprotein processing and the initiation of viral RNA synthesis. In addition, 3Cpro can induce the cleavage of certain cellular factors required for transcription, translation and nucleocytoplasmic trafficking to modulate cell physiology for viral replication. Due to interactions between 3Cpro and these essential factors, 3Cpro is also involved in viral pathogenesis to support efficient infection. Furthermore, based on the structural conservation, the development of irreversible inhibitors and discovery of non-covalent inhibitors for 3Cpro are ongoing and a better understanding of the roles played by 3Cpro may provide insights into the development of potential antiviral treatments. In this review, the current knowledge regarding the structural features, multiple functions in the viral life cycle, pathogen host interaction, and development of antiviral compounds for 3Cpro is summarized.

  19. Amblyomma americanum as a Bridging Vector for Human Infection with Francisella tularensis.

    Directory of Open Access Journals (Sweden)

    Rinosh J Mani

    Full Text Available The γ-proteobacterium Francisella tularensis causes seasonal tick-transmitted tularemia outbreaks in natural rabbit hosts and incidental infections in humans in the south-central United States. Although Dermacentor variabilis is considered a primary vector for F. tularensis, Amblyomma americanum is the most abundant tick species in this endemic region. A systematic study of F. tularensis colonization of A. americanum was undertaken to better understand its potential to serve as an overwintering reservoir for F. tularensis and as a bridging vector for human infections. Colony-reared A. americanum were artificially fed F. tularensis subspecies holarctica strain LVS via glass capillaries and colonization levels determined. Capillary-fed larva and nymph were initially infected with 10(4 CFU/tick which declined prior to molting for both stages, but rebounded post-molting in nymphs and persisted in 53% at 10(3 to 10(8 CFU/nymph at 168 days post-capillary feeding (longest sampling time in the study. In contrast, only 18% of adults molted from colonized nymphs maintained LVS colonization at 10(1 to 10(5 CFU/adult at 168 days post-capillary feeding (longest sampling time. For adults, LVS initially colonized the gut and disseminated to salivary glands by 24 h and had an ID50 of <5CFU in mice. Francisella tularensis infected the ovaries of gravid females, but transmission to eggs was infrequent and transovarial transmission to hatched larvae was not observed. The prolonged persistence of F. tularensis in A. americanum nymphs supports A. americanum as an overwintering reservoir for F. tularensis from which seasonal epizootics may originate; however, although the rapid dissemination of F. tularensis from gut to salivary glands in adults A. americanum is compatible with intermittent feeding adult males acting as bridging vectors for incidental F. tularensis infections of humans, acquisition of F. tularensis by adults may be unlikely based on adult feeding

  20. Determination of Activity of α1-Proteinase Inhibitor by Elastase Kinetic Chromogenic Assay on Microtiter Plate%微量滴定板上的弹性蛋白酶动态显色法检测α1-蛋白酶抑制剂活性

    Institute of Scientific and Technical Information of China (English)

    周志军; 林连珍; 方亮; 周雁翔; 李策生; 彭良俊

    2011-01-01

    Objective To develop an elastase kinetic chromogenic assay on microtiter plate for determination of activity of α1-proteinase inhibitor (α1-PI). Methods Various quantities of α1-PI samples were mixed with a given quantity of elastase on a microtiter plate, and the activity of remained elastase was determined by using an appropriate chromogenic substrate. The A405 value was measured by a microtiter plate reader to analyze the validity of assay and calculate the potency of αt1-PI sample, based on which the condition for assay was optimized. The α1-PI activity was determined by BCS method to confirm the working concentration of sample.The α1-PI activities of 19 samples were determined by BCS method and elastase kinetic chromogenic assay to analyze the correlation between the determination results by two methods. Results The condition for elastase kinetic chromogenic assay was optimized as follows: the volume of sample was 50 μl; the range of standard curve was 2 ~ 14 μg/ml; the dilution and volume of elastase were 1: 1 400 and 100 μl respectively; the time for incubation was 15 min; the dilution and volume of substrate were 1: 10 and 50 μl respectively; the A405 value was continuously monitored for 10 min. The correlation coefficient of determination results of 19 samples determined by two methods was more than 0. 99. Conclusion Compared with those by BCS method, the time for determination of α1-PI activity by elastase kinetic chromogenic assay was shortened, and the variation rate of determination results of the same sample of various dilutions was low. High correlation was observed between the determination results by the two methods, indicating that BCS method might be substituted with elastase kinetic chromogenic assay to decrease the cost for determination.%目的 建立微量滴定板上的弹性蛋白酶动态显色法(以下简称微量滴定板法),用于α_1-蛋白酶抑制剂(α_1-Pro-teinase inhibitor,α_1-PI)的活性检测.方法 以微量

  1. Xiphinema americanum Cobb, 1913 (Dorylaimida: Longidoridae: espécie-praga quarentenária para o Brasil Xiphinema americanum Cobb, 1913 (Dorylaimida: Longidoridae: quarantine pest for Brazil

    Directory of Open Access Journals (Sweden)

    Giovani Greigh de Brito

    2005-02-01

    Full Text Available Diversos aspectos referentes à espécie-praga quarentenária Xiphinema americanum, incluindo características taxonômicas, distribuição geográfica, plantas hospedeiras, aspectos bioecológicos, potencial de transmissão de viroses e medidas de controle, entre outros, foram abordados nesta revisão. A dificuldade encontrada pelos especialistas na correta identificação deste nematóide quarentenário muitas vezes determina que X. americanum seja confundido com outras espécies pertencentes ao mesmo gênero. Esta praga, além de utilizar como hospedeiras várias espécies de plantas e encontrar-se estabelecida em diversas regiões do globo terrestre, constitui-se em um agente transmissor de viroses, dentre as quais algumas são também consideradas quarentenárias para vários países, incluindo o Brasil. Considerando a sua ampla distribuição geográfica somada ao seu alto potencial de transmissão de viroses, o conhecimento dos aspectos abordados nesta revisão pode auxiliar na redução do risco de introdução deste nematóide quarentenário á áreas indenes no Brasil.Different aspects concerning the quarantine pest X. americanum, including taxonomic characteristics, world distribution, host plants, viruses transmission potential, control methods and others, were reviewed. The difficulty in correctly identifying this nematode usually results in its misleading into another species belonging to the same genus. Considering that this pest has almost a worldwide distribution and that it is a virus’s vector, knowledge about it is important to avoid its introduction in new areas, like Brazil.

  2. Purification and characterization of a keratinolytic serine proteinase from Streptomyces albidoflavus.

    Science.gov (United States)

    Bressollier, P; Letourneau, F; Urdaci, M; Verneuil, B

    1999-06-01

    Streptomyces strain K1-02, which was identified as a strain of Streptomyces albidoflavus, secreted at least six extracellular proteases when it was cultured on feather meal-based medium. The major keratinolytic serine proteinase was purified to homogeneity by a two-step procedure. This enzyme had a molecular weight of 18,000 and was optimally active at pH values ranging from 6 to 9.5 and at temperatures ranging from 40 to 70 degrees C. Its sensitivity to protease inhibitors, its specificity on synthetic substrates, and its remarkably high level of NH2-terminal sequence homology with Streptomyces griseus protease B (SGPB) showed that the new enzyme, designated SAKase, was homologous to SGPB. We tested the activity of SAKase with soluble and fibrous substrates (elastin, keratin, and type I collagen) and found that it was very specific for keratinous substrates compared to SGPB and proteinase K.

  3. Purification and characterization of an elastinolytic proteinase secreted by cercariae of Schistosoma mansoni.

    Science.gov (United States)

    McKerrow, J H; Pino-Heiss, S; Lindquist, R; Werb, Z

    1985-03-25

    An elastinolytic proteinase secreted by tissue-invasive larvae of Schistosoma mansoni has been purified to homogeneity. Size-exclusion chromatography and chromatofocusing were used to purify the enzyme 18-fold from crude larval secretions. The native enzyme has a molecular weight of 30,000, a pI of 8, a pH optimum of 9, and a calcium dependence of 2 mM. A second Mr 17,000 form of the enzyme was present in crude secretions and appears to be an autoproteolysis product. The enzyme is a serine proteinase that preferentially binds tetrapeptide inhibitors or substrates with an aromatic or hydrophobic residue at the P-1 site. In addition to being active against elastin, the enzyme degrades Azocoll, gelatin, laminin, fibronectin, keratin, and type IV collagen.

  4. Teor de glicoalcalóides em Solanum americanum infestado por Aphis fabae solanella Glycoalkaloid content in Solanum americanum infested by Aphis fabae solanella

    OpenAIRE

    Marco Antonio Teixeira Zullo; João Paulo Feijão Teixeira; Dayse Soave Spoladore; André Luiz Lourenção; Condorcet Aranha

    1984-01-01

    Solanum americanum Miller, uma fonte conhecida de glicosídios de solassodina, foi encontrada infestada por Aphis fabae solanela Theob. Uma vez que esses glicosídios são matérias-primas potenciais para a síntese de outras substâncias esteroídicas, tornou-se de interesse verificar o efeito de infestação sobre o teor destes glicoalcalóides. Plantas fortemente, moderadamente e não infestadas pelo pulgão foram colhidas no campo, determinando-se o teor de glicoalcalóides esteroídicos nos caules, fo...

  5. Aspects of Biology and Development of Xiphinema americanum and Related Species.

    Science.gov (United States)

    Halbrendt, J M; Brown, D J

    1993-09-01

    Identification of Xiphinema americanum-group nematodes is based on relatively subtle morphological and morphometric differences, many of which overlap. The significance and importance of these separations cannot be assessed without a basic understanding of the biological differences between species. Currently, information is accumulating on Xiphinema biology, development, and genetics that will help to confirm or refute the current systematics of species in this group. Recently, it was demonstrated that Xiphinema species pass through either three or four juvenile stages before becoming adults. This new and fundamental information divides the genus and the X. americanum group into subgroups based on their developmental evolution and provides new insight into the taxonomy and systematic positions of the species.

  6. Morphometric Evidence for Three Juvenile Stages in Some Species of Xiphinema americanum sensu lato.

    Science.gov (United States)

    Halbrendt, J M; Brown, D J

    1992-06-01

    One to two hundred nematodes from each of seven Xiphinema americanum-group populations were measured to determine the range of stylet and body lengths for juveniles and adults. First-stage juveniles were identified by the position of the replacement odontostyle (i.e., the tip of the replacement odontostyle overlapped the base of the odontophore). Nematodes were identified as second stage if the functional odontostyle was the same length as the replacement odontostyle of the first stage. Subsequent stages were similarly identified by establishing the range of corresponding replacement and functional odontostyle lengths. In all populations examined, this procedure created natural divisions that clearly grouped nematodes by stylet and body length. Presumably these groups identified all juvenile and adult stages. Populations of X. americanum, X. rivesi, and X. californicum from the United States had three juvenile stages, but a population of X. pachtaicum from Bulgaria had four juvenile stages.

  7. Tolerância do milheto (Pennisetum americanum ao 2, 4-D Pennisetum americanum tolerance to 2,4-D

    Directory of Open Access Journals (Sweden)

    L.P. Pacheco

    2007-03-01

    Full Text Available Objetivou-se com este trabalho avaliar os efeitos do 2,4-D sobre o crescimento das plantas, a produção de massa seca e verde e a produtividade de grãos do milheto. O experimento foi realizado no período de março a junho de 2006, em Rio Verde-GO, em um Latossolo Vermelho eutroférrico. O milheto (cultivar ADR 500 foi semeado manualmente em área cultivada sob sistema de plantio direto, em espaçamento de 0,45 m, distribuindo-se 12 sementes por metro. Utilizou-se o delineamento de blocos casualizados, em esquema fatorial 4 x 4, sendo avaliadas quatro doses de 2,4-D (0, 335, 670 e 1.005 g ha-1 aplicadas em quatro épocas [10 dias após a emergência das plantas de milheto (DAE (3 folhas; 20 DAE (5 a 6 folhas expandidas; 30 DAE (início de emissão da inflorescência; e 40 DAE (florescimento pleno]. Para evitar a interferência das plantas daninhas na cultura, esta foi capinada manualmente, sempre que necessário. Não se observou nenhum sinal de intoxicação das plantas de milheto pelo 2,4-D aos 15 dias após a aplicação, independentemente da dose ou época de aplicação do herbicida. Todavia, as maiores doses de 2,4-D, em qualquer época de aplicação, provocaram menor acúmulo de massa verde e seca das plantas de milheto, quando se avaliaram os resultados no ponto de rolagem da cultura. O 2,4-D, independentemente da dose utilizada ou época de aplicação, não influenciou a produtividade de grãos do milheto.This study was carried out to evaluate herbicide 2,4-D effects on plant growth, production of dry and green matter and grain productivity. The experiment was carried out from March to June 2006, in Rio Verde,GO in soil classified as Eutroferric Red Latosol. Pennisetum americanum (cultivar ADR 500 was manually sowed in area under no-till system. A space of 0.45 m was used, with 12 seeds being sown per meter. The experiment was arranged in a randomized block design, in a 4 x 4 factorial scheme, with four 2,4-D rates (0. 335, 670 and

  8. Relationship between habitat type, fire frequency, and Amblyomma americanum populations in east-central Alabama.

    Science.gov (United States)

    Willis, Damien; Carter, Robert; Murdock, Chris; Blair, Benjie

    2012-12-01

    Ticks were collected from 20 sites in the Calhoun, Cherokee, and Cleburne Counties in east-central Alabama areas to determine the relationship between plant physiognomy, environmental variables, and tick populations. Sites investigated included various burning regimes, wildland-urban-interface (WUI), a college campus, and an unmanaged area. Amblyomma americanum (L.) (Acari: Ixodidae) dominated the tick population while Ixodes scapularis Say was not encountered. There were complex differences in tick populations among site conditions. After prescribed burning, the tick population size was small but was larger in subsequent 2- and 5-year post-burn sites. An increase in Odocoileus virginianus foraging in recently burned sites is likely responsible for this phenomenon. WUI areas had the largest tick populations likely due to Odocoileus virginianus activity in an area that provides cover, forage, and a connection to a wildlife refuge. It is possible that the likelihood of humans coming in contact with ticks and tick-borne diseases is greater in WUI areas than in unbroken contiguous forest. A. americanum showed a positive correlation with percent cover of grass and leaf litter mass and a negative relationship with pine sapling density. Variables expected to be strongly correlated with A. americanum populations such as soil moisture, canopy closure, and tree density were found to have weak correlations. PMID:23181862

  9. Cloning and tissue expression of cysteine proteinase inhibitor (CPI) gene family inNicotiana tabacum L%烟草半胱氨酸蛋白酶抑制剂(CPI)基因家族的克隆及组织表达谱分析

    Institute of Scientific and Technical Information of China (English)

    林世锋; 元野; 任学良; 邹颉; 黎瑞源; 郭玉双; 赵杰宏; 王仁刚

    2014-01-01

    运用生物信息学方法,结合RT-PCR和SMART RACE技术从烟草(Nicotiana tabacum)中克隆了4个CPI基因的全长cDNA序列,分别命名为NtCPI1、NtCPI2、NtCPI3和NtCPI4, GenBank登陆号分别为KF057988、KF057989、KF057990和KF057991。基因序列分析表明4个基因分别编码98、98、120和123个氨基酸残基的蛋白质,都具有CPI反应位点的保守基序GG、QXVXQ和A/PW,同时具有植物CPI所特有的LARFAV基序,其中NtCPI3和NtCPI4的N端还包含一段27个氨基酸残基组成的信号肽。实时荧光定量PCR试验表明,4个基因的组织表达谱很广,在根、茎、叶和芽组织中都有表达。研究结果为进一步研究半胱氨酸蛋白酶抑制剂在植物中的生理功能奠定了基础。%Full-length cDNAs of fourCPI genes includingNtCPI1、NtCPI2、NtCPI3andNtCPI4were cloned fromNicotiana tabacum L. cv. K326 using RT-PCR and SMART RACE technique. Their sequences were deposited in GenBank with accession number KF057988, KF057989, KF057990 and KF057991. Sequence analysis showed that these four genes were predicted products of 98, 98, 120 and 123 amino acid residues, respectively. In addition to the typical inhibitory motifs, i.e. central signature motif QXVXG, a GG doublet in terminal region, and A/PW residues in C-terminal part. These deduced amino acid sequences contained PhyCys-specific LARFAV-like motif in the N-terminal region, of which a N-terminal signal peptide of 27 residues was found in both NtCPI3 and NtCPI4. Meanwhile, transcripts of these four genes were found in roots, stems, leaves and buds by real-time quantitative PCR, which indicated that they were broadly expressed in tobacco. This study laid foundation for further exploring physiological functions of these cysteine proteinase inhibitor genes in plants.

  10. Kinetics of the inhibition of neutrophil proteinases by recombinant elafin and pre-elafin (trappin-2) expressed in Pichia pastoris.

    Science.gov (United States)

    Zani, Marie-Louise; Nobar, Shila M; Lacour, Sandrine A; Lemoine, Soazig; Boudier, Christian; Bieth, Joseph G; Moreau, Thierry

    2004-06-01

    Elafin and its precursor, trappin-2 or pre-elafin, are specific endogenous inhibitors of human neutrophil elastase and proteinase 3 but not of cathepsin G. Both inhibitors belong, together with secretory leukocyte protease inhibitor, to the chelonianin family of canonical protease inhibitors of serine proteases. A cDNA coding either elafin or its precursor, trappin-2, was fused in frame with yeast alpha-factor cDNA and expressed in the Pichia pastoris yeast expression system. Full-length elafin or full-length trappin-2 were secreted into the culture medium with high yield, indicating correct processing of the fusion proteins by the yeast KEX2 signal peptidase. Both recombinant inhibitors were purified to homogeneity from concentrated culture medium by one-step cationic exchange chromatography and characterized by N-terminal amino acid sequencing, Western blot and kinetic studies. Both recombinant elafin and trappin-2 were found to be fast-acting inhibitors of pancreatic elastase, neutrophil elastase and proteinase 3 with k(ass) values of 2-4 x 10(6) m(-1).s(-1), while dissociation rate constants k(diss) were found to be in the 10(-4) s(-1) range, indicating low reversibility of the complexes. The equilibrium dissociation constant K(i) for the interaction of both recombinant inhibitors with their target enzymes was either directly measured for pancreatic elastase or calculated from k(ass) and k(diss) values for neutrophil elastase and proteinase 3. K(i) values were found to be in the 10(-10) molar range and virtually identical for both inhibitors. Based on the kinetic parameters determined here, it may be concluded that both recombinant elafin and trappin-2 may act as potent anti-inflammatory molecules and may be of therapeutic potential in the treatment of various inflammatory lung diseases.

  11. Properties of a subtilisin-like proteinase from a psychrotrophic Vibrio species comparison with proteinase K and aqualysin I.

    Science.gov (United States)

    Kristjánsson, M M; Magnússon, O T; Gudmundsson, H M; Alfredsson, G A; Matsuzawa, H

    1999-03-01

    An extracellular serine proteinase purified from cultures of a psychrotrophic Vibrio species (strain PA-44) belongs to the proteinase K family of the superfamily of subtilisin-like proteinases. The enzyme is secreted as a 47-kDa protein, but under mild heat treatment (30 min at 40 degrees C) undergoes autoproteolytic cleavage on the carboxyl-side of the molecule to give a proteinase with a molecular mass of about 36 kDa that apparently shares most of the enzymatic characteristics and the stability of the 47-kDa protein. In this study, selected enzymatic properties of the Vibrio proteinase were compared with those of the related proteinases, proteinase K and aqualysin I, as representative mesophilic and thermophilic enzymes, respectively. The catalytic efficiency (kcat/Km) for the amidase activity of the cold-adapted enzyme against succinyl-AAPF-p-nitroanilide was significantly higher than that of its mesophilic and thermophilic counterparts, especially when compared with aqualysin I. The stability of the Vibrio proteinase, both towards heat and denaturants, was found to be significantly lower than of either proteinase K or aqualysin I. One or more disulfide bonds in the psychrotrophic proteinase are important for the integrity of the active enzyme structure, as disulfide cleavage, either by reduction with dithiothreitol or by sulfitolysis, led to a loss in its activity. Under the same conditions, aqualysin I was also partially inactivated by dithiothreitol, but the activity of proteinase K was unaffected. The disulfides of either proteinase K or aqualysin I were not reactive towards sulfitolysis, except under denaturing conditions, while all disulfides of the Vibrio proteinase reacted in absence of a denaturant. The reactivity of the disulfides of the proteins as a function of denaturant concentration followed the order: Vibrio proteinase > proteinase K > aqualysin I. The same order of reactivity was also observed for the inactivation of the enzymes by H2O2

  12. Biases associated with several sampling methods used to estimate abundance of Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae).

    Science.gov (United States)

    Schulze, T L; Jordan, R A; Hung, R W

    1997-11-01

    Several tick sampling methods were evaluated for ixodes scapularis Say and Amblyomma americanum (I.) in oak-dominated mixed hard-wood, pitch pine-dominated, and mixed hardwood and pine forests in coastal New Jersey. Walking surveys were more efficient for collecting I. scapularis adults than dragging by a factor of > 2:1. In contrast, drag sampling yielded nearly twice as many A. americanum adults compared with walking surveys. I. scapularis subadults were rarely collected during walking surveys. A. americanum nymphs were collected from drags approximately 3:1 over walking surveys. Twice as many A. americanum larvae were obtained from drags compared with walking surveys. All developmental stages of A. americanum responded positively to carbon dioxide. Pitfall traps and leaf litter samples collected very few ticks. Tick distribution among habitats varied significantly with the sampling method chosen, and the relative ranking of sites with respect to tick abundance varied depending on the stage of tick sampled. Failure to recognize the biases in these commonly used sampling techniques can potentially lead to incorrect conclusions that can have significant adverse public health consequences. PMID:9439115

  13. Characterization of Xiphinema americanum group species (Nematoda: Dorylaimida) and co-evolution of bacteria from the genus ‘Candidatus Xiphinematobacter’ with these nematodes

    Science.gov (United States)

    The Xiphinema americanum group contains over two-dozen different species of nematode. They are economically important because they vector nepoviruses, which cause damage to several crops. Taxonomic differentiation among species of the X. americanum complex is problematic because many of the species ...

  14. Implantation Serine Proteinases heterodimerize and are critical in hatching and implantation

    Directory of Open Access Journals (Sweden)

    Meng Guoliang

    2006-12-01

    Full Text Available Abstract Background We have recently reported the expression of murine Implantation Serine Proteinase genes in pre-implantation embryos (ISP1 and uterus (ISP1 and ISP2. These proteinases belong to the S1 proteinase family and are similar to mast cell tryptases, which function as multimers. Results Here, we report the purification and initial characterization of ISP1 and 2 with respect to their physico-chemical properties and physiological function. In addition to being co-expressed in uterus, we show that ISP1 and ISP2 are also co-expressed in the pre-implantation embryo. Together, they form a heterodimer with an approximate molecular weight of 63 kD. This complex is the active form of the enzyme, which we have further characterized as being trypsin-like, based on substrate and inhibitor specificities. In addition to having a role in embryo hatching and outgrowth, we demonstrate that ISP enzyme is localized to the site of embryo invasion during implantation and that its activity is important for successful implantation in vivo. Conclusion On the basis of similarities in structural, chemical, and functional properties, we suggest that this ISP enzyme complex represents the classical hatching enzyme, strypsin. Our results demonstrate a critical role for ISP in embryo hatching and implantation.

  15. The embryo's cystatin C and F expression functions as a protective mechanism against the maternal proteinase cathepsin S in mice.

    Science.gov (United States)

    Baston-Buest, D M; Schanz, A; Buest, S; Fischer, J C; Kruessel, J S; Hess, A P

    2010-04-01

    A successful implantation of a mammalian embryo into the maternal endometrium depends on a highly synchronized fetal-maternal dialogue involving chemokines, growth factors, and matrix-modifying enzymes. A growing body of evidence suggests an important role for proteinases playing a role in matrix degeneration and enhancing the embryo's invasive capacity and influencing the mother's immunological status in favor of the conceptus. This study focused on the expression of cathepsin S (CTSS) and its inhibitors in the murine fetal-maternal interface as well as the detection of the cellular sources of either proteinase and inhibitors. Nested RT-PCR for detection of embryonic mRNAs, immunohistochemistry of maternal and fetal tissues in B6C3F1 mice, and FACS analysis for determination of immunocompetent cell population were applied. This study shows that the cysteine proteinase CTSS is upregulated in the stroma of the implantation site, and that pregnancy induces an influx of CTSS-positive uterine natural killer cells. Compared to maternal tissues, the CTSS inhibitors cystatin F and C, but not the proteinase itself, are expressed in blastocysts. In conclusion, CTSS underlies a hormonal regulation in the maternal tissue and therewith most likely supports the embryonic implantation. The invading embryo regulates the depth of its own invasion through the expression of the cathepsin inhibitors and furthermore, interleukin-6 to activate CTSS in maternal tissues. Additionally, the observed decrease in CD3(+) cells leads to the hypothesis that cells of the cytotoxic T-cell group are down-regulated in the decidua to support the implantation and ensure the survival of the embryo.

  16. Structural Studies of the Serine-Carboxyl Proteinase Kumamolisin and the Metallopeptidase Peptidyl-Dipeptidase Dcp

    OpenAIRE

    Comellas Bigler, Mireia

    2007-01-01

    The crystal structure of the serine-carboxyl proteinase kumamolisin was solved in native form and in complex with two aldehyde inhibitors. The structures show a subtilisin-like fold with a modified catalytic triad (Ser-Glu-Asp), which allows proteolytic activity at acidic pH. The crystal structure analysis of the full-length prokumamolisin S278A exhibits an uncleaved linker segment that extends along the active-site cleft in a substrate-like manner. This evidence points to an autocatalytic cl...

  17. Design, synthesis and inhibitory effect of pentapeptidyl chloromethyl ketones on proteinase K.

    Science.gov (United States)

    Kore, Anilkumar R; Shanmugasundaram, Muthian

    2010-12-01

    The synthesis and proteolytic inhibitor function of new modified pentapeptide MeOSuc-AAAPF-CH(2)Cl 6 is described. The efficacy of 6 in inhibiting the proteolytic activity of proteinase K at a concentration of 0.10 mM allows a signal to be obtained for an exogenous target ('Xeno RNA') at 29 PCR cycles (i.e., Ct=29), whereas the control MeOSuc-AAAPV-CH₂Cl 1 requires a 7.5-fold higher concentration (0.75 mM) to produce the same Ct.

  18. Inter- and Intraspecific Variation in Wild-type and Single Female-derived Populations of Xiphinema americanum-group Nematodes.

    Science.gov (United States)

    Halbrendt, J M; Brown, D J

    1994-06-01

    Ten populations of Xiphinema americanum-group nematodes were reared from individual females to evaluate inter- and intraspecific variation under identical host and environmental conditions. Data indicated that morphometric variability of X. americanum was the result of genetic variation rather than phenotypic plasticity and that genetic heterogeneity was greater than previously thought. Morphometrics of single female derived (SFD) populations identified different genotypes present in the field populations. Stylet length was the least variable morphometric character of SFD populations, but collectively stylet measurements of all individuals formed an uninterrupted continuum ranging from 107-148 mum. Range and frequency of stylet measurements of field populations could be accounted for by the relative proportion of different genotypes in the population. Nine SFD populations were identified as X. americanum sensu stricto, and one SFD population was similar to X. californicum.

  19. Antibiotic treatment of the tick vector Amblyomma americanum reduced reproductive fitness.

    Directory of Open Access Journals (Sweden)

    Jianmin Zhong

    Full Text Available BACKGROUND: The lone star tick Amblyomma americanum is a common pest and vector of infectious diseases for humans and other mammals in the southern and eastern United States. A Coxiella sp. bacterial endosymbiont was highly prevalent in both laboratory-reared and field-collected A. americanum. The Coxiella sp. was demonstrated in all stages of tick and in greatest densities in nymphs and adult females, while a Rickettsia sp. was less prevalent and in lower densities when present. METHODOLOGY/PRINCIPAL FINDINGS: We manipulated the numbers of both bacterial species in laboratory-reared A. americanum by injecting engorged nymphs or engorged, mated females with single doses of an antibiotic (rifampin or tetracycline or buffer alone. Burdens of the bacteria after molting or after oviposition were estimated by quantitative polymerase chain reaction with primers and probes specific for each bacterial species or, as an internal standard, the host tick. Post-molt adult ticks that had been treated with rifampin or tetracycline had lower numbers of the Coxiella sp. and Rickettsia sp. and generally weighed less than ticks that received buffer alone. Similarly, after oviposition, females treated previously with either antibiotic had lower burdens of both bacterial species in comparison to controls. Treatment of engorged females with either antibiotic was associated with prolonged time to oviposition, lower proportions of ticks that hatched, lower proportions of viable larvae among total larvae, and lower numbers of viable larvae per tick. These fitness estimators were associated with reduced numbers of the Coxiella sp. but not the Rickettsia sp. CONCLUSION/SIGNIFICANCE: The findings indicate that the Coxiella sp. is a primary endosymbiont, perhaps provisioning the obligately hematophagous parasites with essential nutrients. The results also suggest that antibiotics could be incorporated into an integrated pest management plan for control of these and other

  20. Transmission of Tomato Ringspot Virus by Xiphinema americanum and X. rivesi from New York Apple Orchards.

    Science.gov (United States)

    Georgi, L L

    1988-04-01

    Populations of Xiphinema americanum and X. rivesi were collected from apple orchards in eastern and western New York and tested in the laboratory for ability to transmit tomato ringspot virus (TmRSV) to cucumber and dandelion. Populations varied in the frequency with which they transmitted TmRSV, but this variation did not correspond to variation in disease prevalence in the orchard. The lower prevalence of TmRSV-incited disease in apple trees in western New York cannot be attributed to inability of the local Xiphinema spp. to transmit TmRSV.

  1. The Microbiome of Ehrlichia-Infected and Uninfected Lone Star Ticks (Amblyomma americanum)

    Science.gov (United States)

    Trout Fryxell, R. T.; DeBruyn, J. M.

    2016-01-01

    The Lone Star tick, Amblyomma americanum, transmits several bacterial pathogens including species of Anaplasma and Ehrlichia. Amblyomma americanum also hosts a number of non-pathogenic bacterial endosymbionts. Recent studies of other arthropod and insect vectors have documented that commensal microflora can influence transmission of vector-borne pathogens; however, little is known about tick microbiomes and their possible influence on tick-borne diseases. Our objective was to compare bacterial communities associated with A. americanum, comparing Anaplasma/Ehrlichia -infected and uninfected ticks. Field-collected questing specimens (n = 50) were used in the analyses, of which 17 were identified as Anaplasma/Ehrlichia infected based on PCR amplification and sequencing of groEL genes. Bacterial communities from each specimen were characterized using Illumina sequencing of 16S rRNA gene amplicon libraries. There was a broad range in diversity between samples, with inverse Simpson’s Diversity indices ranging from 1.28–89.5. There were no statistical differences in the overall microbial community structure between PCR diagnosed Anaplasma/Ehrlichia-positive and negative ticks, but there were differences based on collection method (P < 0.05), collection site (P < 0.05), and sex (P < 0.1) suggesting that environmental factors may structure A. americanum microbiomes. Interestingly, there was not always agreement between Illumina sequencing and PCR diagnostics: Ehrlichia was identified in 16S rRNA gene libraries from three PCR-negative specimens; conversely, Ehrlichia was not found in libraries of six PCR-positive ticks. Illumina sequencing also helped identify co-infections, for example, one specimen had both Ehrlichia and Anaplasma. Other taxa of interest in these specimens included Coxiella, Borrelia, and Rickettsia. Identification of bacterial community differences between specimens of a single tick species from a single geographical site indicates that intra

  2. Effects of gamma radiation on spermatogenesis and fertility of male Amblyomma americanum (Acari: Ixodidae)

    International Nuclear Information System (INIS)

    Amblyomma americanum males were treated with 0.5, 1, 2, 3, 4, 8, and 16 krad of gamma radiation. Testes of ticks treated with 2, 3, 4, 8, and 16 krad were smaller than those of ticks irradiated at lower levels and controls. No recognizable alteration in timing of spermatogenesis was noted among the different radiation groups, but severe breakdown and depletion of germinal cells was noted at 4, 8, and 16 krad. Percent hatch of larvae from crosses of irradiated males and untreated females decreased with increasing radiation level. No hatch was observed from eggs of females mated to males treated at 2 krad or higher

  3. The Microbiome of Ehrlichia-Infected and Uninfected Lone Star Ticks (Amblyomma americanum.

    Directory of Open Access Journals (Sweden)

    R T Trout Fryxell

    Full Text Available The Lone Star tick, Amblyomma americanum, transmits several bacterial pathogens including species of Anaplasma and Ehrlichia. Amblyomma americanum also hosts a number of non-pathogenic bacterial endosymbionts. Recent studies of other arthropod and insect vectors have documented that commensal microflora can influence transmission of vector-borne pathogens; however, little is known about tick microbiomes and their possible influence on tick-borne diseases. Our objective was to compare bacterial communities associated with A. americanum, comparing Anaplasma/Ehrlichia -infected and uninfected ticks. Field-collected questing specimens (n = 50 were used in the analyses, of which 17 were identified as Anaplasma/Ehrlichia infected based on PCR amplification and sequencing of groEL genes. Bacterial communities from each specimen were characterized using Illumina sequencing of 16S rRNA gene amplicon libraries. There was a broad range in diversity between samples, with inverse Simpson's Diversity indices ranging from 1.28-89.5. There were no statistical differences in the overall microbial community structure between PCR diagnosed Anaplasma/Ehrlichia-positive and negative ticks, but there were differences based on collection method (P < 0.05, collection site (P < 0.05, and sex (P < 0.1 suggesting that environmental factors may structure A. americanum microbiomes. Interestingly, there was not always agreement between Illumina sequencing and PCR diagnostics: Ehrlichia was identified in 16S rRNA gene libraries from three PCR-negative specimens; conversely, Ehrlichia was not found in libraries of six PCR-positive ticks. Illumina sequencing also helped identify co-infections, for example, one specimen had both Ehrlichia and Anaplasma. Other taxa of interest in these specimens included Coxiella, Borrelia, and Rickettsia. Identification of bacterial community differences between specimens of a single tick species from a single geographical site indicates that

  4. [Characterization of thermal denaturation process of proteinase K by spectrometry].

    Science.gov (United States)

    Zhang, Qi-Bing; Na, Xin-Zhu; Yin, Zong-Ning

    2013-07-01

    The effect of different temperatures on the activity and conformational changes of proteinase K was studied. Methods Proteinase K was treated with different temperatures, then denatured natural substrate casein was used to assay enzyme activity, steady-state and time-resolved fluorescence spectroscopy was used to study tertiary structure, and circular dichroism was used to study secondary structure. Results show with the temperature rising from 25 to 65 degrees C, the enzyme activity and half-life of proteinase K dropped, maximum emission wavelength red shifted from 335 to 354 nm with fluorescence intensity decreasing. Synchronous fluorescence intensity of tryptophan residues decreased and that of tyrosine residues increased. Fluorescence lifetime of tryptophan residues reduced from 4. 427 1 to 4. 032 4 ns and the fraction of alpha-helix dropped. It was concluded that it is simple and accurate to use steady-state/time-resolved fluorescence spectroscopy and circular dichroism to investigate thermal stability of proteinase K. Thermal denaturation of proteinase K followed a three-state process. Fluorescence intensity of proteinase K was affected by fluorescence resonance energy transfer from tyrosine to tryptophan residues. The alpha-helix was the main structure to maintain conformational stability of enzyme active site of proteinase K.

  5. Digestive proteinases from marine organisms and their applications

    Directory of Open Access Journals (Sweden)

    Sappasith Klomklao

    2008-01-01

    Full Text Available Fish viscera have wide biotechnological potential as a source of digestive enzymes, especially proteinases. The biological diversity of fish species provides a wide array of enzymes with unique properties. Fish digestive proteolytic enzymes most commonly found include pepsin and trypsin. Those enzymes from fish viscera may have the advantages for the applications in the food industry since their temperature and other characteristics differ from homologous proteinases from warm-blooded animals. Therefore, digestive proteinases can be isolated as a value-added product from fish viscera and used as the processing aids in food industries to maximize the utilization of marine resources.

  6. Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) infection in Amblyomma americanum (Acari: Ixodidae) at Aberdeen Proving Ground, Maryland.

    Science.gov (United States)

    Stromdahl, E Y; Randolph, M P; O'Brien, J J; Gutierrez, A G

    2000-05-01

    Human monocytic ehrlichiosis (HME) is a sometimes fatal, emerging tick-borne disease caused by the bacterium Ehrlichia chaffeensis. It is frequently misdiagnosed because its symptoms mimic those of the flu. Current evidence indicates that Amblyomma americanum (L.), the lone star tick, is the major vector of HME. To determine if E. chaffeensis is present in ticks at Aberdeen Proving Ground, MD, questing A. americanum ticks were collected from 33 sites. Nucleic acid was extracted from 34 adult and 81 nymphal pools. Sequences diagnostic for E. chaffeensis from three different loci (16S rRNA, 120-kDa protein, and a variable-length polymerase chain reaction [PCR] target, or VLPT) were targeted for amplification by the PCR. Fifty-two percent of the collection sites yielded pools infected with E. chaffeensis, confirming the presence and widespread distribution of E. chaffeensis at Aberdeen Proving Ground. Analysis with the both the 120-kDa protein primers and the VLPT primers showed that genetic variance exists. A novel combination of variance for the two loci was detected in two tick pools. The pathogenic implications of genetic variation in E. chaffeensis are as yet unknown.

  7. Susceptibility of Four Tick Species Amblyomma americanum, Dermacentor variabilis, Ixodes scapularis, and Rhipicephalus sanguineus (Acari: Ixodidae) to Nootkatone

    Science.gov (United States)

    The essential oil nootkatone has shown acaricidal activity on ticks. The toxicity of nootkatone was determined in laboratory assays using a vial coating technique against unfed nymphs of four Ixodid ticks: Amblyomma americanum L., Dermacentor variabilis (Say), Ixodes scapularis Say, and Rhipicepha...

  8. The chemosensory appendage proteome of Amblyomma americanum (Acari: Ixodidae) reveals putative odorant-binding and other chemoreception-related proteins

    Science.gov (United States)

    Proteomic analyses were done on 2 chemosensory appendages of the lone star tick, Amblyomma americanum. Proteins in the fore tarsi, which contain the olfactory Haller's organ, and in the palps, that include gustatory sensilla, were compared with proteins in the third tarsi. Also, male and female tick...

  9. Bacterial proteinases as targets for the development of second-generation antibiotics.

    Science.gov (United States)

    Travis, J; Potempa, J

    2000-03-01

    The emergence of bacterial pathogen resistance to common antibiotics strongly supports the necessity to develop alternative mechanisms for combating drug-resistant forms of these infective organisms. Currently, few pharmaceutical companies have attempted to investigate the possibility of interrupting metabolic pathways other than those that are known to be involved in cell wall biosynthesis. In this review, we describe multiple, novel roles for bacterial proteinases during infection using, as a specific example, the enzymes from the organism Porphyromonas gingivalis, a periodontopathogen, which is known to be involved in the development and progression of periodontal disease. In this manner, we are able to justify the concept of developing synthetic inhibitors against members of this class of enzymes as potential second-generation antibiotics. Such compounds could not only prove valuable in retarding the growth and proliferation of bacterial pathogens but also lead to the use of this class of inhibitors against invasion by other infective organisms. PMID:10708847

  10. Using mitogenomic and nuclear ribosomal sequence data to investigate the phylogeny of the Xiphinema americanum species complex.

    Directory of Open Access Journals (Sweden)

    Inga A Zasada

    Full Text Available Nematodes within the Xiphinema americanum species complex are economically important because they vector nepoviruses which cause considerable damage to a variety of agricultural crops. The taxonomy of X. americanum species complex is controversial, with the number of putative species being the subject of debate. Accurate phylogenetic knowledge of this group is highly desirable as it may ultimately reveal genetic differences between species. For this study, nematodes belonging to the X. americanum species complex, including potentially mixed species populations, were collected from 12 geographically disparate locations across the U.S. from different crops and in varying association with nepoviruses. At least four individuals from each population were analyzed. A portion of the 18S nuclear ribosomal DNA (rDNA gene was sequenced for all individuals while the internal transcribed spacer region 1 (ITS1 of rDNA was cloned and 2 to 6 clones per individual were sequenced. Mitochondrial genomes for numerous individuals were sequenced in parallel using high-throughput DNA sequencing (HTS technology. Phylogenetic analysis of the 18S rDNA revealed virtually identical sequences across all populations. Analysis of ITS1 rDNA sequences revealed several well-supported clades, with some degree of congruence with geographic location and viral transmission, but also numerous presumably paralogous sequences that failed to form clades with other sequences from the same population. Analysis of mitochondrial DNA (mtDNA indicated the presence of three distinct monophyletic clades of X. americanum species complex nematodes. Two clades contained nematodes found in association with nepovirus and the third contained divergent mtDNA sequences from three nematode populations from the western U.S. where nepovirus was absent. The inherent heterogeneity in ITS1 rDNA sequence data and lack of informative sites in 18S rDNA analysis suggests that mtDNA may be more useful in sorting

  11. Using mitogenomic and nuclear ribosomal sequence data to investigate the phylogeny of the Xiphinema americanum species complex.

    Science.gov (United States)

    Zasada, Inga A; Peetz, Amy; Howe, Dana K; Wilhelm, Larry J; Cheam, Daravuth; Denver, Dee R; Smythe, Ashleigh B

    2014-01-01

    Nematodes within the Xiphinema americanum species complex are economically important because they vector nepoviruses which cause considerable damage to a variety of agricultural crops. The taxonomy of X. americanum species complex is controversial, with the number of putative species being the subject of debate. Accurate phylogenetic knowledge of this group is highly desirable as it may ultimately reveal genetic differences between species. For this study, nematodes belonging to the X. americanum species complex, including potentially mixed species populations, were collected from 12 geographically disparate locations across the U.S. from different crops and in varying association with nepoviruses. At least four individuals from each population were analyzed. A portion of the 18S nuclear ribosomal DNA (rDNA) gene was sequenced for all individuals while the internal transcribed spacer region 1 (ITS1) of rDNA was cloned and 2 to 6 clones per individual were sequenced. Mitochondrial genomes for numerous individuals were sequenced in parallel using high-throughput DNA sequencing (HTS) technology. Phylogenetic analysis of the 18S rDNA revealed virtually identical sequences across all populations. Analysis of ITS1 rDNA sequences revealed several well-supported clades, with some degree of congruence with geographic location and viral transmission, but also numerous presumably paralogous sequences that failed to form clades with other sequences from the same population. Analysis of mitochondrial DNA (mtDNA) indicated the presence of three distinct monophyletic clades of X. americanum species complex nematodes. Two clades contained nematodes found in association with nepovirus and the third contained divergent mtDNA sequences from three nematode populations from the western U.S. where nepovirus was absent. The inherent heterogeneity in ITS1 rDNA sequence data and lack of informative sites in 18S rDNA analysis suggests that mtDNA may be more useful in sorting out the

  12. [Inactivation of T4 phage in water environment using proteinase].

    Science.gov (United States)

    Lü, Wen-zhou; Yang, Qing-xiang; Zhang, Yu; Yang, Min; Zhu, Chun-fang

    2004-09-01

    The inactivation effectiveness of proteinase to viruses was investigated by using T4 phage as a model virus. The results showed that the inactivation effectiveness of proteinase to T4 phage was obvious. In the optimum conditions and 67.5 u/mL concentration, the inactivation rate of proteinase K to T4 phage in sterilized water and in sewage achieved 99.4% and 49.4% respectively in an hour, and achieved >99.9% and 81.1% in three hours. The inactivation rate of the industrial proteinase 1398 to T4 phage in sterilized water achieved 74.4% in an hour. The effects of pH and temperature on the inactivation effectiveness was not evident.

  13. Production of a heterologous proteinase A by Saccharomyces kluyveri

    DEFF Research Database (Denmark)

    Møller, K; Tidemand, L D; Winther, Jakob R.;

    2001-01-01

    In order to evaluate the potential of Saccharomyces kluyveri for heterologous protein production, S. kluyveri Y159 was transformed with a S. cerevisiae-based multi-copy plasmid containing the S. cerevisiae PEP4 gene, which encodes proteinase A, under the control of its native promoter. As a refer......In order to evaluate the potential of Saccharomyces kluyveri for heterologous protein production, S. kluyveri Y159 was transformed with a S. cerevisiae-based multi-copy plasmid containing the S. cerevisiae PEP4 gene, which encodes proteinase A, under the control of its native promoter......, compared to a yield of 0.40 g/g in S. cerevisiae. Overexpression of PEP4 led to the secretion of active proteinase A in both S. kluyveri and S. cerevisiae. The yield of active proteinase A during growth on glucose was found to be 3.6-fold higher in S. kluyveri than in the S. cerevisiae reference strain....

  14. Evolutionary patterns of proteinase activity in attine ant fungus gardens

    DEFF Research Database (Denmark)

    Semenova, Tatyana; Hughes, David Peter; Boomsma, Jacobus Jan;

    2011-01-01

    of evolutionary more derived fungal symbionts. This notion is also supported by buffering capacities of fungus gardens at pH 5.2 being remarkably high, and suggests that the fungal symbiont actively helps to maintain garden acidity at this specific level. Metalloproteinases dominated the activity profiles....... Conclusions: Proteinase pH optima and buffering capacities of fungal symbionts appear to have evolved remarkable adaptations to living in obligate symbiosis with farming ants. Although the functional roles of serine and metalloproteinases in fungus gardens are unknown, the differential production...... hypothesized that fungal proteinase activity may have been under selection for efficiency and that different classes of proteinases might be involved. Results: We determined proteinase activity profiles across a wide pH range for fungus gardens of 14 Panamanian species of fungus-growing ants, representing...

  15. Teor de glicoalcalóides em Solanum americanum infestado por Aphis fabae solanella Glycoalkaloid content in Solanum americanum infested by Aphis fabae solanella

    Directory of Open Access Journals (Sweden)

    Marco Antonio Teixeira Zullo

    1984-01-01

    Full Text Available Solanum americanum Miller, uma fonte conhecida de glicosídios de solassodina, foi encontrada infestada por Aphis fabae solanela Theob. Uma vez que esses glicosídios são matérias-primas potenciais para a síntese de outras substâncias esteroídicas, tornou-se de interesse verificar o efeito de infestação sobre o teor destes glicoalcalóides. Plantas fortemente, moderadamente e não infestadas pelo pulgão foram colhidas no campo, determinando-se o teor de glicoalcalóides esteroídicos nos caules, folhas e frutos. Verificou-se que esse teor de glicoalcalóides esteroídicos é dependente do órgão da planta e da fitossanidade do material vegetal. O teor médio de glicoalcalóides em plantas não infestadas é sempre maior que em plantas infestadas, tanto na planta como um todo como em cada órgão examinado. Esses resultados não asseguram que o teor de glicoalcalóides seja responsável pela resistência à infestação, porém que há uma boa correlação entre teor de glicoalcolóides e fitossanidade do material vegetal.Solanum americanum Mill, a known source of solasodina glycosides, was found to be infested by the aphids Aphis fabae solanella Theob. Since these glycosides are potential raw materials for the synthesis of steroidal drugs, it is important to know the effect of infestation on the yield of solasodine glycosides. Plants heavily, moderately and uninfested by the aphids were collected in the field, and the glycoalkaloid content of the stems, leaves, and fruits were determined. It was found that the glycoalkaloid content is dependent on the different parts of the plant and phytosanity of the plant material. The average yield of glycoalkaloids in uninfested plants was always higher than in infested plants, both in the whole plant or in each part of the plant examined. These results cannot assure that the glycoalkaloid content is responsible for the resistance, but that there is a close relationship between glycoalkaloid

  16. Proteinase 3 and prognosis of patients with acute myocardial infarction

    OpenAIRE

    Ng, Leong L.; Khan, Sohail Q; Narayan, Hafid; Quinn, Paulene; Squire, Iain B; Davies, Joan E.

    2010-01-01

    Abstract Background A multimarker approach may be useful for risk stratification in AMI patients, particularly utilising pathways that are pathophysiologically distinct. Aim Our aim was to assess the prognostic value of Proteinase 3 in patients post acute myocardial infarction (AMI). We compared the prognostic value of Proteinase 3, an inflammatory marker to an established marker N-terminal pro-B-type natriuretic peptide (NT-proBNP) post-AMI. Method We recruited 9...

  17. Purification of human leucocyte DNA: proteinase K is not necessary.

    Science.gov (United States)

    Douglas, A M; Georgalis, A M; Benton, L R; Canavan, K L; Atchison, B A

    1992-03-01

    A rapid nontoxic method for the purification of DNA from human leucocytes is described. Preliminary experiments which tested different methods of DNA purification indicated that digestion of proteins with proteinase K was unnecessary. This led to the development of a simple procedure involving lysis of the cells in SDS followed by extraction with 6 M NaCl. The method described overcomes the requirement for lengthy incubations in the presence of expensive proteinase K and subsequent extraction with toxic chemicals.

  18. The effect of calciums on molecular motions of proteinase K.

    Science.gov (United States)

    Liu, Shu-Qun; Tao, Yan; Meng, Zhao-Hui; Fu, Yun-Xin; Zhang, Ke-Qin

    2011-02-01

    The native serine protease proteinase K binds two calcium cations. It has been reported that Ca(2+) removal decreased the enzyme's thermal stability and to some extent the substrate affinity, but has discrepant effects on catalytic activity of the enzyme. Molecular dynamics simulations were performed on the Ca(2+)-bound and Ca(2+)-free proteases to investigate the mechanism by which the calciums affect the structural stability, molecular motions, and catalytic activity of proteinase K. Very similar structural properties were observed between these two forms of proteinase K during simulations; and several long-lived hydrogen bonds and salt bridges common to both forms of proteinase K were found to be crucial in maintaining the local conformations around these two Ca(2+) sites. Although Ca(2+) removal enhanced the overall flexibility of proteinase K, the flexibility in a limited number of segments surrounding the substrate-binding pockets decreased. The largest differences in the equilibrium structures of the two simulations indicate that, upon the removal of Ca(2+), the large concerted motion originating from the Ca1 site can transmit to the substrate-binding regions but not to the catalytic triad residues. In conjunction with the large overlap of the essential subspaces between the two simulations, these results not only provide insight into the dynamics of the underlying molecular mechanism responsible for the unchanged enzymatic activity as well as the decreased thermal stability and substrate affinity of proteinase K upon Ca(2+) removal, but also complement the experimentally determined structural and biochemical data.

  19. The possible involvement of D-amino acids or their metabolites in Arabidopsis cysteine proteinase/cystatin N-dependent proteolytic pathway.

    Science.gov (United States)

    Gholizadeh, A

    2015-01-01

    Cysteine proteinases and their inhibitors 'cystatins' play essential roles in plant growth and development. They are involved in various signaling pathways and in the response to wide ranges of biotic and abiotic environmental stresses. To investigate their possible influence from D-amino acids or their metabolism in vivo, Arabidopsis seedlings were allowed to grow under four physicochemically different D-amino acids including D-aspartate, D-serine, D-alanine and D-phenylalanine containing media. The reverse transcription polymerase chain reaction (R T-PCR) analysis of cysteine proteinase and cystatin gene expressions showed that the addition of D-amino acid to the plant growth media considerably induce the expression of proteinase transcript while decrease the expression level of inhibitor gene in the leaf and root tissues of the test plant in overall. Based on the obtained results the potential impact of D-amino acids or their metabolism on the activity of cysteine proteinase/cystatin-dependent proteolytic apparatus as well as their possible cooperation were predicted and discussed in the plant system.

  20. Localization of Transmissible and Nontransmissible Viruses in the Vector Nematode Xiphinema americanum.

    Science.gov (United States)

    Wang, Shouhua; Gergerich, Rose C; Wickizer, Sandra L; Kim, Kyung S

    2002-06-01

    ABSTRACT The inner lining of the food canal of nematodes that transmit plantinfecting viruses is regarded as the retention region of viruses. To characterize the location of transmissible and nontransmissible viruses in the vector nematode Xiphinema americanum, three nepoviruses, Tobacco ringspot virus (TRSV), Tomato ringspot virus(TomRSV), and Cherry leaf roll virus(CLRV), and one non-nematode-transmissible virus, Squash mosaic virus (SqMV), were evaluated for transmission efficiency and localization sites in the nematode. Transmission trials showed highest transmission efficiency for TomRSV (38% with 1 and 100% with 10 nematodes, respectively), intermediate efficiency for TRSV (27% with 1 and 65% with 10 nematodes, respectively), and no transmission for CLRV and SqMV. Electron microscopy and immunofluorescent labeling revealed that TRSV was primarily localized to the lining of the lumen of the stylet extension and the anterior esophagus, but only rarely in the triradiate lumen. Within a nematode population, particles of TRSV were no longer observed in these three regions 10 weeks after acquisition, and it is assumed that there was gradual and random loss of the virus from these areas. The percentage of nematodes that were labeled by virus-specific immunofluorescent labeling in a population of viruliferous nematodes decreased gradually after TRSV acquisition when the nematodes were placed on a nonhost of the virus, and the loss of immunofluorescent labeling paralleled the decrease in the ability of the nematode population to transmit the virus. TomRSV was localized only in the triradiate lumen based on thin-section electron microscopy. No virus-like particles were observed in any part of the food canal of nematodes that had fed on CLRV-infected plants. Virus-like particles that appeared to be partially degraded were observed only in the triradiate lumen of nematodes that had fed on SqMV-infected plants. These results clarified the status of localization of two

  1. Infection Prevalences of Common Tick-borne Pathogens in Adult Lone Star Ticks (Amblyomma americanum) and American Dog Ticks (Dermacentor variabilis) in Kentucky

    OpenAIRE

    Fritzen, Charissa M.; Huang, Junjun; Westby, Kathleen; Freye, James D.; Dunlap, Brett; Yabsley, Michael J.; Schardein, Mike; Dunn, John R.; Jones, Timothy F.; Moncayo, Abelardo C.

    2011-01-01

    Rocky Mountain spotted fever, Lyme disease, and ehrlichiosis are tick-borne diseases that are reported annually in Kentucky. We conducted a survey to describe infection prevalence of tick-borne pathogens in Amblyomma americanum and Dermacentor variabilis ticks collected in Kentucky. During 2007–2008, we collected 287 ticks (179 D. variabilis and 108 A. americanum) from canine, feral hog, horse, raccoon, white-tailed deer, and human hosts in six counties in Kentucky. Ticks were screened for Ri...

  2. A new approach to identify species in Xiphinema americanum sensu lato.

    Science.gov (United States)

    Prior, T; Hockland, S; Decraemer, W

    2010-01-01

    Four putative species belonging to the X. americanum group are known to transmit American nepoviruses and these nematodes and viruses are listed in European quarantine legislation. Identification of species in this group is therefore of particular importance for phytosanitary purposes, but is problematic because of the similar morphology of the putative species. As part of the Synthesys project BE-TAF 1769, eight collaborative institutions contributed material to Fera for study. Video technology allowed the best records of type material and in conjunction with photographic images created a 'virtual' collection of images that relies less on the deteriorating quality of museum specimens. Revised definitions of lip region and tail shape are considered to be the most stable characters for differentiation. Position of the amphidial aperture, development of the odontostyle collar and some established morphometric characters are considered of limited use although they retain value for latter stages of identification.

  3. Characterization of the bacterial communities of life stages of free living lone star ticks (Amblyomma americanum.

    Directory of Open Access Journals (Sweden)

    Amanda Jo Williams-Newkirk

    Full Text Available The lone star tick (Amblyomma americanum is an abundant and aggressive biter of humans, domestic animals, and wildlife in the southeastern-central USA and an important vector of several known and suspected zoonotic bacterial pathogens. However, the biological drivers of bacterial community variation in this tick are still poorly defined. Knowing the community context in which tick-borne bacterial pathogens exist and evolve is required to fully understand the ecology and immunobiology of the ticks and to design effective public health and veterinary interventions. We performed a metagenomic survey of the bacterial communities of questing A. americanum and tested 131 individuals (66 nymphs, 24 males, and 41 females from five sites in three states. Pyrosequencing was performed with barcoded eubacterial primers targeting variable 16S rRNA gene regions 5-3. The bacterial communities were dominated by Rickettsia (likely R. amblyommii and an obligate Coxiella symbiont, together accounting for 6.7-100% of sequences per tick. DNAs from Midichloria, Borrelia, Wolbachia, Ehrlichia, Pseudomonas, or unidentified Bacillales, Enterobacteriaceae, or Rhizobiales groups were also detected frequently. Wolbachia and Midichloria significantly co-occurred in Georgia (p<0.00001, but not in other states. The significance of the Midichloria-Wolbachia co-occurrence is unknown. Among ticks collected in Georgia, nymphs differed from adults in both the composition (p = 0.002 and structure (p = 0.002 of their bacterial communities. Adults differed only in their community structure (p = 0.002 with males containing more Rickettsia and females containing more Coxiella. Comparisons among adult ticks collected in New York and North Carolina supported the findings from the Georgia collection despite differences in geography, collection date, and sample handling, implying that the differences detected are consistent attributes. The data also suggest that some members of

  4. Aspartic proteinases in the digestive tract of marine decapod crustaceans.

    Science.gov (United States)

    Navarrete del Toro, María de Los Angeles; García-Carreño, Fernando; López, Manuel Díaz; Celis-Guerrero, Laura; Saborowski, Reinhard

    2006-08-01

    Decapod crustaceans synthesize highly active proteolytic enzymes in the midgut gland and release at least a part of them into the stomach where they facilitate the first step in peptide hydrolysis. The most common proteinases in the gastric fluid characterized so far are serine proteinases, that is, trypsin and chymotrypsin. These enzymes show highest activities at neutral or slightly alkaline conditions. The presence of acid proteinases, as they prevail in vertebrates, has been discussed contradictorily yet in invertebrates. In this study, we show that acid aspartic proteinases appear in the gastric fluid of several decapods. Lobsters Homarus gammarus showed the highest activity with a maximum at pH 3. These activities were almost entirely inhibited by pepstatin A, which indicates a high share of aspartic proteinases. In other species (Panulirus interruptus, Cancer pagurus, Callinectes arcuatus and Callinectes bellicosus), proteolytic activities were present at acid conditions but were distinctly lower than in H. gammarus. Zymograms at pH 3 showed in each of the studied species at least one, but mostly two-four bands of activity. The apparent molecular weight of the enzymes ranged from 17.8 to 38.6 kDa. Two distinct bands were identified which were inhibited by pepstatin A. Acid aspartic proteinases may play an important role in the process of extracellular digestion in decapod crustaceans. Activities were significantly higher in clawed lobster than in spiny lobster and three species of brachyurans. Therefore, it may be suggested that the expression of acid proteinases is favored in certain groups and reduced in others. PMID:16788916

  5. Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways

    Energy Technology Data Exchange (ETDEWEB)

    Lund, Leif R; Romer, John; Thomasset, Nicole; Solberg, Helene; Pyke, Charles; Bissell, Mina J; Dano, Keld; Werb, Zena

    1996-01-01

    Postlactational involution of the mammary gland is characterized by two distinct physiological events: apoptosis of the secretory, epithelial cells undergoing programmed cell death, and proteolytic degradation of the mammary gland basement membrane. We examined the spatial and temporal patterns of apoptotic cells in relation to those of proteinases during involution of the BALB/c mouse mammary gland. Apoptosis was almost absent during lactation but became evident at day 2 of involution, when {beta}-casein gene expression was still high. Apoptotic cells were then seen at least up to day 8 of involution, when {beta}-casein gene expression was being extinguished. Expression of sulfated glycoprotein-2 (SGP-2), interleukin-1{beta} converting enzyme (ICE) and tissue inhibitor of metalloproteinases-1 was upregulated at day 2, when apoptotic cells were seen initially. Expression of the matrix metalloproteinases gelatinase A and stromelysin-1 and the serine proteinase urokinase-type plasminogen activator, which was low during lactation, was strongly upregulated in parallel starting at day 4 after weaning, coinciding with start of the collapse of the lobulo-alveolar structures and the intensive tissue remodeling in involution. The major sites of mRNA synthesis for these proteinases were fibroblast-like cells in the periductal stroma and stromal cells surrounding the collapsed alveoli, suggesting that the degradative phase of involution is due to a specialized mesenchymal-epithelial interaction. To elucidate the functional role of these proteinases during involution, at the onset of weaning we treated mice systemically with the glucocorticoid hydrocortisone, which is known to inhibit mammary gland involution. Although the initial wave of apoptotic cells appeared in the lumina of the gland, the dramatic regression and tissue remodeling usually evident by day 5 was substantially inhibited by systemic treatment with hydrocortisone. mRNA and protein for gelatinase A, stromelysin

  6. Developing a rapid throughput screen for detection of nematicidal activity of plant cysteine proteinases: the role of Caenorhabditis elegans cystatins.

    Science.gov (United States)

    Phiri, A M; De Pomerai, D; Buttle, D J; Behnke, J M B

    2014-02-01

    Plant cysteine proteinases (CPs) from papaya (Carica papaya) are capable of killing parasitic nematode worms in vitro and have been shown to possess anthelmintic effects in vivo. The acute damage reported in gastrointestinal parasites has not been found in free-living nematodes such as Caenorhabditis elegans nor among the free-living stages of parasitic nematodes. This apparent difference in susceptibility might be the result of active production of cysteine proteinase inhibitors (such as cystatins) by the free-living stages or species. To test this possibility, a supernatant extract of refined papaya latex (PLS) with known active enzyme content was used. The effect on wild-type (Bristol N2) and cystatin null mutant (cpi-1(-/-) and cpi-2(-/-)) C. elegans was concentration-, temperature- and time-dependent. Cysteine proteinases digested the worm cuticle leading to release of internal structures and consequent death. Both cystatin null mutant strains were highly susceptible to PLS attack irrespective of the temperature and concentration of exposure, whereas wild-type N2 worms were generally resistant but far more susceptible to attack at low temperatures. PLS was able to induce elevated cpi-1 and cpi-2 cystatin expression. We conclude that wild-type C. elegans deploy cystatins CPI-1 and CPI-2 to resist CP attack. The results suggest that the cpi-1 or cpi-2 null mutants (or a double mutant combination of the two) could provide a cheap and effective rapid throughput C. elegans-based assay for screening plant CP extracts for anthelmintic activity.

  7. Atividade potencialmente alelopática do óleo essencial de Ocimum americanum Potentially allelophatic activity of the essential oil of Ocimum americanum

    Directory of Open Access Journals (Sweden)

    A.P.S. Souza Filho

    2009-01-01

    Full Text Available Os óleos essenciais são reconhecidos pelas suas diversificadas ações biológicas. A biodiversidade amazônica é rica em espécies de plantas produtoras de óleos essenciais. Neste trabalho, objetivou-se caracterizar a atividade potencialmente alelopática do óleo essencial de Ocimum americanum (estoraque e determinar seus efeitos sobre a germinação de sementes e o desenvolvimento de duas espécies de plantas daninhas. O óleo essencial foi testado em concentrações variando de 100 a 2.000 mg L-1, considerando seus efeitos sobre a germinação de sementes (25 ºC de temperatura constante e fotoperíodo de 12 horas e o desenvolvimento da radícula e do hipocótilo (25 ºC de temperatura constante e fotoperíodo de 24 horas das plantas daninhas malícia (Mimosa pudica e mata-pasto (Senna obtusifolia. Fatores relacionados a concentração, especificidade das plantas receptoras e parâmetros analisados foram decisivos para os efeitos obtidos. A tendência geral foi de relação positiva entre concentração e efeito inibitório. Malícia foi mais sensível aos efeitos do que mata-pasto. Comparativamente, a germinação, seguida do desenvolvimento da radícula, foi afetada pelo óleo essencial em maior magnitude, ficando o desenvolvimento do hipocótilo como o de menor sensibilidade. Os efeitos observados podem ser atribuídos à presença, no óleo essencial, de monoterpenos, monoterpenos oxigenados, sesquiterpenos, alifáticos e fenilpropanoides, com destaque para os constituintes com atividade alelopática já comprovada, como o limoneno, a cânfora e o linalol.Essential oils are known for their several biological activities. The biodiversity of the Amazon region is rich in essential-oil producing plants.The aim of this work was to study the potentially allelopathic activity of the essential oil of Ocimum americanum and to determine its effects on seed germination and growth of two weed species. Solutions of the essential oil were tested

  8. Silencing of cystatin M in metastatic oral cancer cell line MDA-686Ln by siRNA increases cysteine proteinases and legumain activities, cell proliferation and in vitro invasion.

    NARCIS (Netherlands)

    Vigneswaran, N.; Wu, J.; Nagaraj, N.; James, R.; Zeeuwen, P.L.J.M.; Zacharias, W.

    2006-01-01

    Cystatins are inhibitors of lysosomal cysteine proteinases. Cystatin M demonstrates more diverse tissue distribution, target specificity and biological function than other cystatins from the same family. We utilized small interference RNAs (siRNA) to silence cystatin M gene expression in a metastati

  9. Activity of Hydroxamic Acids from Secale cereale Against the Plant-Parasitic Nematodes Meloidogyne incognita and Xiphinema americanum.

    Science.gov (United States)

    Zasada, I A; Meyer, S L F; Halbrendt, J M; Rice, C

    2005-10-01

    ABSTRACT Cyclic hydroxamic acids are secondary metabolites found in the family Poaceae and have been implicated in the allelopathy of rye (Secale cereale). The toxicity of these compounds against plant-parasitic nematodes is unknown. DIBOA (2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one), DIMBOA (2,4-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one), and their degradation products BOA (benzoxazolin-2(3H)-one) and MBOA (6-methoxy-benzoxazolin-2(3H)-one) were screened in vitro against Meloidogyne incognita second-stage juveniles (J2) and eggs and mixed-stages of Xiphinema americanum. Xiphinema americanum was more sensitive to DIBOA and DIMBOA than M. incognita J2, with a maximum apparent mortality of 96 and 92% compared to 73 and 72% at 90 mug/ml. Eggs of M. incognita were less sensitive to the hydroxamic acids than J2; only DIBOA resulted in a 50% reduction in egg hatch, with a lethal concentration (LC(50)) of 74 mug/ml compared to 21 mug/ml for J2. When M. incognita J2 were exposed to DIBOA for 48 h and the compound was removed and replaced with water, the LC(50) value increased from 21.0 to 40.7 mug/ml. MBOA was not toxic to X. americanum or M. incognita eggs, but was toxic to M. incognita J2, with LC(50) values of 44 and 20 mug/ml before and after the compound was removed and replaced with water. BOA was the least toxic hydroxamic acid tested; it did not reduce M. incognita egg hatch after 1 week of exposure or increase X. americanum mortality after 24 h of exposure. While in vitro studies provide a valuable starting point in determining the toxicity of the chemical component of rye, the relevance of the data to soil remains to be determined.

  10. Localization and accessibility of antigenic sites of the extracellular serine proteinase of Lactococcus lactis

    NARCIS (Netherlands)

    Laan, Harm; Kok, Jan; Haandrikman, Alfred J.; Venema, Gerhardus; Konings, Wilhelmus

    1992-01-01

    Lactococcus lactis strains produce an extracellular subtilisin-related serine proteinase in which immunologically different components can be distinguished. Monoclonal antibodies specific for the different proteinase components have been raised and their epitopes were identified. By Western-blot ana

  11. Involvement of gibberellins in expression of a cysteine proteinase (SH-EP) in cotyledons of Vigna mungo seedlings.

    Science.gov (United States)

    Taneyama, M; Okamoto, T; Yamane, H; Minamikawa, T

    2001-11-01

    The expression of a papain-type proteinase, designated SH-EP, in cotyledons of Vigna mungo seedlings has been shown to require some factors in the embryonic axes. Gibberellin A1 (GA(1)) and GA(20) were identified by GC-MS in embryonic axes of V. mungo seedlings. The level of accumulation of SH-EP in cotyledons of V. mungo seedlings was greatly reduced by treatment of the seeds with uniconazole-P, an inhibitor for GA biosynthesis. The reduced level of accumulation of SH-EP in cotyledons by uniconazole-P was recovered by exogenous application of GA(1) and GA(20) to the seedlings.

  12. Pregnancy zone protein, a proteinase-binding macroglobulin. Interactions with proteinases and methylamine.

    Science.gov (United States)

    Christensen, U; Simonsen, M; Harrit, N; Sottrup-Jensen, L

    1989-11-28

    Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein, strongly related to alpha 2-macroglobulin (alpha 2M). Its properties and its reactions with a number of enzymes, particularly chymotrypsin, and with methylamine have been investigated. It is concluded that native PZP molecules are dimers of disulfide-bridged 180-kDa subunits and that proteinase binding results in covalent 1:1 (tetrameric)PZP-enzyme complexes. Native PZP is unstable, and storage should be avoided, but when kept unfrozen at 0 degree C most PZP preparations stay native 1-3 months. The reaction of PZP with chymotrypsin involves (i) proteolysis of bait regions, (ii) cleavage of beta-cysteinyl-gamma-glutamyl thiol ester groups, (iii) some change of the conformation and quaternary structure of PZP, and (iv) the formation of covalent 1:1 chymotrypsin-PZP(tetramer) complexes in which chymotrypsin is active but shows less activity than free chymotrypsin. The emission spectra of intrinsic fluorescence show significant differences between the PZP-chymotrypsin complex and its native components, whereas no differences are observed between methylamine-reacted PZP and native PZP. Methylamine reacts with the beta-cysteinyl-gamma-glutamyl thiol ester groups of PZP in a second-order process with k = (13.6 +/- 0.5) M-1 s-1, pH 7.6, 25 degrees C. The reaction product is PZP(dimers); no PZP(tetramers) are formed. The proteinase-binding specificity of PZP is far more restricted than that of alpha 2M. Certain chymotrypsin-like and trypsin-like enzymes are bound much less efficiently than is chymotrypsin itself.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Acúmulo de massa seca e de macronutrientes por plantas de Glycine max e Solanum americanum Accumulation of dry mass and macronutrients by Glycine max and Solanum americanum plants

    Directory of Open Access Journals (Sweden)

    S. Bianco

    2012-03-01

    Full Text Available A soja é uma das principais culturas agrícolas do Brasil, sendo a sua produtividade muito influenciada pela competição exercida pelas plantas daninhas. Foram realizados dois experimentos em casa de vegetação, em Jaboticabal, SP, objetivando determinar o acúmulo de massa seca, assim como a distribuição e o acúmulo de macronutrientes em plantas de soja, no período de outubro de 2000 a fevereiro de 2001, e de Solanum americanum, no período de janeiro a maio de 1995. As plantas cresceram em vasos com capacidade de 7 litros, preenchidos com areia de rio lavada e peneirada; elas foram irrigadas diariamente com solução nutritiva. Os tratamentos foram representados pelas épocas de amostragem, realizada a intervalos de 14 dias, iniciando-se 21 dias após a emergência (DAE, até 161 DAE para S. americanum e 119 DAE para soja cv. BR-16 (precoce. O ponto de máximo acúmulo teórico de massa seca deu-se aos 104 DAE para a soja (35,0 g por planta e 143 DAE para S. americanum (179,62 g por planta. Da emergência até 49 e 63 DAE, as folhas apresentaram maior participação no acúmulo de massa seca para soja e S. americanum, respectivamente. Após esses períodos, verificou-se, em ambas as espécies, inversão na representatividade das folhas por caules para a espécie daninha e por caules e, posteriormente, por estruturas reprodutivas, para a cultura. A taxa de absorção diária dos macronutrientes atingiu maiores valores entre 69 e 87 DAE para a soja e entre 105 e 119 DAE para a planta daninha. Considerando a média dos valores de pontos de inflexão observados para a cultura da soja, tem-se que aos 75 DAE uma planta de soja acumula teoricamente 23,9 g de massa seca, 564,4 mg de N, 7,1 mg de P, 490,8 mg de K, 487,0 mg de Ca, 156,6 mg de Mg e 36,0 mg de S. Para o mesmo período, uma planta de S. americanum acumula teoricamente 33,75 g de massa seca, 875,96 mg de N, 88,46 mg de P, 983,54 mg de K, 647,60 mg de Ca, 100,93 mg de Mg e 42,15 mg de

  14. Characterization and gene sequence of the precursor of elafin, an elastase-specific inhibitor in bronchial secretions.

    Science.gov (United States)

    Sallenave, J M; Silva, A

    1993-04-01

    Human bronchial mucous secretions have been shown to contain inhibitors of serine proteinases secreted by neutrophils. The role of these inhibitors is probably to control the enzymes secreted in the airways and in the lung interstitium. Three of these inhibitors have been identified and characterized: alpha 1-proteinase inhibitor, mucus proteinase inhibitor, and elafin. The elafin molecule, a 6.0 kD inhibitor of serine proteinases shows homology with mucus proteinase inhibitor. We recently isolated both molecules in bronchial secretions. In this report, we present evidence for the existence of a precursor of the elafin molecule. We have cloned and sequenced the gene for this precursor and show that it is composed of three exons. The coding information for a 117 amino acid precursor protein of elafin (inclusive of the signal peptide) is contained in the first two exons. This was confirmed at the mRNA and protein levels. By Northern Blot analysis we detected a 800 bp long product, and by immunoaffinity we detected in sputum and in cultured epithelial cell supernatant (NCI-H322 cell line) a 12 kD protein species cross-reacting with anti-elafin IgG. The finding of possible cross-linking function for the precursor in addition to its antiproteinase activity indicates a possible role for this molecule as a cross-linker agent in the extracellular matrix. PMID:8476637

  15. Successful Management of Tendinopathy With Injections of the MMP-inhibitor Aprotinin

    OpenAIRE

    Orchard, John; Massey, Andrew; Brown, Richard; Cardon-Dunbar, Adéline; Hofmann, Jamie

    2008-01-01

    Aprotinin is a broad spectrum proteinase inhibitor (including matrix metalloproteinase [MMP] inhibitor) used for treating patellar and Achilles tendinopathies. One previous randomized control trial demonstrated aprotinin injections superior to both corticosteroid and saline injections in patellar tendinopathy (Level II), whereas results reported for aprotinin treatment in Achilles tendinopathy have been mixed. We performed a case review and followup questionnaire for 430 consecutive patients ...

  16. Molecular and morphological characterisation of Xiphinema americanum group species (Nematoda:Dorylaimida)from California and other regions and co-evolution of bacteria from the genus Candidata Xiphinemobacter with nematodes.

    Science.gov (United States)

    The Xiphinema americanum group is a large species complex containing more than two dozen nematode species. They are economically important because they are vectors of nepoviruses. The species differentiation of X. americanum group is problematic because the species share similar morphological charac...

  17. Multiple pathways for vacuolar sorting of yeast proteinase A

    DEFF Research Database (Denmark)

    Westphal, V; Marcusson, E G; Winther, Jakob R.;

    1996-01-01

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

  18. PLANT PROTEASE INHIBITORS: STRATEGY FOR PEST CONTROL IN CROPS

    Directory of Open Access Journals (Sweden)

    R.S.DHANDE1 N.J.CHIKHALE2

    2014-11-01

    Full Text Available Proteinase inhibitors (PIs are naturally occurring proteins in living organisms and are able to inhibit & control the activity of proteases. PIs are a diverse group of proteins that share a common biochemical activity. The role of plant proteinase inhibitors was investigated by Mickel and Standish in 1947 when they observed the insects larvae were unable to develop normally on soybean products. Subsequently, the soybean trypsin inhibitors were found to be lethal to the flour beetle larvae, Tribolium confusum (Lipke et. al., 1954. Now there are diverse examples of protease inhibitors active against many insect species both in vitro (Pannetier et. al., 1997; Koiwa et. al., 1998 and in vivo (Urwin et. al., 1997; Vain et. al., 1998 bioassays.

  19. Structural characterization of tick cement cones collected from in vivo and artificial membrane blood-fed Lone Star ticks (Amblyomma americanum).

    Science.gov (United States)

    Bullard, Rebekah; Allen, Paige; Chao, Chien-Chung; Douglas, Jessica; Das, Pradipta; Morgan, Sarah E; Ching, Wei-Mei; Karim, Shahid

    2016-07-01

    The Lone Star tick, Amblyomma americanum, is endemic to the southeastern United States and capable of transmitting pathogenic diseases and causing non-pathogenic conditions. To remain firmly attached to the host, the tick secretes a proteinaceous matrix termed the cement cone which hardens around the tick's mouthparts to assist in the attachment of the tick as well as to protect the mouthparts from the host immune system. Cement cones collected from ticks on a host are commonly contaminated with host skin and hair making analysis of the cone difficult. To reduce the contamination found in the cement cone, we have adapted an artificial membrane feeding system used to feed long mouthpart ticks. Cones collected from in vivo and membrane fed ticks are analyzed to determine changes in the cone morphology. Comparisons of the cement cones using light microscopy shows similar structures and color however using scanning electron microscopy the cones have drastically different structures. The in vivo cones contain fibrils, sheets, and are heavily textured whereas cones from membrane fed ticks are remarkably smooth with no distinct structures. Analysis of the secondary protein structures using FTIR-ATR show both in vivo and membrane fed cement cones contain β sheets but only in vivo cement cones contain helical protein structures. Additionally, proteomic analysis using LC-MS/MS identifies many proteins including glycine rich proteins, metalloproteases, and protease inhibitors. Proteomic analysis of the cones identified both secreted and non-secreted tick proteins. Artificial membrane feeding is a suitable model for increased collection of cement cones for proteomic analysis however, structurally there are significant differences.

  20. Structural characterization of tick cement cones collected from in vivo and artificial membrane blood-fed Lone Star ticks (Amblyomma americanum).

    Science.gov (United States)

    Bullard, Rebekah; Allen, Paige; Chao, Chien-Chung; Douglas, Jessica; Das, Pradipta; Morgan, Sarah E; Ching, Wei-Mei; Karim, Shahid

    2016-07-01

    The Lone Star tick, Amblyomma americanum, is endemic to the southeastern United States and capable of transmitting pathogenic diseases and causing non-pathogenic conditions. To remain firmly attached to the host, the tick secretes a proteinaceous matrix termed the cement cone which hardens around the tick's mouthparts to assist in the attachment of the tick as well as to protect the mouthparts from the host immune system. Cement cones collected from ticks on a host are commonly contaminated with host skin and hair making analysis of the cone difficult. To reduce the contamination found in the cement cone, we have adapted an artificial membrane feeding system used to feed long mouthpart ticks. Cones collected from in vivo and membrane fed ticks are analyzed to determine changes in the cone morphology. Comparisons of the cement cones using light microscopy shows similar structures and color however using scanning electron microscopy the cones have drastically different structures. The in vivo cones contain fibrils, sheets, and are heavily textured whereas cones from membrane fed ticks are remarkably smooth with no distinct structures. Analysis of the secondary protein structures using FTIR-ATR show both in vivo and membrane fed cement cones contain β sheets but only in vivo cement cones contain helical protein structures. Additionally, proteomic analysis using LC-MS/MS identifies many proteins including glycine rich proteins, metalloproteases, and protease inhibitors. Proteomic analysis of the cones identified both secreted and non-secreted tick proteins. Artificial membrane feeding is a suitable model for increased collection of cement cones for proteomic analysis however, structurally there are significant differences. PMID:27118479

  1. Immunofluorescent Localization of Tobacco Ringspot Nepovirus in the Vector Nematode Xiphinema americanum.

    Science.gov (United States)

    Wang, S; Gererich, R C

    1998-09-01

    ABSTRACT An indirect immunofluorescent technique was developed to localize tobacco ringspot nepovirus (TRSV) in the vector nematode Xiphinema americanum sensu stricto. A population of this nematode that efficiently transmitted TRSV was given an acquisition access period of 10 days on TRSV-infected cucumber. Treatment of fragments of viruliferous nematodes with a polyclonal antiserum against TRSV followed by fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G resulted in virus-specific bright fluorescence only in the lumen of the stylet extension and esophagus. Virus-specific fluorescent signals were observed in the virus-retention region of 44% of the nematode fragments examined. The percentage of nematodes labeled with virus-specific fluorescence increased as the acquisition access period increased from 0 to 22 days; the increase paralleled the increase in the transmission efficiency of the nematode population. Visualization of the entire virus-retention region of individual nematodes within a population of vector or nonvector nematodes provides a rapid and simple means of monitoring specific attachment of plant viruses.

  2. Seed mucilage from Ocimum americanum linn. as disintegrant in tablets: Separation and evaluation

    Directory of Open Access Journals (Sweden)

    Patel D

    2007-01-01

    Full Text Available Plant products serve as an alternative to synthetic products because of local accessibility, eco-friendly nature and lower prices compared to imported synthetic products. Natural gums and mucilage have been widely explored as pharmaceutical excipients. The present study was undertaken to separate mucilage from the seeds of Ocimum americanum Linn . and explore its use as a tablet disintegrant. Methods for extraction of mucilage from the seeds were developed and the yield by the method C was found to be 14%. The mucilage was evaluated for various parameters as per Indian Pharmacopoeia. The loss on drying, ash value and microbial load were well within the official limits. The disintegrating efficiency of separated mucilage was compared with that of the starch in tablets prepared using lactose, propranolol hydrochloride and polyvinyl pyrrolidone as model diluent, drug and binder, respectively. The disintegration time for tablet formulations prepared using mucilage (10% w/w was less (154 s than that of the tablet formulations prepared using starch as a disintegrant (269 s. The mucilage not interfered with the release of a drug from tablet formulations. Formulated tablets were found stable at 45 o C for 4 weeks without significant change in hardness, disintegration time and in vitro drug release.

  3. "Candidatus Phytoplasma americanum", a phytoplasma associated with a potato purple top wilt disease complex.

    Science.gov (United States)

    Lee, Ing-Ming; Bottner, Kristi D; Secor, Gary; Rivera-Varas, Viviana

    2006-07-01

    Potato purple top wilt (PPT) is a devastating disease that occurs in various regions of North America and Mexico. At least three distinct phytoplasma strains belonging to three different phytoplasma groups (16SrI, 16SrII and 16SrVI) have been associated with this disease. A new disease with symptoms similar to PPT was recently observed in Texas and Nebraska, USA. Two distinct phytoplasma strain clusters were identified. One belongs to the 16SrI phytoplasma group, subgroup A, and the other is a novel phytoplasma that is most closely related to, and shares 96.6 % 16S rRNA gene sequence similarity with, a member of group 16SrXII. Phylogenetic analysis of 16S rRNA gene sequences of the novel PPT-associated phytoplasma strains, previously described 'Candidatus Phytoplasma' organisms and other distinct unnamed phytoplasmas indicated that the novel phytoplasma, termed American potato purple top wilt (APPTW) phytoplasma, represents a distinct lineage and shares a common ancestor with stolbur phytoplasma, "Candidatus Phytoplasma australiense", "Candidatus Phytoplasma japonicum", "Candidatus Phytoplasma fragariae", bindweed yellows phytoplasma (IBS), "Candidatus Phytoplasma caricae" and "Candidatus Phytoplasma graminis". On the basis of unique 16S rRNA gene sequences and biological properties, it is proposed that the APPTW phytoplasma represents "Candidatus Phytoplasma americanum", with APPTW12-NE as the reference strain. PMID:16825635

  4. A serpin-induced extensive proteolytic susceptibility of urokinase-type plasminogen activator implicates distortion of the proteinase substrate-binding pocket and oxyanion hole in the serpin inhibitory mechanism.

    Science.gov (United States)

    Egelund, R; Petersen, T E; Andreasen, P A

    2001-02-01

    The formation of stable complexes between serpins and their target serine proteinases indicates formation of an ester bond between the proteinase active-site serine and the serpin P1 residue [Egelund, R., Rodenburg, K.W., Andreasen, P.A., Rasmussen, M.S., Guldberg, R.E. & Petersen, T.E. (1998) Biochemistry 37, 6375-6379]. An important question concerning serpin inhibition is the contrast between the stability of the ester bond in the complex and the rapid hydrolysis of the acyl-enzyme intermediate in general serine proteinase-catalysed peptide bond hydrolysis. To answer this question, we used limited proteolysis to detect conformational differences between free urokinase-type plasminogen activator (uPA) and uPA in complex with plasminogen activator inhibitor-1 (PAI-1). Whereas the catalytic domain of free uPA, pro-uPA, uPA in complex with non-serpin inhibitors and anhydro-uPA in a non-covalent complex with PAI-1 was resistant to proteolysis, the catalytic domain of PAI-1-complexed uPA was susceptible to proteolysis. The cleavage sites for four different proteinases were localized in specific areas of the C-terminal beta-barrel of the catalytic domain of uPA, providing evidence that the serpin inhibitory mechanism involves a serpin-induced massive rearrangement of the proteinase active site, including the specificity pocket, the oxyanion hole, and main-chain binding area, rendering the proteinase unable to complete the normal hydrolysis of the acyl-enzyme intermediate. The distorted region includes the so-called activation domain, also known to change conformation on zymogen activation.

  5. Identification, classification and expression pattern analysis of sugarcane cysteine proteinases

    Directory of Open Access Journals (Sweden)

    Gustavo Coelho Correa

    2001-12-01

    Full Text Available Cysteine proteases are peptidyl hydrolyses dependent on a cysteine residue at the active center. The physical and chemical properties of cysteine proteases have been extensively characterized, but their precise biological functions have not yet been completely understood, although it is known that they are involved in a number of events such as protein turnover, cancer, germination, programmed cell death and senescence. Protein sequences from different cysteine proteinases, classified as members of the E.C.3.4.22 sub-sub-class, were used to perform a T-BLAST-n search on the Brazilian Sugarcane Expressed Sequence Tags project (SUCEST data bank. Sequence homology was found with 76 cluster sequences that corresponded to possible cysteine proteinases. The alignments of these SUCEST clusters with the sequence of cysteine proteinases of known origins provided important information about the classification and possible function of these sugarcane enzymes. Inferences about the expression pattern of each gene were made by direct correlation with the SUCEST cDNA libraries from which each cluster was derived. Since no previous reports of sugarcane cysteine proteinases genes exists, this study represents a first step in the study of new biochemical, physiological and biotechnological aspects of sugarcane cysteine proteases.Proteinases cisteínicas são peptidil-hidrolases dependentes de um resíduo de cisteína em seu sítio ativo. As propriedades físico-químicas destas proteinases têm sido amplamente caracterizadas, entretanto suas funções biológicas ainda não foram completamente elucidadas. Elas estão envolvidas em um grande número de eventos, tais como: processamento e degradação protéica, câncer, germinação, morte celular programada e processos de senescência. Diferentes proteinases cisteínicas, classificadas pelo Comitê de Nomenclatura da União Internacional de Bioquímica e Biologia Molecular (IUBMB como pertencentes à sub

  6. Purification and partial characterisation of a cathepsin L-like proteinase from sea cucumber (Stichopus japonicus) and its tissue distribution in body wall.

    Science.gov (United States)

    Zhou, Da-Yong; Chang, Xian-Na; Bao, Sha-Sha; Song, Liang; Zhu, Bei-Wei; Dong, Xiu-Ping; Zong, Yuan; Li, Dong-Mei; Zhang, Mao-Mao; Liu, Yu-Xin; Murata, Yoshiyuki

    2014-09-01

    A cathepsin L-like proteinase (CLP) with molecular weight of 30.9 kDa from the gut of sea cucumber (Stichopus japonicas, S. japonicus) was isolated and purified to homogeneity by several chromatographic procedures. The enzyme exhibited optimum activity at pH 5.0-5.5 and 50 °C, and showed thermostability up to 40 °C. The enzyme activity was completely inhibited by Zn(2+), strongly inhibited by Fe(2+) and Cu(2+), drastically reduced by cysteine proteinase inhibitors, but slightly enhanced by thiol-activating agents. The enzyme efficiently hydrolysed the specific substrate of cathepsin L, but hardly hydrolysed the specific substrates for cathepsin B, cathepsin H and cathepsin K. Immunohistochemical studies indicated that the CLP was more abundant in the epidermis rather than in the dermis of S. japonicus body wall. The distribution of CLP showed positive correlation with autolysis rate. Therefore, the relationship between CLP and autolysis deserved further study.

  7. Characterization of a cloned subtilisin-like serine proteinase from a psychrotrophic Vibrio species.

    Science.gov (United States)

    Arnórsdottir, Jóhanna; Smáradóttir, Rúna B; Magnússon, Olafur Th; Thorbjarnardóttir, Sigrídur H; Eggertsson, Gudmundur; Kristjánsson, Magnús M

    2002-11-01

    The gene encoding a subtilisin-like serine proteinase in the psychrotrophic Vibrio sp. PA44 has been successfully cloned, sequenced and expressed in Escherichia coli. The gene is 1593 basepairs and encodes a precursor protein of 530 amino acid residues with a calculated molecular mass of 55.7 kDa. The enzyme is isolated, however, as an active 40.6-kDa proteinase, without a 139 amino acid residue N-terminal prosequence. Under mild conditions the enzyme undergoes a further autocatalytic cleavage to give a 29.7-kDa proteinase that retains full enzymatic activity. The deduced amino acid sequence of the enzyme has high homology to proteinases of the proteinase K family of subtilisin-like proteinases. With respect to the enzyme characteristics compared in this study the properties of the wild-type and recombinant proteinases are the same. Sequence analysis revealed that especially with respect to the thermophilic homologues, aqualysin I from Thermus aquaticus and a proteinase from Thermus strain Rt41A, the cold-adapted Vibrio-proteinase has a higher content of polar/uncharged amino acids, as well as aspartate residues. The thermophilic enzymes had a higher content of arginines, and relatively higher number of hydrophobic amino acids and a higher aliphatic index. These factors may contribute to the adaptation of these proteinases to different temperature conditions.

  8. Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

    Directory of Open Access Journals (Sweden)

    Araki Hiromasa

    2007-04-01

    Full Text Available Abstract Background Proteinase-activated receptors (PARs; PAR1–4 that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT which contributes to the increase in myofibroblast population. Methods EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells. Results Stimulation of PAR with thrombin (1 U/ml or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β. Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. Conclusion PAR4 stimulation of alveolar epithelial cells induced epithelial

  9. Isolation of Tacaribe virus, a Caribbean arenavirus, from host-seeking Amblyomma americanum ticks in Florida.

    Directory of Open Access Journals (Sweden)

    Katherine A Sayler

    Full Text Available Arenaviridae are a family of single stranded RNA viruses of mammals and boid snakes. Twenty-nine distinct mammalian arenaviruses have been identified, many of which cause severe hemorrhagic disease in humans, particularly in parts of sub-Saharan Africa, and in Central and South America. Humans typically become infected with an arenavirus through contact with excreta from infected rodents. Tacaribe virus (TCRV is an arenavirus that was first isolated from bats and mosquitoes during a rabies surveillance survey conducted in Trinidad from 1956 to 1958. Tacaribe virus is unusual because it has never been associated with a rodent host and since that one time isolation, the virus has not been isolated from any vertebrate or invertebrate hosts. We report the re-isolation of the virus from a pool of 100 host-seeking Amblyomma americanum (lone star ticks collected in a Florida state park in 2012. TCRV was isolated in two cell lines and its complete genome was sequenced. The tick-derived isolate is nearly identical to the only remaining isolate from Trinidad (TRVL-11573, with 99.6% nucleotide identity across the genome. A quantitative RT-PCR assay was developed to test for viral RNA in host-seeking ticks collected from 3 Florida state parks. Virus RNA was detected in 56/500 (11.2% of surveyed ticks. As this virus was isolated from ticks that parasitize humans, the ability of the tick to transmit the virus to people should be evaluated. Furthermore, reservoir hosts for the virus need to be identified in order to develop risk assessment models of human infection.

  10. Cystatin F regulates proteinase activity in IL-2-activated natural killer cells.

    Science.gov (United States)

    Maher, Katarina; Konjar, Spela; Watts, Colin; Turk, Boris; Kopitar-Jerala, Natasa

    2014-01-01

    Cystatin F is a unique member of the cystatin family of cysteine protease inhibitors, which is synthesized as an inactive dimer and it is activated by N-terminal cleavage in the endolysosomes. It is expressed in the cells of the immune system: myeloid cells and the cells involved in target cell killing: natural killer (NK) cells and cytotoxic T cells (CTLs). Upon activation of the NK cells with interleukin 2 (IL-2), cystatin F was found upregulated and co-localized in cytotoxic granules with cathepsin C (CatC) and CatV. However, cystatin F inhibits the CatC in cells only when its N-terminal part is processed. Although cystatin F could inhibit both CatV and CatC, the IL-2 stimulation of the YT cells resulted in an increased CatV activity, while the CatC activity was unchanged. The incubation of IL-2 activated NK cells with a cysteine proteinase inhibitor E-64d increased the cystatin F dimer formation. Our results suggest that cystatin F not only inhibits CatV, but it is processed by the CatV in order to inhibit the CatC activity in cytotoxic granules. The regulation of the CatC activity in the cytotoxic granules of the NK cells by the cystatin F could be important for the processing and activation of granule-associated serine proteases - granzymes.

  11. Activation of human tonsil and skin mast cells by agonists of proteinase activated receptor-2

    Institute of Scientific and Technical Information of China (English)

    Shao-heng HE; Hua XIE; Yi-ling FU

    2005-01-01

    Aim: To investigate the effects of the agonists of proteinase activated receptor (PAR)-2,and histamine on degranulation of human mast cells. Methods: Human mast cells were enzymatically dispersed from tonsil and skin tissues. The dis persed cells were then cultured with various stimuli, and tryptase and histamine levels in cell supernatants collected from challenge tubes were measured. Results:PAR-2 agonist peptide SLIGKV provoked a dose-dependent release of histamine from skin mast cells. It also induced tryptase release from tonsil mast cells, tcLIGRLO appeared less potent than SLIGKV in induction of release of histamine and tryptase. Trypsin was able to induce a "bell" shape increase in tryptase release from tonsil mast cells. It was also able to induce a dose-dependent release of histamine from both tonsil and skin mast cells. The actions of trypsin on mast cells were inhibited by soy bean trypsin inhibitor (SBTI) or α1-antitrypsin (α1-AT).Time course study revealed that both stimulated tryptase or histamine release initiated within 10 s and reached their peak release between 4 and 6 min. Pretreatment of cells with metabolic inhibitors or pertussis toxin reduced the ability of mast cells to release tryptase or histamine. Conclusion: It was demonstrated that the in vitro tryptase release properties of human tonsil and skin mast cells suggested a novel type of mast cell heterogeneity. The activation of mast cells by PAR-2 agonists indicated a self-amplification mechanism of mast cell degranulation.

  12. Proteinases of Streptomyces fradiae. I. Preliminary characterization and purification.

    Science.gov (United States)

    Galas, E; Kaluzewska, T

    1989-01-01

    A keratinolytic strain of S. fradiae has been shown to synthesize a complex of extracellular proteinases degrading native keratin proteins, elastin and collagen as well as some globular proteins. These enzymes are characterized by basic optimal pH and are inactivated by pheynlmethylsulfonyl fluoride (PMSF). Using preparative polyacrylamide gel electrophoresis, ion-exchange chromatography and affinity chromatography, 6 fractions of active protein of diversified proteolytic activity have been distinguished in the preparation studied.

  13. Dispersal of Bap-mediated Staphylococcus aureus biofilm by proteinase K.

    Science.gov (United States)

    Kumar Shukla, Sudhir; Rao, Toleti Subba

    2013-02-01

    The dominant role of biofilm-associated protein (Bap) in Staphylococcus aureus biofilm development prompted us to investigate Bap as a potential target for proteinase-mediated biofilm dispersion. Biofilm assay in microtitre plates showed that proteinase K hampered the early adhesion of cells as well as biofilm development. Proteinase K treatment of 24- and 48-h-old biofilms showed enhanced dispersion of bap-positive S. aureus biofilm; however, proteinase K did not affect the bap-negative S. aureus biofilm. When antibiotics were used in combination with proteinase K, significant enhancement in antibiotic action was noticed against bap-positive S. aureus biofilm. This study establishes that antibiotics in combination with proteinase K can be used for controlling S. aureus biofilms in whose development Bap surface protein has a major role. We propose that Bap protein could be a potential target for therapeutic control of S. aureus infections (for example, bovine mastitis).

  14. Expression of serine proteinase P186 of Arthrobotrys oligospora and analysis of its nematode-degrading activity.

    Science.gov (United States)

    Zhao, Hailong; Qiao, Jun; Meng, Qingling; Gong, Shasha; Chen, Cheng; Liu, Tianli; Tian, Lulu; Cai, Xuepeng; Luo, Jianxun; Chen, Chuangfu

    2015-12-01

    The nematode-trapping fungi possess a unique capability of predating and invading nematodes. As a representative nematode-trapping fungus, Arthrobotrys oligospora has been widely used to study the interactions between nematode-trapping fungi and their hosts. Serine proteinase is one of the important virulence factors during process of invasion of the nematode-trapping fungi into nematodes. In this study, using reverse transcription polymerase chain reaction, we amplified the gene sequence of serine proteinase 186 from A. oligospora, cloned it into pPIC9K vector and expressed it in the yeast Pichia pastoris. The expressed recombinant serine proteinase186 (reP186) was purified via Ni-affinity chromatography. The in vitro nematode-degrading activity of reP186 was analyzed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis revealed that reP186 with molecular weight of 33 kDa was successfully obtained. ReP186 was capable of degrading a series of protein substrates including casein, gelatin, bovine serum albumin, denatured collagen and nematode cortical layer. The reP186 exhibited the maximal activity at pH 8.0 and 55 °C and was highly sensitive to the inhibitor, phenylmethanesulfonylfluoride. Treatment of Caenorhabditis elegans and Haemonchus contortus with reP186 for 12, 24 and 36 h, respectively, resulted in 62, 88 and 100 % of killing rates for C. elegans, and 52, 65 and 84 % of killing rates for H. contortus, respectively, indicating a relatively strong nematode-degrading bioactivity of reP186. PMID:26419902

  15. VaSP1, catalytically active serine proteinase from Vipera ammodytes ammodytes venom with unconventional active site triad.

    Science.gov (United States)

    Kurtović, Tihana; Brgles, Marija; Leonardi, Adrijana; Lang Balija, Maja; Sajevic, Tamara; Križaj, Igor; Allmaier, Günter; Marchetti-Deschmann, Martina; Halassy, Beata

    2014-01-01

    VaSP1, a serine proteinase from Vipera ammodytes ammodytes venom, is a glycosylated monomer of 31.5 kDa, as determined by MALDI mass spectrometry, showing multiple isoelectric points between pH 6.5 and pH 8.5. Partial amino acid sequencing of VaSP1 by Edman degradation and MS/MS analysis identified sequences which allowed its classification among the so-called snake venom serine proteinase homologues, members of the peptidase S1 family, however being devoid of the canonical catalytic triad. Only few representatives of this group have been identified so far with just two of them characterised in detail at the protein level. Despite substitution of His57 with Arg, VaSP1 possesses proteolytic activity which can be inhibited by Pefabloc, benzamidine, Zn²⁺ ions, DTT and trypsin inhibitor II, a Kunitz/BPTI group member. It hydrolyses N(α)-benzoyl-Phe-Val-Arg-p-NA, exhibiting Michaelis-Menten behaviour with K(m) = 48.2 μM and V(m) = 0.019 nM s⁻¹. The pH for optimal activity on tested substrate is around 9.0. VaSP1 also cleaves insulin B-chain, digesting it at positions His¹⁰-Leu¹¹, Ala¹⁴-Leu¹⁵ and Tyr¹⁶-Leu¹⁷. Furthermore, the novel serine proteinase is active towards wide array of proteins involved in haemostasis where its degradation of fibrinogen, fibrin, prothrombin, factor X and plasminogen in vivo probably results in depletion of coagulation factors in blood circulation. The possibility that VaSP1 possesses anticoagulant properties has been further indicated by its ability to prolong prothrombin time and activated partial thromboplastin time.

  16. Trichoderma harzianum transformant has high extracellular alkaline proteinase expression during specific mycoparasitic interactions

    Directory of Open Access Journals (Sweden)

    Goldman Maria Helena S.

    1998-01-01

    Full Text Available The mycoparasite Trichoderma harzianum produces an alkaline proteinase that may be specifically involved in mycoparasitism. We have constructed transformant strains of this fungus that overexpress this alkaline proteinase. Some of the transformants were assessed for alkaline proteinase activity, and those with higher activity than the wild type were selected for further studies. One of these transformant strains produced an elevated and constitutive pbr1 mRNA level during mycoparasitic interactions with Rhizoctonia solani.

  17. Efficacy of amitraz collars on white-tailed deer, Odocoileus virginianus (Zimmerman) (Artiodactyla: Cervidae) against free-living populations of Lone Star Ticks, Amblyomma americanum (L.) (Acari: Ixodidae)

    Science.gov (United States)

    Collars containing the acaricide amitraz were fitted around necks of white-tailed deer, Odocoileus virginianus (Zimmermann) confined in a 38.8 ha deer-fenced, densely vegetated plot in south Texas to determine efficacy in controlling free-living populations of lone star ticks, Amblyomma americanum (...

  18. Mechanism of Excretion of a Bacterial Proteinase: Factors Controlling Accumulation of the Extracellular Proteinase of a Sarcina Strain (Coccus P)

    Energy Technology Data Exchange (ETDEWEB)

    BISSELL, MINA J.; TOSI, ROBERTO; GORINI, LUIGI

    1970-06-29

    It has been known that the extracellular proteinase of Coccus P is found only in cultures grown in the presence of Ca{sup 2+}. It is now shown that this cation is required neither for synthesis, excretion, or activation of a zymogen nor as a prosthetic factor necessary for enzymatic activity. The only function of Ca{sup 2+} is to stabilize the active structure of the enzyme molecule, presumably by substituting for absence of S-S bridges. In the absence of Ca{sup 2+} , the excreted proteinase undergoes rapid autodigestion and, instead of the active protein, its hydrolytic products are accumulated in the culture fluid. In minimal medium and under conditions of enzyme stability [presence of Ca{sup 2+} and Ficoll (Pharmacia)], Coccus P accumulates the proteinase at a gradually reduced speed although the rate of cultural growth remains constant. It is shown that this decline in rate of accumulation is caused by the excreted proteinase itself, possibly acting on its own precursor emerging from the cell in a form susceptible to proteolytic attack and not amenable to Ca{sup 2+} protection. A proteinase precursor is actually demonstrable in a calciumless culture at the onset of the enzyme accumulation which follows Ca{sup 2+} addition. It is suggested that excreted proteins require an unfolded (or incompletely folded) structure to cross the cell envelope. The proteinase excreted by a Sarcina strain (Coccus P) is found only in cultures containing Ca{sup 2+} ions (1), a feature common to proteinases of other bacteria (4, 12, 18) and to other excreted enzymes (14). Among the nontoxic divalent cations, Ca{sup 2+} is rather specific in this effect. Other ions such as Mn{sup 2+} or Mg{sup 2+}, the latter being present in all media as an indispensible growth factor, are ineffective. Addition of Ca{sup 2+} to the proteolytically inactive supernatant fluid of a calcium- free culture does not result in the appearance of the missing enzyme activity. The early assumption that Ca{sup 2

  19. A new crystal form of proteinase A, a non-pepsin-type acid proteinase from Aspergillus niger var. macrosporus.

    Science.gov (United States)

    Tanokura, M; Sasaki, H; Muramatsu, T; Iwata, S; Hamaya, T; Takizawa, T; Takahashi, K

    1993-10-01

    Proteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase, whose catalytic residues and mechanism remain to be elucidated. A new form of proteinase A crystals more suitable for crystallography than that obtained previously was prepared from an ammonium sulfate solution at pH 3.5 by the hanging-drop vapor diffusion method. The space group of the crystals was P2(1)2(1)2(1), with unit cell dimensions of a = 69.75 +/- 0.06 A, b = 87.55 +/- 0.05 A, and c = 60.83 +/- 0.04 A. On the assumption of two enzyme molecules per asymmetric unit, the calculated volume to unit protein mass ratio (Vm) was 2.08 A3/Da. By assuming the specific volume to be 0.74 cm3/g, the solvent content (Vso1) was estimated to be 41%, i.e., much larger than that of the crystal form obtained previously at pH 2.0 (Vso1 = 26%). Diffraction data were collected up to a resolution higher than 1.6 A, using the Weissenberg camera for macromolecular crystallography with synchrotron radiation.

  20. Human neutrophil elastase inhibitors in innate and adaptive immunity.

    Science.gov (United States)

    Fitch, P M; Roghanian, A; Howie, S E M; Sallenave, J-M

    2006-04-01

    Recent evidence shows that human neutrophil elastase inhibitors can be synthesized locally at mucosal sites. In addition to efficiently targeting bacterial and host enzymes, they can be released in the interstitium and in the lumen of mucosa, where they have been shown to have antimicrobial activities, and to activate innate immune responses. This review will address more particularly the pleiotropic functions of low-molecular-mass neutrophil elastase inhibitors [SLPI (secretory leucocyte proteinase inhibitor) and elafin] and, more specifically, their role in the development of the adaptive immune response. PMID:16545094

  1. Expression of bovine genes associated with local and systemic immune response to infestation with the Lone Star tick, Amblyomma americanum.

    Science.gov (United States)

    Brannan, Jaime L; Riggs, Penny K; Olafson, Pia U; Ivanov, Ivan; Holman, Patricia J

    2014-10-01

    The Lone Star tick, Amblyomma americanum Linnaeus 1758 (Acari; Ixodidae), causes considerable production losses to the southern U.S. cattle industry due to reduced weight, infertility, secondary infections at bite wound sites, damaged hides, and potentially death, as these ticks tend to infest livestock in large numbers. Increasing environmental concerns, along with the potential for chemical residue in food products, have led to more emphasis on alternative tick control strategies, such as selective breeding practices and anti-tick vaccines. To enable progress toward these goals, a better understanding of bovine host immune mechanisms elicited by ticks is needed. In this study, 7 calves were phenotyped as susceptible, moderately resistant, or highly resistant to adult A. americanum ticks. Tick bite-site biopsies and blood leukocytes were collected at multiple time points throughout 3 successive tick infestations. Gene expression at tick bite-site biopsies was assessed by microarray analysis over 3 time points for each phenotype group. Quantitative reverse transcriptase-PCR expression analysis evaluated 11 candidate genes in tick bite-site biopsies, and 6 in blood leukocytes. Regression curve estimates calculated from the expression values generated by qRT-PCR in tick bite-sites identified correlations between several candidate genes. Increased expression of IGHG1, IL6, IL1α, and IL1RN in bovine tick bite-site biopsies suggests that Th2 differentiation may be important for the local bovine response to A. americanum ticks. Strong correlations in expression for IL1α and IL1β, for IL1α and IL1RN, and for IL1α and TLR4 were found in biopsies from the tick-resistant phenotypes. The up-regulation of IL12 and IL23 in blood leukocytes from Lone Star tick-infested calves of all phenotypes suggests a possible systemic recruitment of memory T cells. This study provides novel insight concerning the bovine immune response to Lone Star ticks and a basis for future

  2. An aspartic proteinase gene family in the filamentous fungus Botrytis cinerea contains members with novel features

    NARCIS (Netherlands)

    Have, ten A.; Dekkers, E.; Kay, J.; Phylip, L.H.; Kan, van J.A.L.

    2004-01-01

    Botrytis cinerea, an important fungal plant pathogen, secretes aspartic proteinase (AP) activity in axenic cultures. No cysteine, serine or metalloproteinase activity could be detected. Proteinase activity was higher in culture medium containing BSA or wheat germ extract, as compared to minimal medi

  3. [Activity of Ca(2+)-dependent neutral proteinases in rat organs under cobalt and mercury chloride injection].

    Science.gov (United States)

    Kaliman, P A; Samokhin, A A; Samokhina, L M

    2003-01-01

    The activity of Ca(2+)-dependent neutral proteinases in rats under cobalt and mercury chloride injection was investigated. The calpains activity increase in the lungs, heart, liver and kidneys was revealed after 2 h cobalt chloride action. The mercury chloride gives a reliable increase of calcium-dependent neutral proteinases only in the kidneys. PMID:14574747

  4. Use of a cysteine proteinase from Carica candamarcensis as a protective agent during DNA extraction

    Directory of Open Access Journals (Sweden)

    M.S. Genelhu

    1998-09-01

    Full Text Available We describe the use of a plant cysteine proteinase isolated from latex of Carica candamarcensis as a protective agent during isolation of bacterial DNA following growth in culture of these cells. Between 100 to 720 units of proteinase (1 µg = 6 units afforded good DNA protection when incubated with various kinds of microorganisms. Agarose gel electrophoresis showed that the resulting DNA was similar in size to DNA preparations obtained by treatment with proteinase K. The viability of the resulting material was checked by PCR amplification using species-specific primers. After standing at room temperature (25oC for 35 days, the enzyme lost 10% of its initial activity. The enzyme stability and good yield of DNA suggest the use of this proteinase as an alternative to proteinase K.

  5. Use of a cysteine proteinase from Carica candamarcensis as a protective agent during DNA extraction.

    Science.gov (United States)

    Genelhu, M S; Zanini, M S; Veloso, I F; Carneiro, A M; Lopes, M T; Salas, C E

    1998-09-01

    We describe the use of a plant cysteine proteinase isolated from latex of Carica candamarcensis as a protective agent during isolation of bacterial DNA following growth in culture of these cells. Between 100 to 720 units of proteinase (1 microgram = 6 units) afforded good DNA protection when incubated with various kinds of microorganisms. Agarose gel electrophoresis showed that the resulting DNA was similar in size to DNA preparations obtained by treatment with proteinase K. The viability of the resulting material was checked by PCR amplification using species-specific primers. After standing at room temperature (25 degrees C) for 35 days, the enzyme lost 10% of its initial activity. The enzyme stability and good yield of DNA suggest the use of this proteinase as an alternative to proteinase K.

  6. Mucolysis of the colonic mucus barrier by faecal proteinases: inhibition by interacting polyacrylate.

    Science.gov (United States)

    Hutton, D A; Pearson, J P; Allen, A; Foster, S N

    1990-03-01

    1. Mucolytic (mucus solubilizing) activity in human faeces has been characterized with both purified human and pig colonic mucin and shown to be mediated by proteolysis. 2. Mucolytic activity was demonstrated by: (i) a drop in mucin viscosity; (ii) a substantial reduction in mucin size, from polymer to degraded subunit, as assessed by Sepharose CL-2B gel filtration; (iii) formation of new N-terminal peptides. 3. Mucolytic activity was also followed in faecal extracts by its proteolytic activity using standard succinyl albumin substrate. Proteolysis extended over the pH range 4.5-11.0. Proteolysis was inhibited at pH 7.5 by soybean trypsin inhibitor and phenylmethanesulphonyl fluoride, suggesting the presence of serine proteinases. 4. The polyacrylate carbomer (934P) inhibited both mucolysis of pig colonic mucin and proteolysis of succinyl albumin. 5. Interaction between the polyacrylate (carbomer 934P) and purified human and pig colonic mucin was demonstrated by a marked synergistic increase in solution viscosity (360% above control). 6. The results demonstrate the presence of a mucolytic activity in the human colonic lumen that has the potential to degrade the mucus barrier, and that polyacrylates inhibit this mucolysis and interact to strengthen the colonic mucus barrier. Polyacrylates may therefore have therapeutic potential in inflammatory bowel disease where luminal proteolytic activity can be raised. PMID:2156646

  7. Proteinase 3 contributes to transendothelial migration of NB1-positive neutrophils.

    Science.gov (United States)

    Kuckleburg, Christopher J; Tilkens, Sarah B; Santoso, Sentot; Newman, Peter J

    2012-03-01

    Neutrophil transmigration requires the localization of neutrophils to endothelial cell junctions, in which receptor-ligand interactions and the action of serine proteases promote leukocyte diapedesis. NB1 (CD177) is a neutrophil-expressed surface molecule that has been reported to bind proteinase 3 (PR3), a serine protease released from activated neutrophils. PR3 has demonstrated proteolytic activity on a number of substrates, including extracellular matrix proteins, although its role in neutrophil transmigration is unknown. Recently, NB1 has been shown to be a heterophilic binding partner for the endothelial cell junctional protein, PECAM-1. Disrupting the interaction between NB1 and PECAM-1 significantly inhibits neutrophil transendothelial cell migration on endothelial cell monolayers. Because NB1 interacts with endothelial cell PECAM-1 at cell junctions where transmigration occurs, we considered that NB1-PR3 interactions may play a role in aiding neutrophil diapedesis. Blocking Abs targeting the heterophilic binding domain of PECAM-1 significantly inhibited transmigration of NB1-positive neutrophils through IL-1β-stimulated endothelial cell monolayers. PR3 expression and activity were significantly increased on NB1-positive neutrophils following transmigration, whereas neutrophils lacking NB1 demonstrated no increase in PR3. Finally, using selective serine protease inhibitors, we determined that PR3 activity facilitated transmigration of NB1-positive neutrophils under both static and flow conditions. These data demonstrate that PR3 contributes in the selective recruitment of the NB1-positive neutrophil population. PMID:22266279

  8. Circulating ADAM17 Level Reflects Disease Activity in Proteinase-3 ANCA-Associated Vasculitis.

    Science.gov (United States)

    Bertram, Anna; Lovric, Svjetlana; Engel, Alissa; Beese, Michaela; Wyss, Kristin; Hertel, Barbara; Park, Joon-Keun; Becker, Jan U; Kegel, Johanna; Haller, Hermann; Haubitz, Marion; Kirsch, Torsten

    2015-11-01

    ANCA-associated vasculitides are characterized by inflammatory destruction of small vessels accompanied by enhanced cleavage of membrane-bound proteins. One of the main proteases responsible for ectodomain shedding is disintegrin and metalloproteinase domain-containing protein 17 (ADAM17). Given its potential role in aggravating vascular dysfunction, we examined the role of ADAM17 in active proteinase-3 (PR3)-positive ANCA-associated vasculitis (AAV). ADAM17 concentration was significantly increased in plasma samples from patients with active PR3-AAV compared with samples from patients in remission or from other controls with renal nonvascular diseases. Comparably, plasma levels of the ADAM17 substrate syndecan-1 were significantly enhanced in active AAV. We also observed that plasma-derived ADAM17 retained its specific proteolytic activity and was partly located on extracellular microparticles. Transcript levels of ADAM17 were increased in blood samples of patients with active AAV, but those of ADAM10 or tissue inhibitor of metalloproteinases 3, which inhibits ADAMs, were not. We also performed a microRNA (miR) screen and identified miR-634 as significantly upregulated in blood samples from patients with active AAV. In vitro, miR-634 mimics induced a proinflammatory phenotype in monocyte-derived macrophages, with enhanced expression and release of ADAM17 and IL-6. These data suggest that ADAM17 has a prominent role in AAV and might account for the vascular complications associated with this disease. PMID:25788529

  9. Purification of a cysteine protease inhibitor from larval hemolymph of the Tobacco Hornworm (Manduca sexta) and functional expression of the recombinant protein.

    Science.gov (United States)

    A cysteine protease inhibitor (CPI) with an apparent molecular mass of 11.5 kDa was purified from larval hemolymph of the tobacco hornworm (Manduca sexta) by gel filtration of Sephadex G-50 followed by hydrophobic and ion-exchange column chromatographies. The purified cysteine proteinase inhibitor, ...

  10. Understanding binding affinity : A combined isothermal titration calorimetry/molecular dynamics study of the binding of a series of hydrophobically modified benzamidinium chloride inhibitors to trypsin

    NARCIS (Netherlands)

    Talhout, Reinskje; Villa, Alessandra; Mark, AE; Engberts, JBFN

    2003-01-01

    The binding of a series of p-alkylbenzamidinium chloride inhibitors to the serine proteinase trypsin over a range of temperatures has been studied using isothermal titration (micro)calorimetry and molecular dynamics simulation techniques. The inhibitors have small structural variations at the para p

  11. Occurrence of novel verrucomicrobial species, endosymbiotic and associated with parthenogenesis in Xiphinema americanum-group species (Nematoda, Longidoridae).

    Science.gov (United States)

    Vandekerckhove, T T; Willems, A; Gillis, M; Coomans, A

    2000-11-01

    Numerous micro-organisms have been described as cytoplasmic symbionts of eukaryotes. Many so-called obligate endosymbionts rely exclusively on maternal (vertical or transovarial) transmission to maintain themselves, rendering them dependent on the host sex ratio, which they would tend to manipulate to their own advantage. The latter phenomenon is often associated with the presence of Wolbachia pipientis (alpha-Proteobacteria) in arthropods and nematodes. A potentially similar situation was discovered involving members of a new clade of Verrucomicrobia, another main line of descent in the Bacteria. Nematode species of the Xiphinema americanum group (Nematoda, Longidoridae), viz. Xiphinema americanum, Xiphinema rivesi and Xiphinema brevicollum, each harbour their own specific verrucomicrobial endosymbionts. They are exclusively maternally inherited and their hosts reproduce by thelytokous (mother-to-daughter) parthenogenesis, males being extremely rare. A new genus, 'Candidatus Xiphinematobacter' gen. nov., along with three new candidate verrucomicrobial species, 'Candidatus Xiphinematobacter americani' sp. nov., 'Candidatus Xiphinematobacter rivesi' sp. nov. and 'Candidatus Xiphinematobacter brevicolli' sp. nov., are described on the basis of transmission electron microscopy, scanning electron microscopy, DAPI (4',6-diamidino-2-phenylindole) epifluorescence microscopy and 16S rDNA sequence analysis. These are the first endosymbiotic species described among the Verrucomicrobia. They share a mean 16S rDNA similarity of about 93%, whereas similarity to their closest relative, clone WCHD3-88, is less than 87%. Thus, the endosymbionts form a homogeneous clade for which the new candidate genus 'Candidatus Xiphinematobacter' gen. nov. is proposed. The type species is 'Candidatus Xiphinematobacter brevicolli' sp. nov.

  12. Tissue inhibitor of metalloproteinase 1 (TIMP-1) as a biomarker in gastric cancer

    DEFF Research Database (Denmark)

    Grunnet, Mie; Mau-Sørensen, Morten; Brünner, Nils

    2013-01-01

    The value of Tissue Inhibitor of MetalloProteinase-1 (TIMP-1) as a biomarker in patients with gastric cancer (GC) is widely debated. The aim of this review is to evaluate available literature describing the association between levels of TIMP-1 in tumor tissue and/or blood and the prognosis...

  13. Understanding enzymic binding affinity : thermodynamics of binding of benzamidinium chloride inhibitors to trypsin

    NARCIS (Netherlands)

    Talhout, Reinskje

    2003-01-01

    Understanding enzymic binding affinity is of fundamental scientific importance as well as a prerequisite for structure-based drug design. In this study, the interactions of the serine proteinase trypsin with several artificial, benzamidinium-based inhibitors have been studied in aqueous solutions. I

  14. Metzincin Proteases and their Inhibitors, Foes or Friends in Nervous System Physiology?

    OpenAIRE

    Rivera, Santiago; Khrestchatisky, Michel; Kaczmarek, Leszek; Rosenberg, Gary A; Jaworski, Diane M.

    2010-01-01

    Members of the metzincin family of metalloproteinases have long been considered merely degradative enzymes for extracellular matrix molecules. Recently, however, there has been growing appreciation for these proteinases and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), as fine modulators of nervous system physiology and pathology. Present all along the phylogenetic tree, in all neural cell types, from the nucleus to the synapse and in the extracellular space, m...

  15. Comparative speed of kill of sarolaner (Simparica™) and afoxolaner (NexGard®) against induced infestations of Amblyomma americanum on dogs

    OpenAIRE

    Six, Robert H.; Everett, William R.; Chapin, Sara; Mahabir, Sean P.

    2016-01-01

    Background The lone star tick, Amblyomma americanum, infests dogs and cats in North America and is the vector of the pathogens that cause monocytic and granulocytic ehrlichiosis in dogs and humans. A parasiticide’s speed of kill is important to minimize the direct and deleterious effects of tick infestation and especially to reduce the risk of transmission of tick-borne pathogens. In this study, speed of kill of a novel orally administered isoxazoline parasiticide, sarolaner (Simparica™ chewa...

  16. [Phospholipase, proteinase and hemolytic activities of Candida albicans isolates obtained from clinical specimens].

    Science.gov (United States)

    Yenişehirli, Gülgün; Bulut, Yunus; Tunçoglu, Ebru

    2010-01-01

    This study was aimed to determine the phospholipase, proteinase and hemolytic activities of Candida albicans strains isolated from clinical specimens. A total of 147 C. albicans strains isolated from blood (n = 29), respiratory specimens (n = 44), urine (n = 52), pus (n = 17) and stool (n = 5) were included in the study. Proteinase and phospholipase activities were determined in 81% and 76% of C. albicans isolates, respectively. All C. albicans isolates revealed beta-hemolytic activity on Sabouraud dextrose agar supplemented with 7% fresh sheep blood and 3% glucose. Phospholipase and proteinase positivity were highest among the respiratory isolates. Proteinase activity of respiratory (93%) and blood (83%) isolates were statistically significantly higher than that of urine (77%; p = 0.032), pus (65%; p = 0.007) and stool isolates (60%; p = 0.026). While phospholipase activity showed statistically significant difference between respiratory (84%) and pus (53%) isolates (p = 0.014), no statistically significant difference was determined for blood (79%), urine (75%) and stool (80%) isolates (p > 0.05). Two blood isolates with 4+ proteinase activity and 3 urine isolates with 3+ proteinase activity were phospholipase negative. One urine isolate with 4+ phospholipase activity and 4 with 3+ phospholipase activity were proteinase negative. Phospholipase and proteinase negative 1 isolate from stool and 1 isolate from pus were found to have 4+ hemolytic activity. In conclusion, besides proteinase and phospholipase enzyme activities, hemolytic activity may play an important role for the C.albicans infections. The pathogenetic role of these virulence factors should be evaluated by further clinical studies.

  17. The mitochondrial genome of Xiphinema americanum sensu stricto (Nematoda: Enoplea): considerable economization in the length and structural features of encoded genes.

    Science.gov (United States)

    He, Y; Jones, J; Armstrong, M; Lamberti, F; Moens, M

    2005-12-01

    The complete sequence of the mitochondrial genome of the plant parasitic nematode Xiphinema americanum sensu stricto has been determined. At 12626bp it is the smallest metazoan mitochondrial genome reported to date. Genes are transcribed from both strands. Genes coding for 12 proteins, 2 rRNAs and 17 putative tRNAs (with the tRNA-C, I, N, S1, S2 missing) are predicted from the sequence. The arrangement of genes within the X. americanum mitochondrial genome is unique and includes gene overlaps. Comparisons with the mtDNA of other nematodes show that the small size of the X. americanum mtDNA is due to a combination of factors. The two mitochondrial rRNA genes are considerably smaller than those of other nematodes, with most of the protein encoding and tRNA genes also slightly smaller. In addition, five tRNAs genes are absent, lengthy noncoding regions are not present in the mtDNA, and several gene overlaps are present.

  18. Proteinase K processing of rabbit muscle creatine kinase

    DEFF Research Database (Denmark)

    Leydier, C; Andersen, Jens S.; Couthon, F;

    1997-01-01

    Proteinase K cleaves selectively both cytosolic and mitochondrial isoforms of creatine kinase leading to the appearance of two fragments, a large N-terminal one (K1) and a small C-terminal peptide (K2) which remain associated together. The loss of enzymatic activity correlates with the extent...... of monomer cleavage. N-terminal sequencing of the K2 fragments from rabbit cytosolic and pig mitochondrial creatine kinase shows that these peptides begin with A328 and A324, respectively. Electrospray ionization mass spectrometry demonstrates that K2 peptide is composed of 53 residues (A328-K380). However......, the C-terminal end of the K1 fragment is not A327 as expected, but D325. Thus, the amino acids residues T326 and A327 have been eliminated by the protease....

  19. Purification and Characterization of an Extracellular Proteinase from Brevibacterium linens ATCC 9174

    OpenAIRE

    Rattray, F P; Bockelmann, W; Fox, P. F.

    1995-01-01

    An extracellular serine proteinase from Brevibacterium linens ATCC 9174 was purified to homogeneity. pH and temperature optima were 8.5 and 50(deg)C, respectively. The results for the molecular mass of the proteinase were 56 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 126 kDa by gel filtration, indicating that the native enzyme exists as a dimer. Mg(sup2+) and Ca(sup2+) activated the proteinase, as did NaCl; however, Hg(sup2+), Fe(sup2+), and Zn(sup2+) caused strong i...

  20. Purification and Characterization of an Extracellular Proteinase from Brevibacterium-Linens ATCC-9174

    DEFF Research Database (Denmark)

    Rattray, F P; Bockelmann, W; Fox, P F

    1995-01-01

    An extracellular serine proteinase from Brevibacterium linens ATCC 9174 was purified to homogeneity. pH and temperature optima were 8,5 and 50 degrees C, respectively. The results for the molecular mass of the proteinase were 56 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis...... and 126 kDa by gel filtration, indicating that the native enzyme exists as a dimer. Mg2+ and Ca2+ activated the proteinase, as did NaCl; however, Hg2+ Fe2+, and Zn2+ caused strong inhibition. The sequence of the first 20 N-terminal amino acids was NH2-Ala-Lys- Asn...

  1. Identification of amylase inhibitor deficient mutants in pigeonpea (Cajanus cajan (L.) Millisp.).

    Science.gov (United States)

    Chougule, N P; Giri, A P; Hivrale, V K; Chhabda, P J; Kachole, M S

    2004-06-01

    We have developed and analyzed several mutant lines (M6 generation) of pigeonpea (Cajanus cajan (L.) Millsp.) for the content of defensive proteins and antinutritional factors. Inhibitors of proteinase and of amylase, lectins, and raffinose family oligosaccharides were analyzed in mature seeds of different pigeonpea accessions (untreated) and compared with mutant lines. Proteinase inhibitor profiles were similar in terms of number and intensities of activity bands but they differ marginally in the activity units in pigeonpea accessions and mutants. Pigeonpea mutants showed significant differences in amylase inhibitor profiles as well as activity units from those of pigeonpea accessions. Interestingly, two mutants (A6-5-1 and A7-3-2) were identified to have absence of amylase inhibitor isoforms. Hemagglutinating activity and raffinose family oligosaccharides content were found to be significantly higher in mutants than in accessions. It is evident from the results that proteinase inhibitors of pigeonpea are stable while amylase inhibitors, lectins, and raffinose family oligosaccharides show altered expression upon mutagen treatments. These mutants will be ideal candidates for further evaluation. PMID:15260142

  2. Kinetics and mechanism of proteinase-binding of pregnancy zone protein (PZP). Appearance of sulfhydryl groups in reactions with proteinases.

    Science.gov (United States)

    Christensen, U; Sottrup-Jensen, L; Simonsen, M

    1992-01-01

    Proteinase binding by pregnancy zone protein (PZP), an alpha-macroglobulin involves bait region cleavages, association of dimeric-PZP into tetrameric and reaction of internal gamma-glutamyl-beta-cysteinyl thiol esters of PZP with proteinase side chains. The product is an equimolar enzyme-PZP(tetramer) covalently linked complex with four free sulfhydryl groups. The kinetics of the appearances of sulfhydryl groups during the reaction of PZP with chymotrypsin has been investigated using stopped-flow and conventional mixing techniques over a broad concentration range. Thiol ester cleavages followed double exponential decays corresponding with two steps. The faster one resulted in the appearance of three sulfhydryl groups with an observed rate constant, k(obs) = k1.1 + k1.2 delta E, dependent on the excess concentration of chymotrypsin, delta E, and k1.1 = 0.03 s-1 and k1.2 = 4 x 10(4) M-1 s-1. The last sulfhydryl group appeared in a slower step, with similar concentration dependence and k2.1 approximately 0.003 s-1 and k2.2 approximately 5 x 10(3) M-1s-1. Covalent binding of the enzyme apparently was simultaneous with the faster thiol ester cleavage step. Based on these and previous results a model of the reaction mechanism of the proteinase binding reaction of PZP is proposed. It consists of four major steps: (i) Bait region cleavage of PZP-dimers by the enzyme, (ii) fast association of enzyme-PZP(dimer) species with native PZP or with another enzyme-PZP(dimer) compound resulting in release of one of the associated enzyme molecules (iii) reaction of an average of three thiol esters of the enzyme-PZP(tetramer) intermediate with the associated internal enzyme molecule or with an external one. In this step one enzyme molecule becomes covalently linked to the PZP-(tetramer), three sulfhydryl groups appear and the enzymic activity of the bound enzyme molecule decreases to the level of that of the final complex. (iv) Hydrolysis of the last thiol ester and in the presence of

  3. Inheritance and Expression of Potato Proteinase Inhibitor Gene Ⅱ (pinⅡ) in Transgenic Rice

    Institute of Scientific and Technical Information of China (English)

    CHENG Zhong-yi; XUE Qing-zhong

    2003-01-01

    The inheritance and expression of bar gene and pinⅡ gene were studied in three transgenic ricelines and their F2 hybrid populations, which were created through hybridization with a PGMS line, ZAU11S.By Basta painting, PCR analysis and determining of the inhibitory trypsin activity, the results show that bargene and pinⅡ gene in rice F2 population fit the simple Mendel's low of inheritance and close linkage, but afew plants in F2 have not sufficiently expressed. The wound inducible pin Ⅱ gene has an expression regulatedspatially and temporally, and the signal transduction pathway is not only upward, but also downward. The in-ducible expression of pinⅡ in different rice transgenic lines is not completely coincident.

  4. Allelopathic potential of Crinum americanum under different extractions conditionPotencial alelopático de Crinum americanum L. sob diferentes condições de extração

    Directory of Open Access Journals (Sweden)

    Maria Inês Salgueiro Lima

    2011-07-01

    Full Text Available The long-term use reduces the efficiency of the traditional herbicides. The use of plant-extracted natural substances is an alternative more efficient and less aggressive to the environment. The allelochemicals of several species are considered to be possible plants for developing an organic herbicide. One of the major problems in this sense is obtaining botanical material in commercial quantities. Improving the extraction techniques is an important step, once it reduces the amount of botanical material needed. In this survey was tested several methods of aqueous extractions of dried leaves of Crinum americanum L., an Amarilidaceae with strong allelopathic properties. The time of extraction is not important for the allelopathic potential of the extracts. On the other hand, when the temperature during extraction was 40 e 60oC, leaded to extracts with stronger allelopathic potential. It is possible to improve the allelopathic potential using the filtered matter more than once.O uso prolongado torna os herbicidas tradicionais cada vez menos eficientes no combate às plantas daninhas. O uso de substâncias naturais extraídas das plantas é uma alternativa eficiente e menos agressiva ao meio ambiente. Os aleloquímicos de muitas espécies são possíveis matérias primas para o desenvolvimento de herbicidas orgânicos. Um dos principais problemas encontrados para a produção desses herbicidas é a obtenção de material botânico em quantidade comercial. Melhorar as técnicas de extração é um passo importante, uma vez que reduz a quantidade de matéria prima necessária. Nesse trabalho foram testados diversos tipos de extrações aquosas de folhas secas de Crinum americanum L., uma Amarilidacea com forte potencial alelopático. O tempo de extração é pouco importante para o potencial alelopático do extrato. Por outro lado, temperaturas de extração de 40 e 60oC levaram à produção de extratos que inibiram mais eficientemente a germina

  5. Isolation and characterization of an ovoinhibitor, a multidomain Kazal-like inhibitor from Turkey (Meleagris gallopavo) seminal plasma.

    Science.gov (United States)

    Słowińska, Mariola; Liszewska, Ewa; Nynca, Joanna; Bukowska, Joanna; Hejmej, Anna; Bilińska, Barbara; Szubstarski, Jarosław; Kozłowski, Krzysztof; Jankowski, Jan; Ciereszko, Andrzej

    2014-11-01

    Turkey seminal plasma contains three serine proteinase inhibitors. Two of them, with low molecular masses (6 kDa), were identified as single-domain Kazal-type inhibitors responsible for regulating acrosin activity. Our experimental objective was to isolate and characterize the inhibitor with the high molecular weight from turkey seminal plasma. The inhibitor was purified using hydrophobic interaction and affinity chromatography. Pure preparations of the inhibitor were used for identification by mass spectrometry, for determination of physicochemical properties (molecular weight, pI, and content and composition of the carbohydrate component), for kinetic studies, and for antibacterial tests. Gene expression and immunohistochemical detection of the inhibitor were analyzed in the testis, epididymis, and ductus deferens. The inhibitor with a high molecular weight from turkey seminal plasma was identified as an ovoinhibitor, which was found in avian semen for the first time. The turkey seminal plasma ovoinhibitor was a six-tandem homologous Kazal-type domain serine proteinase inhibitor that targeted multiple proteases, including subtilisin, trypsin, and elastase, but not acrosin. Our results suggested that hepatocyte growth factor activator was a potential target proteinase for the ovoinhibitor in turkey seminal plasma. The presence of the ovoinhibitor within the turkey reproductive tract suggested that its role was to maintain a microenvironment for sperm in the epididymis and ductus deferens. The turkey seminal plasma ovoinhibitor appeared to play a significant role in an antibacterial semen defense against Bacillus subtilis and Staphylococcus aureus.

  6. Crystallization and preliminary X-ray investigation of proteinase A, a non-pepsin-type acid proteinase from Aspergillus niger var. macrosporus.

    Science.gov (United States)

    Tanokura, M; Matsuzaki, H; Iwata, S; Nakagawa, A; Hamaya, T; Takizawa, T; Takahashi, K

    1992-01-01

    Proteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase distinctly different in various properties from the family of pepsin-type aspartic proteinases, and so far it remains unknown which residues participate in the catalysis of the enzyme and how the mechanism operates. The acid proteinase A was crystallized from an ammonium sulfate solution by the hanging-drop vapor diffusion method. The space group of the crystals was P2(1)2(1)2(1) with unit cell dimensions of a = 54.7 A, b = 70.4 A and c = 38.0 A. On the assumption that there is one enzyme molecule in the asymmetric unit, the calculated ratio of volume to unit protein mass (Vm) was 1.64 A3 per dalton. Diffraction data were collected up to a resolution higher than 1.5 A, using the Weissenberg camera for macromolecular crystallography with synchrotron radiation. The crystal of proteinase A is, therefore, suitable for the structural analysis with a high resolution.

  7. Implantation serine proteinase 2 is a monomeric enzyme with mixed serine proteolytic activity and can silence signalling via proteinase activated receptors.

    Science.gov (United States)

    Sharma, Navneet; Fahr, Jochen; Renaux, Bernard; Saifeddine, Mahmoud; Kumar, Rajeev; Nishikawa, Sandra; Mihara, Koichiro; Ramachandran, Rithwik; Hollenberg, Morley D; Rancourt, Derrick E

    2013-12-01

    Implantation serine proteinase 2 (ISP2), a S1 family serine proteinase, is known for its role in the critical processes of embryo hatching and implantation in the mouse uterus. Native implantation serine proteinases (ISPs) are co-expressed and co-exist as heterodimers in uterine and blastocyst tissues. The ISP1-ISP2 enzyme complex shows trypsin-like substrate specificity. In contrast, we found that ISP2, isolated as a 34 kDa monomer from a Pichia pastoris expression system, exhibited a mixed serine proteolytic substrate specificity, as determined by a phage display peptide cleavage approach and verified by the in vitro cleavage of synthetic peptides. Based upon the peptide sequence substrate selectivity, a database search identified many potential ISP2 targets of physiological relevance, including the proteinase activated receptor 2 (PAR2). The in vitro cleavage studies with PAR2-derived peptides confirmed the mixed substrate specificity of ISP2. Treatment of cell lines expressing proteinase-activated receptors (PARs) 1, 2, and 4 with ISP2 prevented receptor activation by either thrombin (PARs 1 and 4) or trypsin (PAR2). The disarming and silencing of PARs by ISP2 may play a role in successful embryo implantation.

  8. Studies on prekallikrein of bovine plasma. II. Activation of prekallikrein with proteinases and properties of kallikrein activated by bovine Hageman factor.

    Science.gov (United States)

    Takahashi, H; Nagasawa, S; Suzuki, T

    1980-01-01

    Activation of bovine plasma prekallikrein was investigated with several proteinases. Highly purified bovine plasma prekallikrein was rapidly activated to kallikrein [EC 3.4.21.8] by bovine activated Hageman factor, trypsin [EC 3.4.21.4] and Pronase P (proteinases from Streptomyces griseus) and more gradually by papain [EC 3.4.22.2] and ficin [EC 3.4.22.3]. Activation of prekallikrein was also observed with bovine plasmin [EC 3.4.21.7], but not with bovine clotting factors Xa (Stuart factor) [EC 3.4.21.6] and IXa (Christmas factor) or thrombin [EC 3.4.21.5]. Urokinase [EC 3.4.99.26], Reptilase, collagenase [EC 3.4.24.3], elastase [EC 3.4.21.11], alpha-chymotrypsin [EC 3.4.21.1], Nagarse [EC 3.4.21.14], and stem bromelain [EC 3.4.22 4] did not convert prekallikrein to kallikrein. Plasma kallikrein activated to Hageman factor released kinin rapidly from bovine high molecular weight (HMW) kininogen. However, from bovine low molecular weight (LMW) kininogen, liberation of kinin was extremely slow. The kallikrein activity was inhibited by soybean trypsin inhibitor (SBTI), Trasylol, diisopropylfluorophosphate (DFP), and N-alpha-tosyl-L-lysine chloromethylketone (TLCK), but not by egg-white trypsin inhibitor (EWTI), lima bean trypsin inhibitor (LBTI), heparin or hexadimethrine bromide (Polybrene). The kallikrein formed an enzyme-inhibitor complex with SBTI and Trasylol, but not with LBTI. Prekallikrein did not react with SBTI. Prekallikrein consists of a single polypeptide chain of molecular weight about 90,000, as estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Activation of prekallikrein by Hageman factor was found to involve cleavage of the single peptide bond on the disulfide-bridged polypeptide chain, and no change of molecular weight was observed during the activation. The peptide bond cleaved in prekallikrein by the activation was an Arg-X peptide bond on a disulfide-bridged polypeptide chain.

  9. A novel Entamoeba histolytica cysteine proteinase, EhCP4, is key for invasive amebiasis and a therapeutic target.

    Science.gov (United States)

    He, Chen; Nora, George P; Schneider, Eric L; Kerr, Iain D; Hansell, Elizabeth; Hirata, Ken; Gonzalez, David; Sajid, Mohammed; Boyd, Sarah E; Hruz, Petr; Cobo, Eduardo R; Le, Christine; Liu, Wei-Ting; Eckmann, Lars; Dorrestein, Pieter C; Houpt, Eric R; Brinen, Linda S; Craik, Charles S; Roush, William R; McKerrow, James; Reed, Sharon L

    2010-06-11

    Entamoeba histolytica cysteine proteinases (EhCPs) play a key role in disrupting the colonic epithelial barrier and the innate host immune response during invasion of E. histolytica, the protozoan cause of human amebiasis. EhCPs are encoded by 50 genes, of which ehcp4 (ehcp-a4) is the most up-regulated during invasion and colonization in a mouse cecal model of amebiasis. Up-regulation of ehcp4 in vivo correlated with our finding that co-culture of E. histolytica trophozoites with mucin-producing T84 cells increased ehcp4 expression up to 6-fold. We have expressed recombinant EhCP4, which was autocatalytically activated at acidic pH but had highest proteolytic activity at neutral pH. In contrast to the other amebic cysteine proteinases characterized so far, which have a preference for arginine in the P2 position, EhCP4 displayed a unique preference for valine and isoleucine at P2. This preference was confirmed by homology modeling, which revealed a shallow, hydrophobic S2 pocket. Endogenous EhCP4 localized to cytoplasmic vesicles, the nuclear region, and perinuclear endoplasmic reticulum (ER). Following co-culture with colonic cells, EhCP4 appeared in acidic vesicles and was released extracellularly. A specific vinyl sulfone inhibitor, WRR605, synthesized based on the substrate specificity of EhCP4, inhibited the recombinant enzyme in vitro and significantly reduced parasite burden and inflammation in the mouse cecal model. The unique expression pattern, localization, and biochemical properties of EhCP4 could be exploited as a potential target for drug design.

  10. Effect of the Solvent Temperatures on Dynamics of Serine Protease Proteinase K

    OpenAIRE

    Peng Sang; Qiong Yang; Xing Du; Nan Yang; Li-Quan Yang; Xing-Lai Ji; Yun-Xin Fu; Zhao-Hui Meng; Shu-Qun Liu

    2016-01-01

    To obtain detailed information about the effect of the solvent temperatures on protein dynamics, multiple long molecular dynamics (MD) simulations of serine protease proteinase K with the solute and solvent coupled to different temperatures (either 300 or 180 K) have been performed. Comparative analyses demonstrate that the internal flexibility and mobility of proteinase K are strongly dependent on the solvent temperatures but weakly on the protein temperatures. The constructed free energy la...

  11. Sap6, a secreted aspartyl proteinase, participates in maintenance the cell surface integrity of Candida albicans

    OpenAIRE

    Buu, Leh-Miauh; Chen, Yee-Chun

    2013-01-01

    Background The polymorphic species Candida albicans is the major cause of candidiasis in humans. The secreted aspartyl proteinases (Saps) of C. albicans, encoded by a family of 10 SAP genes, have been investigated as the virulent factors during candidiasis. However, the biological functions of most Sap proteins are still uncertain. In this study, we applied co-culture system of C. albicans and THP-1 human monocytes to explore the pathogenic roles and biological functions of Sap proteinases. R...

  12. Biodistribution, pharmacokinetics and toxicity of a Vasconcellea cundinamarcensis proteinase fraction with pharmacological activity

    Directory of Open Access Journals (Sweden)

    Fernanda O. Lemos

    2016-02-01

    Full Text Available Abstract Prior studies demonstrate that a proteinase fraction from Vasconcellea cundinamarcensis V.M. Badillo, Caricaceae, exhibits wound healing activity in gastric and cutaneous models and antitumoral/antimetastatic effects. Here, we present the toxicity, pharmacokinetics and biodistribution data for this proteinase fraction following a single dose into Swiss mice by i.v., s.c. or p.o. routes. The i.v. and s.c. toxicity assays demonstrate that proteinase fraction at ≤20 mg/kg is non-lethal after single injection, while parental administration (p.o. of ≤300 mg/kg does not cause death. Based on p.o. acute toxicity dose using Organisation for Economic Cooperation and Development protocols, proteinase fraction ranks as Class IV “harmful” substance. Proteinase fraction shows high uptake determined as Kp (distribution tissue/blood in organs linked to metabolism and excretion. Also, high bioavailability (≈100% was observed by s.c. administration. The blood contents following i.v. dose fits into a pharmacokinetic bi-compartmental model, consisting of high removal constants – kel 0.22 h−1 and kd 2.32 h−1and a half-life – t½ = 3.13 h. The Ames test of proteinase fraction (0.01–1% demonstrates absence of mutagenic activity. Likewise, genotoxic evaluation of proteinase fraction (5 or 10 mg/kg, i.p. shows no influence in micronuclei frequency. In conclusion, the acute doses for proteinase fraction lack mutagenic and genotoxic activity, clearing the way for clinical assays.

  13. Neutrophil-derived Oxidants and Proteinases as Immunomodulatory Mediators in Inflammation

    OpenAIRE

    V. Witko-Sarsat; B. Descamps-Latscha

    1994-01-01

    Neutrophils generate potent microbicidal molecules via the oxygen-dependent pathway, leading to the generation of reactive oxygen intermediates (ROI), and via the non-oxygen dependent pathway, consisting in the release of serine proteinases and metalloproteinases stored in granules. Over the past years, the concept has emerged that both ROI and proteinases can be viewed as mediators able to modulate neutrophil responses as well as the whole inflammatory process. This is w...

  14. Antibody in sera of patients infected with Trichomonas vaginalis is to trichomonad proteinases.

    OpenAIRE

    Alderete, J F; Newton, E.; C. Dennis; Neale, K A

    1991-01-01

    BACKGROUND--A recent report demonstrated the immunogenic character of the cysteine proteinases of Trichomonas vaginalis. It was of interest, therefore, to examine for the presence of serum anti-proteinase antibody among patients with trichomoniasis. METHODS--An immunoprecipitation assay was used involving protein A-bearing Staphylococcus aureus first coated with the IgG fraction of goat anti-human Ig and then mixed with individual sera of patients to bind human antibody. These antibody-coated...

  15. Equistatin, a protease inhibitor from the sea anemone Actinia equina, is composed of three structural and functional domains

    NARCIS (Netherlands)

    Strukelj, B.; Lenarcic, B.; Gruden, K.; Pungercar, J.; Rogelj, B.; Turk, V.; Bosch, D.; Jongsma, M.A.

    2000-01-01

    A cDNA encoding a precursor of equistatin, a potent cysteine and aspartic proteinase inhibitor, was isolated from the sea anemone Actinia equina. The deduced amino acid sequence of a 199-amino-acid residue mature protein with 20 cysteine residues, forming three structurally similar thyroglobulin typ

  16. Interpain A, a cysteine proteinase from Prevotella intermedia, inhibits complement by degrading complement factor C3.

    Directory of Open Access Journals (Sweden)

    Michal Potempa

    2009-02-01

    Full Text Available Periodontitis is an inflammatory disease of the supporting structures of the teeth caused by, among other pathogens, Prevotella intermedia. Many strains of P. intermedia are resistant to killing by the human complement system, which is present at up to 70% of serum concentration in gingival crevicular fluid. Incubation of human serum with recombinant cysteine protease of P. intermedia (interpain A resulted in a drastic decrease in bactericidal activity of the serum. Furthermore, a clinical strain 59 expressing interpain A was more serum-resistant than another clinical strain 57, which did not express interpain A, as determined by Western blotting. Moreover, in the presence of the cysteine protease inhibitor E64, the killing of strain 59 by human serum was enhanced. Importantly, we found that the majority of P. intermedia strains isolated from chronic and aggressive periodontitis carry and express the interpain A gene. The protective effect of interpain A against serum bactericidal activity was found to be attributable to its ability to inhibit all three complement pathways through the efficient degradation of the alpha-chain of C3 -- the major complement factor common to all three pathways. P. intermedia has been known to co-aggregate with P. gingivalis, which produce gingipains to efficiently degrade complement factors. Here, interpain A was found to have a synergistic effect with gingipains on complement degradation. In addition, interpain A was able to activate the C1 complex in serum, causing deposition of C1q on inert and bacterial surfaces, which may be important at initial stages of infection when local inflammatory reaction may be beneficial for a pathogen. Taken together, the newly characterized interpain A proteinase appears to be an important virulence factor of P. intermedia.

  17. Elafin/elastase-specific inhibitor in bronchoalveolar lavage of normal subjects and farmer's lung.

    Science.gov (United States)

    Tremblay, G M; Sallenave, J M; Israél-Assayag, E; Cormier, Y; Gauldie, J

    1996-10-01

    Secretory leukocyte proteinase inhibitor (SLPI) and alpha1-proteinase inhibitor (alpha1(PI)) cannot fully explain the total neutrophil elastase (NE) inhibitory capacity detected in bronchoalveolar lavage (BAL) fluid, suggesting the existence of other NE inhibitor(s). In the present study, we measured the concentrations of elafin, a newly described, low-molecular-weight serine proteinase inhibitor, SLPI, and alpha1(PI) in BAL fluids from eight healthy subjects, 13 asymptomatic farmers, seven farmers with active farmer's lung (FL), and seven farmers with previous (Ex) FL. In addition to SLPI and alpha1(PI), elafin was present in BAL fluids from control subjects and asymptomatic farmers, 13 (7-31) and 12 (7-67) mmol/mol of albumin (median and range) respectively. Elafin concentration increased significantly to 105 (38-207) mmol/mol of albumin in farmers with active FL and was also elevated in farmers with Ex FL. Elafin levels were highly correlated with lung inflammatory cell numbers, especially lymphocytes, and the decrease in single-breath diffusion capacity (DLCO). Elafin and SLPI were linked to yet uncharacterized proteins in BAL fluids. In conclusion, elafin is a constituent of BAL fluid from normal subjects and is found in enhanced concentrations in FL and in farmers with lymphocytic alveolitis. This suggests that elafin may play a role in lung homeostasis and inflammation. PMID:8887613

  18. Biochemical, immunological and kinetic characterisation of thiol protease inhibitor (cystatin) from liver.

    Science.gov (United States)

    Shah, Aaliya; Priyadarshini, Medha; Khan, Mohd Shahnawaz; Aatif, Mohammad; Amin, Fakhra; Bano, Bilqees

    2013-10-01

    Regulation of the cysteine protease activity is imperative for proper functioning of the various organ systems. Elevated activities of cysteine proteinases due to impaired regulation by the endogenous cysteine proteinase inhibitors (cystatins) have been linked to liver malignancies. To gain an insight into these regulatory processes, it is essential to purify and characterise the inhibitors, cystatins. Present study was undertaken to purify the inhibitor from the liver. The purification was accomplished in four steps: alkaline treatment, ammonium sulphate fractionation, acetone precipitation and gel filtration column (Sephacryl S-100 HR). The eluted protein exhibited inhibitory activity towards papain, and its purity was further reaffirmed using western blotting and immunodiffusion. The purified inhibitor (liver cystatin (LC)) was stable in the pH range of 6-8 and temperature up to 45 °C. In view of the significance of kinetics parameters for drug delivery, the kinetic parameters of liver cystatin were also determined. LC showed the greatest affinity for papain followed by ficin and bromelain. UV and fluorescence spectroscopy results showed that binding of LC with thiol proteases induced changes in the environment of aromatic residues. Recent advances in the field of proteinase inhibitors have drawn attention to the possible use of this collected knowledge to control pathologies.

  19. Neuronal projections from the Haller's organ and palp sensilla to the synganglion of Amblyomma americanum§.

    Science.gov (United States)

    Borges, Lígia Miranda Ferreira; Li, Andrew Yongsheng; Olafson, Pia Untalan; Renthal, Robert; Bauchan, Gary Roy; Lohmeyer, Kimberly Hutchison; León, Adalberto Angel Pérez de

    2016-06-14

    The present study was conducted to elucidate the neuronal pathways between peripheral olfactory and taste sensilla and the synganglion in an Ixodidae tick species. The tarsus of the front legs (olfactory nerves) and the fourth palpal segment (gustatory nerves) of unfed Amblyomma americanum males and females were excised. A neuronal tracer, dextran tetramethylrhodamine, was used for filling of the sensory neurons. The synganglion preparations were examined using a confocal microscope. Neuronal arborizations from the Haller's organ were confined to the olfactory lobes and the first pedal ganglion. The estimated number of olfactory glomeruli ranged from 16 to 22 per olfactory lobe in the females. The number of glomeruli was not counted in males because they were densely packed. Sensory neurons associated with sensilla at the distal end of the palpal organ projected into the palpal ganglion in the synganglion through the palpal nerve. Gustatory sensory neurons associated with palpal sensilla projected into a commissure with several bulges, which are confined in the palpal ganglion. The findings of distinct projection patterns of sensory neurons associated with the Haller's organ and palpal organ in the lone star tick from this study advanced our knowledge on mechanisms of sensory information processing in ticks.

  20. Evaluation of DEET and eight essential oils for repellency against nymphs of the lone star tick, Amblyomma americanum (Acari: Ixodidae).

    Science.gov (United States)

    Meng, Hao; Li, Andrew Y; Costa Junior, Livio M; Castro-Arellano, Ivan; Liu, Jingze

    2016-02-01

    DEET and Eight commercially available essential oils (oregano, clove, thyme, vetiver, sandalwood, cinnamon, cedarwood, and peppermint) were evaluated for repellency against host-seeking nymphs of the lone star tick, Amblyomma americanum. Concentration-repellency response was established using the vertical paper bioassay technique for each essential oil and compared with that of N,N-diethyl-3-methyl benzamide (DEET), a standard repellent compound present in many commercial repellent formulations. The effective concentration of DEET that repels 50% of ticks (EC50) was estimated at 0.02 mg/cm(2), while EC50s of the essential oils fall between 0.113 and 0.297 mg/cm(2). Based on EC50 estimates, oregano essential oil was the most effective among all essential oils tested, followed by clove, thyme, vetiver, sandalwood, cinnamon, cedarwood, and peppermint oils. None of the tested essential oils demonstrated a level of tick repellency found with DEET. Results from this study illustrated the challenge in search for more effective natural tick repellents. PMID:26590930

  1. "Purification and evaluation of somatic, excretory-secretory and Cysteine proteinase antigens of Fasciola Hepatica using IgG-ELISA in diagnosing Fascioliasis "

    Directory of Open Access Journals (Sweden)

    "Rokni MB

    2001-08-01

    Full Text Available Fasciolosis, or liver fluke disease, caused by parasites of the genus Fasciola is emerging as an important disease in man and animals, in the world and Iran, particularly in nortern parts. The economical losses in domestic animals are considerable. In the recent decade there were two major outbreaks of human fasciolosis in the Caspian region, northern part of Iran with 7000-10000 infected cases. Sicne it is impossible to diagnose fasciolosis in acute phase using coprological methods and even in chronic phases its sensitivity is low, evaluating and establishing a reliable and cost-effetive test is indispensable and notewortly.In the present survey, we produced and examined the sensitivity and specificity of liver fluke homogenate (LFH , excretory-secetory (ES and cysteine proteinase (CP antigens of F. hepatica using IgG-ELISA test. A 25-27 kilo Dalton coomassie blue-stained band was observed and using of specific inhibitors indicated that this antigen belongs to the class of cysteine proteinase. The sensitivity of LFH, ES and CP antigen in IgG-ELISa was 100% for each, while their specificity was 97.8%, 98.8% and 98.8% respectively. There was a significant difference in mean OD values between cases of proven fasciolosis and other true negative cases, including healthy control individuals and patients with other parasitic diseases.This present report is the first to demonstrate the purification and evaluation of F. hepatica cysteine proteinase antigen by IgG-ELISA test for the diagnosis of fasciolosis in Iran. In conclusion, the IgG-ELISa using ES and CP show high sensitivity and specificity and would be a valuable tool to diagnose human fasciolosis in Iran, particularly in endemic areas.

  2. Lipases and proteinases in milk : occurrence, heat inactivation, and their importance for the keeping quality of milk products

    OpenAIRE

    Driessen, F.M.

    1983-01-01

    The occurrence and heat inactivation of native and bacterial lipases and proteinases in milk were studied.Production of these enzymes by Gram-negative psychrotrophic bacteria in milk was found to take place towards the end of exponential growth and in the stationary growth phase.Kinetics of heat inactivation in milk of milk lipoprotein lipase, alkaline milk proteinase and lipases and proteinases of some Gram-negative bacteria are given.The effects of residual lipolytic and proteolytic activit...

  3. The kinetics of proteinase K digestion of linear prion polymers.

    Science.gov (United States)

    Masel, J; Jansen, V A

    1999-09-22

    Transmissible spongiform encephalopathies such as scrapie are caused by a protein-only infectious agent, known as a prion. It is not clear how a protein can be capable of replicating itself, and the mechanism remains controversial. One influential model hypothesizes that prions are nucleated, macroscopically linear polymers. We investigated the theoretical kinetics of this model and derived predictions which could be used to test the model. In the model, the polymerization and depolymerization rates are independent polymer size. This leads to an exponential size distribution at equilibrium. In agreement with a prediction stemming from this size distribution, the average size of PrP-res polymers was proportional to the square root of the concentration of PrP-res in a published study of in vitro conversion. Prion digestion by proteinase K (PK) is predicted to be biphasic. The second phase of digestion should be virtually independent of the PK concentration and should depend on the initial size distribution of prion polymers. For initially equilibrated polymers with an exponential size distribution, phase two digestion is exponential at a predicted rate. This rate varies in a defined way with the concentration used for equilibration and with other parameters which affect the average polymer size.

  4. Screening and purification of a novel trypsin inhibitor from Prosopis juliflora seeds with activity toward pest digestive enzymes.

    Science.gov (United States)

    Sivakumar, S; Franco, O L; Tagliari, P D; Bloch, C; Mohan, M; Thayumanavan, B

    2005-08-01

    Several pests are capable of decreasing crop production causing severe economical and social losses. Aiming to find novel molecules that could impede the digestion process of different pests, a screening of alpha-amylase and trypsin-like proteinase inhibitors was carried out in Prosopis juliflora, showing the presence of both in dry seeds. Furthermore, a novel trypsin inhibitor, with molecular mass of 13,292 Da, was purified showing remarkable in vitro activity against T. castaneum and C. maculatus.

  5. Human Tissue Factor Pathway Inhibitor-2 is Internalized by Cells and Translocated to the Nucleus by the Importin System

    OpenAIRE

    Kempaiah, Prakasha; Chand, Hitendra S.; Kisiel, Walter

    2008-01-01

    Tissue factor pathway inhibitor-2 (TFPI-2) is a serine proteinase inhibitor that induces caspase-mediated apoptosis when offered to a variety of tumor cells. In order to investigate the mechanism of TFPI-2-induced apoptosis, we initially studied the uptake and trafficking of TFPI-2 by HT-1080 cells. Exogenously offered TFPI-2 was rapidly internalized and distributed in both the cytosolic and nuclear fractions. Nuclear localization of TFPI-2 was also detected in a variety of endothelial cells ...

  6. Momordica charantia trypsin inhibitor Ⅱ inhibits growth and development of Helicoverpa armigera

    Institute of Scientific and Technical Information of China (English)

    Manasi Alok Telang; Prashant Pyati; Mohini Sainani; Vidya Shrikant Gupta; Ashok Prabhakar Giri

    2009-01-01

    Bitter gourd (Momordica charantia L.) seeds contain several squash-type serine proteinase inhibitors (PIs),which inhibit the digestive proteinases of the polyphagous insect pest Helicoverpa armigera.In the present work isolation of a DNA sequence encoding the mature peptide of a trypsin inhibitor McTI-Ⅱ,its cloning and expression as a recombinant protein using Pichia pastoris have been reported.Recombinant McTI-Ⅱinhibited bovine trypsin at 1:1 molar ratio,as expected,but did not inhibit chymotrypsin or elastase.McTI-Ⅱalso strongly inhibited trypsin-like proteinases (81% inhibition) as well as the total proteolytic activity of digestive proteinases (70% inhibition) from the midgut of H.armigera larvae.The insect larvae fed with McTI-Ⅱ-incorporated artificial diet suffered over 70% reduction in the average larval weight after 12 days of feeding.Moreover,ingestion of McTI-Ⅱresulted in 23% mortality in the larval population.The strong antimetabolic activity of McTI-Ⅱtoward H.armigera indicates its probable use in developing insect tolerance in susceptible plants.

  7. A Dynamic Population Model to Investigate Effects of Climate and Climate-Independent Factors on the Lifecycle of Amblyomma americanum (Acari: Ixodidae).

    Science.gov (United States)

    Ludwig, Antoinette; Ginsberg, Howard S; Hickling, Graham J; Ogden, Nicholas H

    2016-01-01

    The lone star tick, Amblyomma americanum, is a disease vector of significance for human and animal health throughout much of the eastern United States. To model the potential effects of climate change on this tick, a better understanding is needed of the relative roles of temperature-dependent and temperature-independent (day-length-dependent behavioral or morphogenetic diapause) processes acting on the tick lifecycle. In this study, we explored the roles of these processes by simulating seasonal activity patterns using models with site-specific temperature and day-length-dependent processes. We first modeled the transitions from engorged larvae to feeding nymphs, engorged nymphs to feeding adults, and engorged adult females to feeding larvae. The simulated seasonal patterns were compared against field observations at three locations in United States. Simulations suggested that 1) during the larva-to-nymph transition, some larvae undergo no diapause while others undergo morphogenetic diapause of engorged larvae; 2) molted adults undergo behavioral diapause during the transition from nymph-to-adult; and 3) there is no diapause during the adult-to-larva transition. A model constructed to simulate the full lifecycle of A. americanum successfully predicted observed tick activity at the three U.S. study locations. Some differences between observed and simulated seasonality patterns were observed, however, identifying the need for research to refine some model parameters. In simulations run using temperature data for Montreal, deterministic die-out of A. americanum populations did not occur, suggesting the possibility that current climate in parts of southern Canada is suitable for survival and reproduction of this tick.

  8. A dynamic population model to investigate effects of climate and climate-independent factors on the lifecycle of the tick Amblyomma americanum (Acari: Ixodidae)

    Science.gov (United States)

    Ludwig, Antoinette; Ginsberg, Howard; Hickling, Graham J.; Ogden, Nicholas H.

    2015-01-01

    The lone star tick, Amblyomma americanum, is a disease vector of significance for human and animal health throughout much of the eastern United States. To model the potential effects of climate change on this tick, a better understanding is needed of the relative roles of temperature-dependent and temperature-independent (day-length-dependent behavioral or morphogenetic diapause) processes acting on the tick lifecycle. In this study, we explored the roles of these processes by simulating seasonal activity patterns using models with site-specific temperature and day-length-dependent processes. We first modeled the transitions from engorged larvae to feeding nymphs, engorged nymphs to feeding adults, and engorged adult females to feeding larvae. The simulated seasonal patterns were compared against field observations at three locations in United States. Simulations suggested that 1) during the larva-to-nymph transition, some larvae undergo no diapause while others undergo morphogenetic diapause of engorged larvae; 2) molted adults undergo behavioral diapause during the transition from nymph-to-adult; and 3) there is no diapause during the adult-to-larva transition. A model constructed to simulate the full lifecycle of A. americanum successfully predicted observed tick activity at the three U.S. study locations. Some differences between observed and simulated seasonality patterns were observed, however, identifying the need for research to refine some model parameters. In simulations run using temperature data for Montreal, deterministic die-out of A. americanum populations did not occur, suggesting the possibility that current climate in parts of southern Canada is suitable for survival and reproduction of this tick.

  9. Susceptibility of four tick species, Amblyomma americanum, Dermacentor variabilis, Ixodes scapularis, and Rhipicephalus sanguineus (Acari: Ixodidae), to nootkatone from essential oil of grapefruit.

    Science.gov (United States)

    Flor-Weiler, Lina B; Behle, Robert W; Stafford, Kirby C

    2011-03-01

    Toxicity of nootkatone was determined in laboratory assays against unfed nymphs of Amblyomma americanum L., Dermacentor variabilis (Say), Ixodes scapularis Say, and Rhipicephalus sanguineus Latreille. We determined the 50% lethal concentration (LC50) and 90% lethal concentration (LC90) of nootkatone by recording tick mortality 24 h after exposure in treated glass vials. Nymphs were susceptible to nootkatone with LC50 values of 0.352, 0.233, 0.169, and 0.197 microg/cm2, and LC90 values of 1.001, 0.644, 0.549, and 0.485 microg/cm2 for A. americanum, D. variabilis, I. scapularis, and R. sanguineus, respectively. The LC50 value for R. sanquineus was not significantly different from D. variabilis or I. scapularis. Other LC50 comparisons were significantly different. The LC90 for A. americanum was higher when compared with the three other tick species, which were not significantly different. Because nootkatone is volatile, we measured the amount of nootkatone recovered from duplicate-treated vials before tick exposure and from vials after tick exposure. Nootkatone recovered from vials before exposure ranged from 82 to 112% of the expected amounts. The nootkatone recovered after the 24-h exposure period ranged from 89% from vials coated with higher concentrations of nootkatone, down to 29% from vials coated with low nootkatone concentrations. Determination of the nootkatone residue after vial coating demonstrated loss of the active compound while verifying the levels of tick exposure. Toxicity of low concentrations of nootkatone to the active questing stage of ticks reported in this study provides a reference point for future formulation research to exploit nootkatone as a safe and environment-friendly tick control. PMID:21485368

  10. Suppression of host-seeking Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) nymphs after dual applications of plant-derived acaricides in New Jersey.

    Science.gov (United States)

    Jordan, Robert A; Dolan, Marc C; Piesman, Joseph; Schulze, Terry L

    2011-04-01

    We evaluated the ability of dual applications of natural, plant-derived acaricides to suppress nymphal Ixodes scapularis Say and Amblyomma americanum (L.) (Acari: Ixodidae) in a Lyme disease endemic area of New Jersey. An aqueous formulation of 2% nootkatone provided >90% control of I. scapularis through 7 d. Control declined to 80.9% at 14 d, and a second application was made that provided >95% control through the remaining 4 wk of the nymphal season. Nootkatone provided >90% control of A. americanum through 35 d postapplication. Applications of 2% carvacrol and EcoTrol T&O resulted in rapid knockdown of both tick species, but control declined significantly to 76.7 and 73.7%, respectively, after 14 d when a second application was made that extended control of both tick species to between 86.2 and 94.8% at 21 d. Subsequently, control declined steadily in all plots by 42 d postapplication except for I. scapularis in carvacrol-treated plots, where levels of control >90% were observed through 35 d. Of the three compounds tested, 2% nootkatone provided the most consistent results, with 96.5 and 91.9% control of I. scapularis and A. americanum through 42 and 35 d, respectively. The ability of plant-derived natural products to quickly suppress and maintain significant control of populations of these medically important ticks may represent a future alternative to the use of conventional synthetic acaricides. In addition, the demonstrated efficacy of properly-timed backpack sprayer application may enable homeowner access to these minimal-risk acaricides. PMID:21510219

  11. Comparison of flagging, walking, trapping, and collecting from hosts as sampling methods for northern deer ticks, Ixodes dammini, and lone-star ticks, Amblyomma americanum (Acari:Ixodidae).

    Science.gov (United States)

    Ginsberg, H S; Ewing, C P

    1989-09-01

    Ticks were sampled by flagging, collecting from the investigator's clothing (walking samples), trapping with dry-ice bait, and collecting from mammal hosts on Fire Island, NY, U.S.A. The habitat distribution of adult deer ticks, Ixodes dammini, was the same in simultaneous collections from the investigator's clothing and from muslin flags. Walking and flagging samples can both be biased by differences between investigators, so the same person should do comparative samples whenever possible. Walking samples probably give a more accurate estimate than flagging samples of the human risk of encountering ticks. However, ticks (such as immature I. dammini) that seek hosts in leaf litter and ground-level vegetation are poorly sampled by walking collections. These ticks can be sampled by flagging at ground level. Dry-ice-baited tick-traps caught far more lone-star ticks, Amblyomma americanum, than deer ticks, even in areas where deer ticks predominated in flagging samples. In comparisons of tick mobility in the lab, nymphal A. americanum were more mobile than nymphal I. dammini in 84% of the trials. Therefore, the trapping bias may result from increased trap encounter due to more rapid movement by A. americanum, although greater attraction to carbon dioxide may also play a role. Tick traps are useful for intraspecific between-habitat comparisons. Early in their seasonal activity period, larval I. dammini were better represented in collections from mouse hosts than in flagging samples. Apparently, sampling from favored hosts can detect ticks at low population levels, but often cannot be used to get accurate estimates of pathogen prevalence in questing ticks. PMID:2806016

  12. Short-term variability of biomarkers of proteinase activity in patients with emphysema associated with type Z alpha-1-antitrypsin deficiency

    Directory of Open Access Journals (Sweden)

    Nieuwenhuizen Willem

    2005-05-01

    Full Text Available Abstract Background The burden of proteinases from inflammatory cells in the lung of subjects with type Pi ZZ of alpha-1-antitrypsin deficiency is higher than in those without the deficiency. Cross-sectional studies have shown increased levels of biomarkers of extracellular matrix degradation in vivo. Longitudinal variability of these biomarkers is unknown but desirable for clinical studies with proteinase inhibitors. Methods We measured three different types of biomarkers, including desmosines, elastase-formed fibrinogen fragments and heparan sulfate epitope JM403, in plasma and urine for a period of 7 weeks in a group of 12 patients who participated in a placebo-controlled study to assess the safety of a single inhalation of hyaluronic acid. Results Effect of study medication on any of the biomarkers was not seen. Baseline desmosines in plasma and urine correlated with baseline CO diffusion capacity (R = 0.81, p = 0.01 and R = 0.65, p = 0.05. Mean coefficient of variation within patients (CVi for plasma and urine desmosines was 18.7 to 13.5%, respectively. Change in urinary desmosine levels correlated significantly with change in plasma desmosine levels (R = 0.84, p Conclusion We found acceptable variability in our study parameters, indicating the feasibility of their use in an evaluation of biochemical efficacy of alpha-1-antitrypsin augmentation therapy in Pi Z subjects.

  13. Determination of germ tube, phospholipase, and proteinase production by bloodstream isolates of Candida albicans

    Directory of Open Access Journals (Sweden)

    Antonella Souza Mattei

    2013-06-01

    Full Text Available Introduction Candida albicans is a commensal and opportunistic agent that causes infection in immunocompromised individuals. Several attributes contribute to the virulence and pathogenicity of this yeast, including the production of germ tubes (GTs and extracellular hydrolytic enzymes, particularly phospholipase and proteinase. This study aimed to investigate GT production and phospholipase and proteinase activities in bloodstream isolates of C. albicans. Methods One hundred fifty-three C. albicans isolates were obtained from blood samples and analyzed for GT, phospholipase, and proteinase production. The assays were performed in duplicate in egg yolk medium containing bovine serum albumin and human serum. Results Detectable amounts of proteinase were produced by 97% of the isolates, and 78% of the isolates produced phospholipase. GTs were produced by 95% of the isolates. A majority of the isolates exhibited low levels of phospholipase production and high levels of proteinase production. Conclusions Bloodstream isolates of C. albicans produce virulence factors such as GT and hydrolytic enzymes that enable them to cause infection under favorable conditions.

  14. NaCl-activated extracellular proteinase from Virgibacillus sp. SK37 isolated from fish sauce fermentation.

    Science.gov (United States)

    Sinsuwan, S; Rodtong, S; Yongsawatdigul, J

    2007-06-01

    Virgibacillus sp. SK37 exhibited high extracellular proteolytic activity in skim milk broth containing 10% NaCl. Optimum conditions of the crude proteinase were at pH 8.0 and 65 degrees C. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-AMC, suggesting the serine proteinase with a subtilisin-like characteristic. Proteolytic activity increased with NaCl concentration up to 20%. Ca(2+) activated the enzyme activity but reduced enzyme stability at 65 degrees C. Several proteinases with dominant molecular mass (MW) of 81, 67, 63, 50, 38, and 18 kDa were detected on native-polyacrylamide gel electrophoresis (native-PAGE) activity staining in the absence and presence of 25% NaCl. These results demonstrated that Virgibacillus sp. SK37 produced salt-activated extracellular proteinases. Virgibacillus sp. SK37 could be a promising strain for starter culture development used in fish sauce fermentation. PMID:17995713

  15. Neutrophil elastase and proteinase 3 trafficking routes in myelomonocytic cells

    Energy Technology Data Exchange (ETDEWEB)

    Kaellquist, Linda; Rosen, Hanna [Department of Hematology, BMC C14, Lund University, SE-221 84 Lund (Sweden); Nordenfelt, Pontus [Section for Clinical and Experimental Infection Medicine, Department of Clinical Sciences, Lund University, SE-221 84 Lund (Sweden); Calafat, Jero; Janssen, Hans [Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 1211066, Amsterdam (Netherlands); Persson, Ann-Maj [Department of Hematology, BMC C14, Lund University, SE-221 84 Lund (Sweden); Hansson, Markus, E-mail: Markus.Hansson@med.lu.se [Department of Hematology, BMC C14, Lund University, SE-221 84 Lund (Sweden); Olsson, Inge [Department of Hematology, BMC C14, Lund University, SE-221 84 Lund (Sweden)

    2010-11-15

    Neutrophil elastase (NE) and proteinase 3 (PR3) differ in intracellular localization, which may reflect different trafficking mechanisms of the precursor forms when synthesized at immature stages of neutrophils. To shed further light on these mechanisms, we compared the trafficking of precursor NE (proNE) and precursor PR3 (proPR3). Like proNE [1], proPR3 interacted with CD63 upon heterologous co-expression in COS cells but endogenous interaction was not detected although cell surface proNE/proPR3/CD63 were co-endocytosed in myelomonocytic cells. Cell surface proNE/proPR3 turned over more rapidly than cell surface CD63 consistent with processing/degradation of the pro-proteases but recycling of CD63. Colocalization of proNE/proPR3/CD63 with clathrin and Rab 7 suggested trafficking through coated vesicles and late endosomes. Partial caveolar trafficking of proNE/CD63 but not proPR3 was suggested by colocalization with caveolin-1. Blocking the C-terminus of proNE/proPR3 by creating a fusion with FK506 binding protein inhibited endosomal re-uptake of proNE but not proPR3 indicating 'pro{sub C}'-peptide-dependent structural/conformational requirements for proNE but not for proPR3 endocytosis. The NE aminoacid residue Y199 of a proposed NE sorting motif that interacts with AP-3 [2] was not required for proNE processing, sorting or endocytosis in rat basophilic leukemia (RBL) cells expressing heterologous Y199-deleted proNE; this suggests operation of another AP-3-link for proNE targeting. Our results show intracellular multi-step trafficking to be different between proNE and proPR3 consistent with their differential subcellular NE/PR3 localization in neutrophils.

  16. Biological roles of cysteine proteinases in the pathogenesis of Trichomonas vaginalis.

    Science.gov (United States)

    Hernández, Hilda M; Marcet, Ricardo; Sarracent, Jorge

    2014-01-01

    Human trichomonosis, infection with Trichomonas vaginalis, is the most common non-viral sexually transmitted disease in the world. The host-parasite interaction and pathophysiological processes of trichomonosis remain incompletely understood. This review focuses on the advancements reached in the area of the pathogenesis of T. vaginalis, especially in the role of the cysteine proteinases. It highlights various approaches made in this field and lists a group of trichomonad cysteine proteinases involved in diverse processes such as invasion of the mucous layer, cytoadherence, cytotoxicity, cytoskeleton disruption of red blood cells, hemolysis, and evasion of the host immune response. A better understanding of the biological roles of cysteine proteinases in the pathogenesis of this parasite could be used in the identification of new chemotherapeutic targets. An additional advantage could be the development of a vaccine in order to reduce transmission of T. vaginalis.

  17. A new method of research on molecular evolution of pro-teinase superfamily

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The molecular evolutionary tree, also known as a phylogenetic tree, of the serine proteinase superfamily was constructed by means of structural alignment. Three-dimensional structures of proteins were aligned by the SSAP program of Orengo and Taylor to obtain evolutionary dis-tances. The resulting evolutionary tree provides a topology graph that can reflect the evolution of structure and function of homology proteinase. Moreover, study on evolution of the serine proteinase superfamily can lead to better under-standing of the relationship and evolutionary difference among proteins of the superfamily, and is of significance to protein engineering, molecular design and protein structure prediction. Structure alignment is one of the useful methods of research on molecular evolution of protein.

  18. Modification of standard proteinase K/phenol method for extraction of DNA from small tumour biopsies.

    Science.gov (United States)

    Pitera, R; Pitera, J E; Mufti, G J; Salisbury, J R

    1993-09-01

    The standard proteinase K/phenol DNA isolation method was found to produce unsatisfactory yields of DNA from small tissue biopsies (less than 50 mg). The influences of the volume of cell lysis buffer and the amount of proteinase K on the final DNA yield and quality were studied, and an improved method was devised and compared with both the standard procedure and a phenol-free protocol. The optimal volume of cell lysis buffer was found to be 200 microliters per mg of tissue while the optimal amount of proteinase K was 60 micrograms per mg of tissue. A mean yield of 12 mu/mg tissue of pure, high molecular weight DNA was achieved from 50 frozen samples prepared by crushing. Yields from 20 microns thick cryostat sections reached 30 micrograms/mg.

  19. Toll-like receptors recognize distinct proteinase-resistant glycoconjugates in Campylobacter jejuni and Escherichia coli.

    Science.gov (United States)

    Phongsisay, Vongsavanh; Hara, Hiromitsu; Fujimoto, Shuji

    2015-03-01

    Campylobacter jejuni causes gastroenteritis and autoimmune neuropathy Guillain-Barré syndrome. The mechanism by which C. jejuni infection results in such the hyperimmunity is not completely understood. Host immunity plays an important role in the disease pathogenesis; however, little is known how immune system recognizes this human pathogen. In this study, we report that Toll-like receptors recognize distinct proteinase K-resistant glycoconjugates in C. jejuni and Escherichia coli. Lipopolysaccharide is solely proteinase-resistant glycoconjugate in E. coli. In contrast, C. jejuni possesses at least five different components that are resistant to proteinase digestion and are capable of inducing NF-κB activation through TLR2 and TLR4. Possession of multiple activators of Toll-like receptors may be the unique strategy of C. jejuni to trigger hyperimmunity.

  20. Human placental extract mediated inhibition of proteinase K: implications of heparin and glycoproteins in wound physiology.

    Science.gov (United States)

    Sharma, Kanika; Mukherjee, Chaitali; Roy, Siddhartha; De, Debashree; Bhattacharyya, Debasish

    2014-09-01

    Efficient debridement of the wound bed following the removal of microbial load prevents its progression into a chronic wound. Bacterial infection and excessive proteolysis characterize impaired healing and therefore, their inhibition might restore the disturbed equilibrium in the healing process. Human placental extract exhibits reversible, non-competitive inhibition towards Proteinase K, a microbial protease, by stabilizing it against auto-digestion. Scattering and fluorescence studies followed by biochemical analysis indicated the involvement of a glycan moiety. Surface plasmon resonance demonstrated specific interaction of heparin with Proteinase K having Kd in μM range. Further, Proteinase K contains sequence motifs similar to other heparin-binding proteins. Molecular docking revealed presence of clefts suitable for binding of heparin-derived oligosaccharides. Comprehensive analysis of this inhibitory property of placental extract partly explains its efficacy in curing wounds with common bacterial infections.

  1. Coronavirus 3CL(pro) proteinase cleavage sites: Possible relevance to SARS virus pathology

    DEFF Research Database (Denmark)

    Kiemer, Lars; Lund, Ole; Brunak, Søren;

    2004-01-01

    . Prompted by this, we set out to analyse and predict cleavage by the coronavirus main proteinase using computational methods. Results: We retrieved sequence data on seven fully sequenced coronaviruses and identified the main 3CL proteinase cleavage sites in polyproteins using alignments. A neural network...... was trained to recognise the cleavage sites in the genomes obtaining a sensitivity of 87.0% and a specificity of 99.0%. Several proteins known to be cleaved by other viruses were submitted to prediction as well as proteins suspected relevant in coronavirus pathology. Cleavage sites were predicted in proteins...

  2. Proteinase, phospholipase, hyaluronidase and chondroitin-sulphatase production by Malassezia pachydermatis.

    Science.gov (United States)

    Coutinho, S D; Paula, C R

    2000-02-01

    The production of four functional enzyme categories was investigated in 30 strains of Malassezia pachydermatis isolated from dogs with otitis or dermatitis. The most appropriate reading intervals for these assays were determined with the aid of statistical comparisons. All strains produced proteinase and chondroitin-sulphatase; hyaluronidase and phospholipase were produced by all skin isolates (15/15) and 14 out of 15 ear canal isolates. Strains from ear canals did not differ significantly as a group from skin strains in quantitative production of any of the four enzymes; production of proteinase and chondroitin-sulphatase in particular was markedly uniform. PMID:10746230

  3. Putrescine-dependent re-localization of TvCP39, a cysteine proteinase involved in Trichomonas vaginalis cytotoxicity.

    Directory of Open Access Journals (Sweden)

    Bertha Isabel Carvajal-Gamez

    Full Text Available Polyamines are involved in the regulation of some Trichomonas vaginalis virulence factors such as the transcript, proteolytic activity, and cytotoxicity of TvCP65, a cysteine proteinase (CP involved in the trichomonal cytotoxicity. In this work, we reported the putrescine effect on TvCP39, other CP that also participate in the trichomonal cytotoxicity. Parasites treated with 1,4-diamino-2-butanone (DAB (an inhibitor of putrescine biosynthesis, diminished the amount and proteolytic activity of TvCP39 as compared with untreated parasites. Inhibition of putrescine biosynthesis also reduced ∼ 80% the tvcp39 mRNA levels according to RT-PCR and qRT-PCR assays. Additionally, actinomycin D-treatment showed that the tvcp39 mRNA half-life decreased in the absence of putrescine. However, this reduction was restored by exogenous putrescine addition, suggesting that putrescine is necessary for tvcp39 mRNA stability. TvCP39 was localized in the cytoplasm but, in DAB treated parasites transferred into exogenous putrescine culture media, TvCP39 was re-localized to the nucleus and nuclear periphery of trichomonads. Interestingly, the amount and proteolytic activity of TvCP39 was recovered as well as the tvcp39 mRNA levels were restored when putrescine exogenous was added to the DAB-treated parasites. In conclusion, our data show that putrescine regulate the TvCP39 expression, protein amount, proteolytic activity, and cellular localization.

  4. Putrescine-dependent re-localization of TvCP39, a cysteine proteinase involved in Trichomonas vaginalis cytotoxicity.

    Science.gov (United States)

    Carvajal-Gamez, Bertha Isabel; Quintas-Granados, Laura Itzel; Arroyo, Rossana; Vázquez-Carrillo, Laura Isabel; Ramón-Luing, Lucero De los Angeles; Carrillo-Tapia, Eduardo; Alvarez-Sánchez, María Elizbeth

    2014-01-01

    Polyamines are involved in the regulation of some Trichomonas vaginalis virulence factors such as the transcript, proteolytic activity, and cytotoxicity of TvCP65, a cysteine proteinase (CP) involved in the trichomonal cytotoxicity. In this work, we reported the putrescine effect on TvCP39, other CP that also participate in the trichomonal cytotoxicity. Parasites treated with 1,4-diamino-2-butanone (DAB) (an inhibitor of putrescine biosynthesis), diminished the amount and proteolytic activity of TvCP39 as compared with untreated parasites. Inhibition of putrescine biosynthesis also reduced ∼ 80% the tvcp39 mRNA levels according to RT-PCR and qRT-PCR assays. Additionally, actinomycin D-treatment showed that the tvcp39 mRNA half-life decreased in the absence of putrescine. However, this reduction was restored by exogenous putrescine addition, suggesting that putrescine is necessary for tvcp39 mRNA stability. TvCP39 was localized in the cytoplasm but, in DAB treated parasites transferred into exogenous putrescine culture media, TvCP39 was re-localized to the nucleus and nuclear periphery of trichomonads. Interestingly, the amount and proteolytic activity of TvCP39 was recovered as well as the tvcp39 mRNA levels were restored when putrescine exogenous was added to the DAB-treated parasites. In conclusion, our data show that putrescine regulate the TvCP39 expression, protein amount, proteolytic activity, and cellular localization.

  5. Experimental use of two standard tick collection methods to evaluate the relative effectiveness of several plant-derived and synthetic repellents against Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae).

    Science.gov (United States)

    Schulze, Terry L; Jordan, Robert A; Dolan, Marc C

    2011-12-01

    We used two standard tick collection methods to test the relative effectiveness of two natural product compounds (nootkatone and carvacrol, classified as an eremophilene sesquiterpene and a monoterpene, respectively, that are derived from botanical sources) with commercially-available plant-derived (EcoSMART Organic Insect Repellent, comprised of plant essential oils) and permethrin-based (Repel Permanone) repellents against Ixodes scapularis Say and Amblyomma americanum (L.). Cloth drags were equally effective in sampling both species of host-seeking nymphs, whereas CO, traps attracted primarily A. americanum. All four repellents performed well on drags, with nootkatone and Permanone Repel (100% repelled through 14 d) slightly more effective than carvacrol and EcoSMART (90.7% and 97.7% repelled at 14 d, respectively) at repelling I. scapularis nymphs. Although the same trend in percent repellency was noted in the CO2 trap trial against both A. americanum nymphs and adults, EcoSMART outperformed Permanone in repelling A. Americanum nymphs after 14 d in the drag trial. Generally, the effectiveness of all repellents tested declined over time. The use of tick drags and CO2 traps was rapid, inexpensive, and easy to use in determining the relative effectiveness of repellents in the field. PMID:22299371

  6. Trypsin inhibitors from Capsicum baccatum var. pendulum leaves involved in Pepper yellow mosaic virus resistance.

    Science.gov (United States)

    Moulin, M M; Rodrigues, R; Ribeiro, S F F; Gonçalves, L S A; Bento, C S; Sudré, C P; Vasconcelos, I M; Gomes, V M

    2014-01-01

    Several plant organs contain proteinase inhibitors, which are produced during normal plant development or are induced upon pathogen attack to suppress the enzymatic activity of phytopathogenic microorganisms. In this study, we examined the presence of proteinase inhibitors, specifically trypsin inhibitors, in the leaf extract of Capsicum baccatum var. pendulum inoculated with PepYMV (Pepper yellow mosaic virus). Leaf extract from plants with the accession number UENF 1624, which is resistant to PepYMV, was collected at 7 different times (0, 24, 48, 72, 96, 120, and 144 h). Seedlings inoculated with PepYMV and control seedlings were grown in a growth chamber. Protein extract from leaf samples was partially purified by reversed-phase chromatography using a C2/C18 column. Residual trypsin activity was assayed to detect inhibitors followed by Tricine-SDS-PAGE analysis to determine the N-terminal peptide sequence. Based on trypsin inhibitor assays, trypsin inhibitors are likely constitutively synthesized in C. baccatum var. pendulum leaf tissue. These inhibitors are likely a defense mechanism for the C. baccatum var. pendulum- PepYMV pathosystem. PMID:25501145

  7. Role of Protease-Inhibitors in Ocular Diseases.

    Science.gov (United States)

    Pescosolido, Nicola; Barbato, Andrea; Pascarella, Antonia; Giannotti, Rossella; Genzano, Martina; Nebbioso, Marcella

    2014-01-01

    It has been demonstrated that the balance between proteases and protease-inhibitors system plays a key role in maintaining cellular and tissue homeostasis. Indeed, its alteration has been involved in many ocular and systemic diseases. In particular, research has focused on keratoconus, corneal wounds and ulcers, keratitis, endophthalmitis, age-related macular degeneration, Sorsby fundus dystrophy, loss of nerve cells and photoreceptors during optic neuritis both in vivo and in vitro models. Protease-inhibitors have been extensively studied, rather than proteases, because they may represent a therapeutic approach for some ocular diseases. The protease-inhibitors mainly involved in the onset of the above-mentioned ocular pathologies are: α2-macroglobulin, α1-proteinase inhibitor (α1-PI), metalloproteinase inhibitor (TIMP), maspin, SERPINA3K, SERPINB13, secretory leukocyte protease inhibitor (SLPI), and calpeptin. This review is focused on the several characteristics of dysregulation of this system and, particularly, on a possible role of proteases and protease-inhibitors in molecular remodeling that may lead to some ocular diseases. Recently, researchers have even hypothesized a possible therapeutic effect of the protease-inhibitors in the treatment of injured eye in animal models. PMID:25493637

  8. Role of Protease-Inhibitors in Ocular Diseases

    Directory of Open Access Journals (Sweden)

    Nicola Pescosolido

    2014-12-01

    Full Text Available It has been demonstrated that the balance between proteases and protease-inhibitors system plays a key role in maintaining cellular and tissue homeostasis. Indeed, its alteration has been involved in many ocular and systemic diseases. In particular, research has focused on keratoconus, corneal wounds and ulcers, keratitis, endophthalmitis, age-related macular degeneration, Sorsby fundus dystrophy, loss of nerve cells and photoreceptors during optic neuritis both in vivo and in vitro models. Protease-inhibitors have been extensively studied, rather than proteases, because they may represent a therapeutic approach for some ocular diseases. The protease-inhibitors mainly involved in the onset of the above-mentioned ocular pathologies are: α2-macroglobulin, α1-proteinase inhibitor (α1-PI, metalloproteinase inhibitor (TIMP, maspin, SERPINA3K, SERPINB13, secretory leukocyte protease inhibitor (SLPI, and calpeptin. This review is focused on the several characteristics of dysregulation of this system and, particularly, on a possible role of proteases and protease-inhibitors in molecular remodeling that may lead to some ocular diseases. Recently, researchers have even hypothesized a possible therapeutic effect of the protease-inhibitors in the treatment of injured eye in animal models.

  9. Monomeric 55-kDa guanidinobenzoatase switches to a serine proteinase activity upon tetramerization. Tetrameric proteinase SP 220 K appears as the native form.

    Science.gov (United States)

    Poustis-Delpont, C; Thaon, S; Auberger, P; Gerardi-Laffin, C; Sudaka, P; Rossi, B

    1994-05-20

    Guanidinobenzoatases are cell surface enzymes present in cells capable of migration or remodeling. The guanidinobenzoatase purified to homogeneity from human renal carcinoma did not display gelatinase activity under the 55-kDa form (Poustis-Delpont, C., Descomps, R., Auberger, P., Delque-Bayer, P., Sudaka, P., and Rossi, B. (1992) Cancer Res. 52, 3622-3628). We bring new insights into the structure-activity relationships of this enzyme using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, [3H]diisopropyl fluorophosphate labeling, gelatin zymography, and immunodetection using a polyclonal antibody raised against the 55-kDa entity. Upon aggregation into a 220-kDa form, the enzyme exhibited [3H]diisopropyl fluorophosphate labeling and diisopropyl fluorophosphate-inhibitable gelatinase activity whereas its capability to cleave p-nitrophenyl p'-guanidinobenzoate as a substrate was abolished. Thus, the guanidinobenzoatase property appears as a feature of a 55-kDa inactive form of a serine proteinase subunit. After boiling in the presence of sodium dodecyl sulfate (3% w/v), the 220-kDa entity subjected to SDS-polyacrylamide gel electrophoresis could be dissociated into a 55-kDa protein as shown by silver staining. The resulting 55-kDa band remained [3H]diisopropyl fluorophosphate-labeled and reacted with anti-55-kDa guanidinobenzoatase antibodies, strongly suggesting that the 220-kDa proteinase was a noncovalently associated tetramer. Interestingly, Triton X-100 extracts of renal carcinoma plasma membranes exhibited a 220-kDa serine proteinase activity, as expressed in gelatin zymography, which was barely detectable in the non-tumoral counterpart. It is noteworthy that an anti-55-kDa guanidinobenzoatase reactive 220-kDa species was also observed in renal carcinoma plasma membranes extracts as assessed by Western blot, whereas it was hardly visible in the non-tumoral counterpart. No signal was immunodetected at M(r) 55,000 in renal carcinoma and kidney cortex

  10. Random substitution of large parts of the propeptide of yeast proteinase A

    DEFF Research Database (Denmark)

    van den Hazel, H B; Kielland-Brandt, Morten; Winther, Jakob R.

    1995-01-01

    The yeast aspartic protease, proteinase A, has a 54 amino-acid propeptide, which is removed during activation of the zymogen in the vacuole. Apart from being involved inhibition/activation, the propeptide has been shown to be essential for formation of a stable active enzyme (van den Hazel, H. B....

  11. Proteinase from germinating bean cotyledons. Evidence for involvement of a thiol group in catalysis.

    Science.gov (United States)

    Csoma, C; Polgár, L

    1984-09-15

    To degrade storage proteins germinating seeds synthesize proteinases de novo that can be inhibited by thiol-blocking reagents [Baumgartner & Chrispeels (1977) Eur. J. Biochem. 77, 223-233]. We have elaborated a procedure for isolation of such a proteinase from the cotyledons of Phaseolus vulgaris. The purification procedure involved fractionation of the cotyledon homogenate with acetone and with (NH4)2SO4 and successive chromatographies on DEAE-cellulose, activated thiol-Sepharose Sepharose and Sephacryl S-200. The purified enzyme has an Mr of 23,400, proved to be highly specific for the asparagine side chain and blocking of its thiol group resulted in loss of the catalytic activity. The chemical properties of the thiol group of the bean enzyme were investigated by acylation with t-butyloxycarbonyl-L-asparagine p-nitro-phenyl ester and by alkylations with iodoacetamide and iodoacetate. Deviations from normal pH-rate profile were observed, which indicated that the thiol group is not a simple functional group, but constitutes a part of an interactive system at the active site. The pKa value for acylation and the magnitude of the rate constant for alkylation with iodoacetate revealed that the bean proteinase possesses some properties not shared by papain and the other cysteine proteinases studied to date. PMID:6385962

  12. Subunit structure of karatasin, the proteinase isolated from Bromelia plumieri (karatas).

    Science.gov (United States)

    Montes, C; Amador, M; Cuevas, D; Cordoba, F

    1990-01-01

    Close to 15% of the karatasin proteinase activity in the fruit juice of Bromelia plumieri (karatas) is present outside dialysis Visking tubing in 7 days in 0.2 M acetate buffer (pH) 3.5 or 6.5) containing phenyl mercuric acetate. The small proteinase(s), distinct from the 85% activity in juice due to nondialysable karatasin with a reported Mr of 24,868, separates across Spectrapore (13 kDa) membranes but not across Spectrapore with 3.5 kDa average pore diameter. The dialyzed proteinase is named karatasin-D (K-D). Purified non-Dialysable karatasin can be dissociated to what seems to be K-D by incubation in a buffer solution, containing SDS and 2-mercaptoethanol with phenyl mercuric acetate, in dialysis experiments for 8 days at room temperature using Spectrapore 13 kDa tubing. Thus, native karatasin in B. plumieri fruit juice seem to be the result of association of 2 small molecular mass K-D subunits, linked together by disulfide bonds and electrostatic forces, in equilibrium with small amounts of free K-D molecules. The amino acid composition and partial sequence of karatasin up to the 14th position from the amino terminus have discrete analogies with papain and with stem bromelain.

  13. Fluorometric determination of acid proteinase activity in Candida albicans strains from diabetic patients with vulvovaginal candidiasis.

    Science.gov (United States)

    Yildirim, Zuhal; Kilic, Nedret; Kalkanci, Ayse

    2011-09-01

    Vulvovaginal candidiasis is one of the most frequent disorders in obstetrics and gynaecology. Approximately three-quarters of all adult women experience at least one episode of vulvovaginal candidiasis during their life span. Diabetes mellitus (DM) increases the rate of vaginal colonisation and infection with Candida species. The secreted acid proteinase might be especially relevant in the pathogenesis of vulvovaginal candidiasis. The aim of this study was to determine the acid proteinase activity in the samples of Candida albicans from diabetic patients with vulvovaginal candidiasis by a fluorometric method. Vaginal swabs were taken from 33 women (aged between 22 and 57 years) having symptoms of vaginitis. Patients were divided into three groups: control group, controlled diabetic group and uncontrolled diabetic group. The proteinase activity in the culture supernatants was determined by a modified fluorometric method. Acid proteinase activities were significantly increased in the uncontrolled diabetic group in comparison with both the control group and the controlled diabetic group (P albicans pathogenesis in diabetic patients. Improving glucose control may reduce the risk of Candida colonisation and potentially symptomatic infection, among women with diabetes and hence may be useful even for weaker enzyme activity measurements.

  14. Recombinant Cysteine Proteinase from Leishmania (Leishmania) chagasi Implicated in Human and Dog T-Cell Responses

    OpenAIRE

    da Costa Pinheiro, Paulo Henrique; de Souza Dias, Suzana; EULÁLIO, Kelsen Dantas; Mendonça, Ivete L.; Katz, Simone; Barbiéri, Clara Lúcia

    2005-01-01

    High in vitro lymphoproliferative responses were induced in humans and dogs by a recombinant Leishmania (Leishmania) chagasi cysteine proteinase, with secretion of IFN-γ in asymptomatic subjects or of IFN-γ, interleukin 4 (IL-4), and IL-10 in oligosymptomatic subjects. In contrast, responses of symptomatic patients and dogs were lower, with production of IL-4 and IL-10.

  15. Detergents modify proteinase K resistance of PrP Sc in different transmissible spongiform encephalopathies (TSEs).

    Science.gov (United States)

    Breyer, Johanna; Wemheuer, Wiebke M; Wrede, Arne; Graham, Catherine; Benestad, Sylvie L; Brenig, Bertram; Richt, Jürgen A; Schulz-Schaeffer, Walter J

    2012-05-25

    Prion diseases are diagnosed by the detection of their proteinase K-resistant prion protein fragment (PrP(Sc)). Various biochemical protocols use different detergents for the tissue preparation. We found that the resistance of PrP(Sc) against proteinase K may vary strongly with the detergent used. In our study, we investigated the influence of the most commonly used detergents on eight different TSE agents derived from different species and distinct prion disease forms. For a high throughput we used a membrane adsorption assay to detect small amounts of prion aggregates, as well as Western blotting. Tissue lysates were prepared using DOC, SLS, SDS or Triton X-100 in different concentrations and these were digested with various amounts of proteinase K. Detergents are able to enhance or diminish the detectability of PrP(Sc) after proteinase K digestion. Depending on the kind of detergent, its concentration - but also on the host species that developed the TSE and the disease form or prion type - the detectability of PrP(Sc) can be very different. The results obtained here may be helpful during the development or improvement of a PrP(Sc) detection method and they point towards a detergent effect that can be additionally used for decontamination purposes. A plausible explanation for the detergent effects described in this article could be an interaction with the lipids associated with PrP(Sc) that may stabilize the aggregates.

  16. Subcellular location of the helper component-proteinase of Cowpea Aphid-Borne Mosaic Virus

    NARCIS (Netherlands)

    Mlotshwa, S.; Verver, J.; Sithole-Niang, I.; Gopinath, K.; Carette, J.; Kammen, van A.; Wellink, J.

    2002-01-01

    The helper component-proteinase (HC-Pro) of Cowpea aphid-borne mosaic virus (CABMV) was expressed in Escherichia coli and used to obtain HC-Pro antiserum that was used as an analytical tool for HC-Pro studies. The antiserum was used in immunofluorescence assays to study the subcellular location of H

  17. Proteinase-activated receptor 2 modulates neuroinflammation in experimental autoimmune encephalomyelitis and multiple sclerosis

    NARCIS (Netherlands)

    F. Noorbakhsh (Farshid); K. Tsutsui (Kazuyoshi); N. Vergnolle (Nathalie); L.A. Boven (Leonie); S.F. Shariat (Shahrokh); M. Vodjgani (Mohammed); K.G. Warren (Kenneth); P. Andrade-Gordon (Patricia); N.K. Hollenberg (Norman); C. Power (Christopher)

    2006-01-01

    textabstractThe proteinase-activated receptors (PARs) are widely recognized for their modulatory properties of inflammation and neurodegeneration. We investigated the role of PAR2 in the pathogenesis of multiple sclerosis (MS) in humans and experimental autoimmune encephalomyelitis (EAE) in mice. PA

  18. Isolation and characterization of a proteinase K sensitive PrPSc fraction

    Science.gov (United States)

    Recent studies have shown that a sizeable fraction of PrPSc present in prion-infected tissues is,contrary to previous conceptions, sensitive to digestion by proteinase K (PK). This finding has important implications in the context of diagnosis of prion disease, as PK has been extensively used in att...

  19. Proteinase K and the structure of PrPse: the good, the bad, and the ugly

    Science.gov (United States)

    Infectious proteins (prions) are, ironically, defined by their resistance to proteolytic digestion. A defining characteristic of the transmissible isoform of the prion protein (PrPSc) is its partial resistance to proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunod...

  20. Activities of amylase, proteinase, and lipase enzymes from Lactococcus chungangensis and its application in dairy products.

    Science.gov (United States)

    Konkit, Maytiya; Kim, Wonyong

    2016-07-01

    Several enzymes are involved in the process of converting milk to lactic acid and coagulated milk to curd and, therefore, are important in dairy fermented products. Amylase, proteinase, and lipase are enzymes that play an important role in degrading milk into monomeric molecules such as oligosaccharides, amino acids, and fatty acids, which are the main molecules responsible for flavors in cheese. In the current study, we determined the amylase, proteinase, and lipase activities of Lactococcus chungangensis CAU 28(T), a bacterial strain of nondairy origin, and compared them with those of the reference strain, Lactococcus lactis ssp. lactis KCTC 3769(T), which is commonly used in the dairy industry. Lactococcus chungangensis CAU 28(T) and L. lactis ssp. lactis KCTC 3769(T) were both found to have amylase, proteinase, and lipase activities in broth culture, cream cheese, and yogurt. Notably, the proteinase and lipase activities of L. chungangensis CAU 28(T) were higher than those of L. lactis ssp. lactis KCTC 3769(T), with proteinase activity of 10.50 U/mL in tryptic soy broth and 8.64 U/mL in cream cheese, and lipase activity of 100 U/mL of tryptic soy broth, and 100 U/mL of cream cheese. In contrast, the amylase activity was low, with 5.28 U/mL in tryptic soy broth and 8.86 U/mL in cream cheese. These enzyme activities in L. chungangensis CAU 28(T) suggest that this strain has potential to be used for manufacturing dairy fermented products, even though the strain is of nondairy origin. PMID:27108177

  1. Nuclear magnetic resonance studies on the pKa values and interaction of ionizable groups in bromelain inhibitor VI from pineapple stem.

    Science.gov (United States)

    Hatano, Ken-ichi; Kojima, Masaki; Tanokura, Masaru; Takahashi, Kenji

    2003-01-01

    Bromelain inhibitor VI (BI-VI), a cysteine proteinase inhibitor from pineapple stem, is a unique double-chain molecule composed of two distinct domains A and B. In order to clarify the molecular mechanism of the proteinase-inhibitor interaction, we investigated the electrostatic properties of this inhibitor. The inhibitory activity toward bromelain was revealed to be maximal at pH 3-4 and the gross conformation to be stable over a wide range of pH. Based on these results, pH titration experiments were performed on the proton resonances of BI-VI in the pH range of 1.5-9.9, and pKa values (pKexp) were determined for all carboxyl groups and alpha-amino groups. The pKexp were also compared with theoretical values calculated from the NMR-derived structures of BI-VI. The electrostatic surface potential map constructed using the pKexp values revealed that BI-VI possesses continuous negatively charged and scattered positively charged regions on the molecular surface and both regions appear to serve for docking properly with a basic target enzyme. Furthermore, it was suggested that the ionic interaction of the inhibitor with the target enzyme is primarily important for the inhibition, which seems to involve some carboxyl groups in the inhibitor and a thiol group in the proteinase.

  2. Urinary trypsin inhibitor - an experimental and clinical study

    Energy Technology Data Exchange (ETDEWEB)

    Berling, B.M.

    1991-12-31

    The urinary trypsin inhibitor (UTI) is an acid stable proteinase inhibitor present in blood and urine. It was purified from urine using affinity chromatography, ion exchange chromatography and gel filtration. Two forms of UTI were present in urine, A and B. A radioimmunoassay for measurement of UTI in urine and plasma was performed. The normal level of UTI in plasma and serum was about 2 mg/l. The normal excretion in urine was about 8 mg per 24 hours. The plasma and urine levels of UTI were studied in patients with acute pancreatitis and in patients undergoing cholecystectomy. Uremic patients had a marked increase of UTI in plasma compatible with decreased glomerular filtration. In samples from healthy persons as well as from patients only inhibitor A was found. Inhibitor B has recently been renamed bikunin because of its two Kunitz-type inhibiting domains. Inhibitor A might be called tetrakunin. Radioactively labeled UTI (inhibitor A) was injected intravenously in three male volunteers. The plasma half-life of {sup 125}I UTI was 2 hours. Free biologically active inhibitor was found in the urine during the first four hours after injection. The organ distribution of intravenously injected {sup 125}I UTI was studied in rats. Fifteen minutes after injection the major part of the radioactivity was found in the kidneys, suggesting that the kidneys are the primary site of UTI metabolism. Using immunohistochemical techniques UTI was found in the proximal tubules of the normal human kidney further indicating the tubular reabsorption and methabolisms of UTI.

  3. Urinary trypsin inhibitor - an experimental and clinical study

    International Nuclear Information System (INIS)

    The urinary trypsin inhibitor (UTI) is an acid stable proteinase inhibitor present in blood and urine. It was purified from urine using affinity chromatography, ion exchange chromatography and gel filtration. Two forms of UTI were present in urine, A and B. A radioimmunoassay for measurement of UTI in urine and plasma was performed. The normal level of UTI in plasma and serum was about 2 mg/l. The normal excretion in urine was about 8 mg per 24 hours. The plasma and urine levels of UTI were studied in patients with acute pancreatitis and in patients undergoing cholecystectomy. Uremic patients had a marked increase of UTI in plasma compatible with decreased glomerular filtration. In samples from healthy persons as well as from patients only inhibitor A was found. Inhibitor B has recently been renamed bikunin because of its two Kunitz-type inhibiting domains. Inhibitor A might be called tetrakunin. Radioactively labeled UTI (inhibitor A) was injected intravenously in three male volunteers. The plasma half-life of 125I UTI was 2 hours. Free biologically active inhibitor was found in the urine during the first four hours after injection. The organ distribution of intravenously injected 125I UTI was studied in rats. Fifteen minutes after injection the major part of the radioactivity was found in the kidneys, suggesting that the kidneys are the primary site of UTI metabolism. Using immunohistochemical techniques UTI was found in the proximal tubules of the normal human kidney further indicating the tubular reabsorption and methabolisms of UTI

  4. TISSUE INHIBITOR OF METALLOPROTEINASE 1, MATRIX METALLOPROTEINASE 9, ALPHA-1 ANTITRYPSIN, METALLOTHIONEIN AND UROKINASE TYPE PLASMINOGEN ACTIVATOR RECEPTOR IN SKIN BIOPSIES FROM PATIENTS AFFECTED BY AUTOIMMUNE BLISTERING DISEASES

    Directory of Open Access Journals (Sweden)

    Ana Maria Abreu Velez

    2013-07-01

    Full Text Available Introduction: Proteinases and proteinase inhibitors have been described to play a role in autoimmune skin blistering diseases. We studied skin lesional biopsies from patients affected by several autoimmune skin blistering diseases for proteinases and proteinase inhibitors. Methods: We utilized immunohistochemistry to evaluate biopsies for alpha-1-antitrypsin, human matrix metalloproteinase 9 (MMP9, human tissue inhibitor of metalloproteinases 1 (TIMP-1, metallothionein and urokinase type plasminogen activator receptor (uPAR. We tested 30 patients affected by endemic pemphigus, 30 controls from the endemic area, and 15 normal controls. We also tested 30 biopsies from patients with bullous pemphigoid (BP, 20 with pemphigus vulgaris (PV, 8 with pemphigus foliaceus, and 14 with dermatitis herpetiformis (DH. Results: Contrary to findings in the current literature, most autoimmune skin blistering disease biopsies were negative for uPAR and MMP9. Only some chronic patients with El Bagre-EPF were positive to MMP9 in the dermis, in proximity to telocytes. TIMP-1 and metallothionein were positive in half of the biopsies from BP patients at the basement membrane of the skin, within several skin appendices, in areas of dermal blood vessel inflammation and within dermal mesenchymal-epithelial cell junctions.

  5. Anesthetic activity of the essential oil of Ocimum americanum in Rhamdia quelen (Quoy & Gaimard, 1824 and its effects on stress parameters

    Directory of Open Access Journals (Sweden)

    Lenise de Lima Silva

    2015-12-01

    Full Text Available ABSTRACT The aim of this study was to evaluate the anesthetic activity of the essential oil (EO of Ocimum americanum L. in silver catfish (Rhamdia quelen . In the first experiment, the depressor effects and chemical composition of the leaf EO (LEO and inflorescence EO (IEO were compared. Juveniles (n = 10 were placed in aquaria containing different concentrations of EO (25 - 500 mg L-1 to determine the point at which anesthesia was induced and the length of the recovery period. In the following experiment, the effects of 300 and 500 mg L-1 LEO exposure on stress parameters (plasma cortisol, glucose and sodium levels after air exposure for 1 min were assayed. Fish (n = 10 per sampling time were sampled immediately or transferred to anesthetic-free aquaria until sampling (15, 30, 60 or 240 min. LEO was composed mainly of β-linalool and 1,8-cineole in similar proportions, whereas IEO showed β-linalool as major compound. Anesthesia was obtained in silver catfish with 200-500 mg L-1 between 4-8 min for LEO and 6-16 min for IEO. Lower EO concentrations did not reach anesthetic stage up to 30 min. LEO used as anesthetic prevented the cortisol increase and sodium loss induced by aerial exposure. Glucose levels were raised in catfish exposed to LEO compared to basal group (not air exposed in almost all observation times. EO of O. americanum obtained from leaves was considered suitable to anesthetic procedures due to its fast induction and handling-induced stress prevention.

  6. Study on purification and characterization of a serine proteinase from the skeletal muscle of blue scad(Decapterus maruadsi)%蓝圆鲹肌肉中丝氨酸蛋白酶的分离纯化及性质研究

    Institute of Scientific and Technical Information of China (English)

    王梦想; 钟婵; 蔡秋凤; 刘光明; 苏文金; 曹敏杰

    2012-01-01

    鱼类死后肌肉容易发生软化现象。研究表明,这与肌肉中的丝氨酸蛋白酶有着密切的关系。本研究通过硫酸铵盐析、DEAE-Sephacel、Q-Sepharose及Capto Q等柱层析相结合的方法,从蓝圆鲹肌肉中纯化得到一种具有分解明胶能力的丝氨酸蛋白酶,SDS-PAGE结果显示其分子量约为60ku,该酶最适温度及最适pH分别为40℃和9.0。丝氨酸蛋白酶抑制剂Pefabloc SC、Benzamidine、MBTI、PMSF和LBTI均能明显的抑制该酶的活性,而其他蛋白酶抑制剂对其活性没有明显的影响。底物特异性表明其能有效的降解丝氨酸蛋白酶荧光底物Boc-Leu-Lys-Arg-MCA,但进一步研究发现,该酶对I型胶原蛋白及明胶有明显的分解能力,同时对肌球蛋白重链也有一定的分解作用,说明该酶可能参与鱼肉保鲜中肌肉软化的过程。%Some researches revealed that the tenderization of fish muscle during postmortem was caused by the endogenous proteinase especially serine proteinase.A collagenolytic serine proteinase was purified from blue scad skeletal muscle to homogeneity by ammonium sulfate fractionation and chromatographies including DEAE-Sephacel,Q-Sepharose and Capto Q.The molecular weight of the enzyme was 60ku as detected by SDS-PAGE.The optimal pH and temperature of the purified enzyme were 9.0 and 40℃,respectively.The enzyme activity was inhibited by serine proteinase inhibitors such as Pefabloc SC,Benzamidine,MBTI,PMSF and LBTI.However,other proteinase inhibitors had no effect on serine proteinase.Substrate specificity experiment demonstrated that the enzyme showed high specificity towards Boc-Leu-Lys-Arg-MCA.Furthermore,the enzyme effectively hydrolyzed gelatin,native type-I collagen and myofibrillar proteins such as myosin heavy chain(MHC),these datum suggested that this enzyme might play an important role during postmortem tenderization of fish muscle.

  7. Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1

    OpenAIRE

    Sakaguchi, Masayoshi; Osaku, Kanae; Maejima, Susumu; Ohno, Nao; Sugahara, Yasusato; Oyama, Fumitaka; Kawakita, Masao

    2014-01-01

    The proteinase K subfamily enzymes, thermophilic Aqualysin I (AQN) from Thermus aquaticus YT-1 and psychrophilic serine protease (VPR) from Vibrio sp. PA-44, have six and seven salt bridges, respectively. To understand the possible significance of salt bridges in the thermal stability of AQN, we prepared mutant proteins in which amino acid residues participating in salt bridges common to proteinase K subfamily members and intrinsic to AQN were replaced to disrupt the bridges one at a time. Di...

  8. Effect of insulin on the mRNA expression of procollagen N-proteinases in chondrosarcoma OUMS-27 cells

    OpenAIRE

    Akyol, Sumeyya; Cömertoğlu, İsmail; FIRAT, RIDVAN; Çakmak, Özlem; YUKSELTEN, YUNUS; ERDEN, GÖNÜL; Ugurcu, Veli; Demircan,Kadir

    2015-01-01

    Chondrosarcoma is one of the most common bone tumors, and at present, there is no non-invasive treatment option for this cancer. The chondrosarcoma OUMS-27 cell line produces proteoglycan and type II, IX, and XI collagens, which constitutes cartilage tissue. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) proteases are a group of secreted proteases, which include the procollagen N-proteinases ADAMTS-2, -3 and -14. These procollagen N-proteinases perform a role in the p...

  9. Effect of added proteinases and level of starter culture on the formation of biogenic amines in raw milk Manchego cheese.

    Science.gov (United States)

    Fernández-García, E; Tomillo, J; Núñez, M

    1999-11-15

    The influence of two proteinases (Bacillus subtilis neutral proteinase and Micrococcus sp. cysteine proteinase) and two starter culture levels (0.1% and 1%) on biogenic amine formation has been studied in raw ewes' milk Manchego cheese. Amino acid decarboxylating micro-organisms were determined on tyrosine enriched selective media. Biogenic amines were analysed by capillary electrophoresis in citrate buffer at pH 3.6. Addition of proteinases and level of starter culture did not influence the population of micro-organisms with amino acid decarboxylating activity, which represented on average 1% of the bacterial population in 30-day-old cheeses. Tyramine and histamine were detected in all batches of cheese from day 30. Concentrations of tyramine and histamine were higher in cheeses made from milk with neutral proteinase (up to 356 and 284 mg kg(-1), respectively, after 90 days) than in cheeses made from milk with cysteine proteinase (up to 269 and 189 mg kg(-1), respectively) or with no proteinase added (up to 305 and 226 mg kg(-1), respectively). Formation of tyramine and histamine was also favoured in cheeses made with 1% starter culture with respect to cheeses made with only 0.1% starter culture, probably due to the higher pH values of the former cheeses. After 90 days of ripening, concentrations of 10-20 mg kg(-1) phenylethylamine were observed in 9 of the 12 batches, and levels < 10 mg kg(-1) tryptamine were only detected in 3 batches, with no significant relationship between the concentration of these amines and proteinase addition or level of starter culture. PMID:10733250

  10. [Effect of Azospirillum lectins on the Activity of Proteolytic Enzymes and Their Inhibitors in Wheat Seedling Roots].

    Science.gov (United States)

    Alen'kina, S A; Nikitina, V E

    2015-01-01

    The lectins of associative nitrogen-fixing strains Azospirillum brasilense Sp7 and Sp245 were shown to exerte a multidirectional effect on the activity of acidic (pH 3.5), neutral (6.8), and alkaline (pH 7.8) proteinases. The lectin of the epiphytic A. brasilense Sp7 decreased proteolytic activity at all pH values, whereas the lectin of the endophytic A. brasilense Sp245 activated neutral and alkaline proteinases, while not affecting the alkaline ones. Experiments with protease inhibitors made it possible to conclude that the lectins of the studied A. brasilense strains alter the ratio between the activities of different protease types in germinating seeds. The activity of trypsin inhibitors in wheat seedling roots was found to increase in the presence of the lectins. Our results indicate a broader spectrum of effects of azospirilla lectins on the host plant organism.

  11. The amino acid sequence of a 20 kDa bifunctional subtilisin/alpha-amylase inhibitor from bran [correction of brain] of rice (Oryza sativa L.) seeds.

    Science.gov (United States)

    Ohtsubo, K; Richardson, M

    1992-08-31

    A 20 kDa bifunctional inhibitor of the microbial proteinase, subtilisin, and the alpha-amylase from the larvae of the red flour beetle (Tribolium castaneum) was purified from bran of rice seeds by saline extraction, precipitation with ammonium sulphate, ion-exchange chromatography on DEAE-Cellulose and Toyopearl CM-650, and preparative HPLC on Vydac C18. The complete primary structure was determined by automatic degradation of the intact, reduced and S-alkylated protein, and by manual DABITC/PITC micro-sequencing of peptides obtained from the protein following separate enzymic digestions with trypsin, pepsin, chymotrypsin, elastase and the protease from S. aureus V8. The protein sequence, which contained 176 residues, showed strong homology with similar bifunctional inhibitors previously isolated from wheat and barley which are related to the Kunitz family of proteinase inhibitors from legume seeds. PMID:1511747

  12. Hydrolytic activity of Virgibacillus sp. SK37, a starter culture of fish sauce fermentation, and its cell-bound proteinases.

    Science.gov (United States)

    Sinsuwan, Sornchai; Rodtong, Sureelak; Yongsawatdigul, Jirawat

    2012-08-01

    Fish sauce production relies on a natural fermentation process requiring 12-18 months for process completion. Virgibacillus sp. SK37 has been shown to be a potential strain for fish sauce acceleration. However, hydrolytic activity of proteinases bound at cell surface of this strain has not been well elucidated. Addition of 0.2 % CaCl(2) (w/w) in conjunction with starter cultures of Virgibacillus sp. SK 37 increased protein hydrolysis as measured by α-amino group content throughout fermentation (P Virgibacillus sp. SK 37 were extracted into a free form by incubating the washed cells in Ca(2+)-free buffer at 37 °C for 2 h. Cell-bound proteinases revealed molecular mass of 19, 20, 22, 32, 34, and 44 kDa based on a synthetic peptide zymogram. The proteinases showed subtilisin-like serine characteristics with the highest activity at 50 °C and pH 8 and 11. Activity of the extracted proteinases increased ~4 times at ≥100 mM CaCl(2). In addition, CaCl(2) enhanced thermal stability of the extracted proteinases. Enzymes showed proteolytic activity in either the absence or presence of 10 and 25 % NaCl toward fish muscle, soy protein isolate, and casein substrates. Cell-bound proteinases were likely to play an important role in protein hydrolysis during fish sauce fermentation. PMID:22806191

  13. The procollagen N-proteinases ADAMTS2, 3 and 14 in pathophysiology.

    Science.gov (United States)

    Bekhouche, Mourad; Colige, Alain

    2015-01-01

    Collagen fibers are the main components of most of the extracellular matrices where they provide a structural support to cells, tissues and organs. Fibril-forming procollagens are synthetized as individual chains that associate to form homo- or hetero-trimers. They are characterized by the presence of a central triple helical domain flanked by amino and carboxy propeptides. Although there are some exceptions, these two propeptides have to be proteolytically removed to allow the almost spontaneous assembly of the trimers into collagen fibrils and fibers. While the carboxy-propeptide is mainly cleaved by proteinases from the tolloid family, the amino-propeptide is usually processed by procollagen N-proteinases: ADAMTS2, 3 and 14. This review summarizes the current knowledge concerning this subfamily of ADAMTS enzymes and discusses their potential involvement in physiopathological processes that are not directly linked to fibrillar procollagen processing. PMID:25863161

  14. Modified TB rapid test by proteinase K for rapid diagnosis of pleural tuberculosis.

    Science.gov (United States)

    Yari, Shamsi; Hadizadeh Tasbiti, Alireza; Ghanei, Mostafa; Shokrgozar, Mohammad Ali; Fateh, Abolfazl; Yari, Fatemeh; Bahrmand, Ahmadreza

    2016-03-01

    The diagnosis of pleural tuberculosis continues to be a challenge due to the low sensitivity of traditional diagnostic methods. Better and more rapid tests are needed for diagnosis of pleural TB. In this study, pleural fluids were tested with rapid test to determine Mycobacterium tuberculosis (MTB antigen). Affinity chromatography was used to purify specific polyclonal antibodies against MTB antigen. Pleural samples after decontamination were treated with proteinase K. Rapid test for pleural fluids was prepared by specific antibody. Rapid test was performed on 85 pleural fluid patients. The patients had a mean age of 46.55 ± 15.96 years and 38 were men. The performance of rapid test, using proteinase K, was found to be the most impressive: sensitivity 93%, specificity 94%, PPV 90%, and NPV 96% compared with adenosine deaminase test (ADA), PCR, smear, and culture. The present study did demonstrate that modified TB rapid test can substantially improve the diagnosis of extrapulmonary TB.

  15. Enhanced response of a proteinase K-based conductometric biosensor using nanoparticles.

    Science.gov (United States)

    Nouira, Wided; Maaref, Abderrazak; Elaissari, Hamid; Vocanson, Francis; Siadat, Maryam; Jaffrezic-Renault, Nicole

    2014-07-23

    Proteinases are involved in a multitude of important physiological processes, such as protein metabolism. For this reason, a conductometric enzyme biosensor based on proteinase K was developed using two types of nanoparticles (gold and magnetic). The enzyme was directly adsorbed on negatively charged nanoparticles and then deposited and cross-linked on a planar interdigitated electrode (IDE). The biosensor was characterized with bovine serum albumin (BSA) as a standard protein. Higher sensitivity was obtained using gold nanoparticles. The linear range for BSA determination was then from 0.5 to 10 mg/L with a maximum response of 154 µs. These results are greater than that found without any nanoparticles (maximum response of 10 µs). The limit of detection (LOD) was 0.3 mg/L. An inter-sensor reproducibility of 3.5% was obtained.

  16. Seed-specific aspartic proteinase FeAP12 from buckwheat (Fagopyrum esculentum Moench

    Directory of Open Access Journals (Sweden)

    Timotijević Gordana S.

    2010-01-01

    Full Text Available Aspartic proteinase gene (FeAP12 has been isolated from the cDNA library of developing buckwheat seeds. Analysis of its deduced amino acid sequence showed that it resembled the structure and shared high homology with typical plant aspartic proteinases (AP characterized by the presence of a plant-specific insert (PSI, unique among APs. It was shown that FeAP12 mRNA was not present in the leaves, roots, steam and flowers, but was seed-specifically expressed. Moreover, the highest levels of FeAP12 expression were observed in the early stages of seed development, therefore suggesting its potential role in nucellar degradation.

  17. Human seminal proteinase and prostate-specific antigen are the same protein

    Indian Academy of Sciences (India)

    Abdul Waheed; Md Imtaiyaz Hassan; Robert L Van Etten; Faizan Ahmad

    2008-06-01

    Human seminal proteinase and prostate-specific antigen (PSA) were each isolated from human seminal fluid and compared. Both are glycoproteins of 32–34 kDa with protease activities. Based on some physicochemical, enzymatic and immunological properties, it is concluded that these proteins are in fact identical. The protein exhibits properties similar to kallikrein-like serine protease, trypsin, chymotrypsin and thiol acid protease. Tests of the activity of the enzyme against some potential natural and synthetic substrates showed that bovine serum albumin was more readily hydrolysed than casein. The results of this study should be useful in purifying and assaying this protein. Based on published studies and the present results, the broad proteolytic specificity of human seminal proteinase suggests a role for this protein in several physiological functions.

  18. Enhanced Response of a Proteinase K-Based Conductometric Biosensor Using Nanoparticles

    Directory of Open Access Journals (Sweden)

    Wided Nouira

    2014-07-01

    Full Text Available Proteinases are involved in a multitude of important physiological processes, such as protein metabolism. For this reason, a conductometric enzyme biosensor based on proteinase K was developed using two types of nanoparticles (gold and magnetic. The enzyme was directly adsorbed on negatively charged nanoparticles and then deposited and cross-linked on a planar interdigitated electrode (IDE. The biosensor was characterized with bovine serum albumin (BSA as a standard protein. Higher sensitivity was obtained using gold nanoparticles. The linear range for BSA determination was then from 0.5 to 10 mg/L with a maximum response of 154 µs. These results are greater than that found without any nanoparticles (maximum response of 10 µs. The limit of detection (LOD was 0.3 mg/L. An inter-sensor reproducibility of 3.5% was obtained.

  19. The nematicidal effect of cysteine proteinases on the root knot nematode Meloidogne incognita

    OpenAIRE

    Gorny, Samuel Victor

    2013-01-01

    Despite current control measures, plant parasitic nematodes are estimated to be responsible for > $100 billion of damage to worldwide crop production per annum. Current nematicides are highly toxic, and due to health and environmental safety concerns, many are being withdrawn from the market under directive 914/414/EEC. Alternative control strategies are urgently required. The cysteine proteinases papain, actinidain and recombinant endoproteinase B isoform 2 (R.EP-B2) have been demonstrate...

  20. Cloning and expression of an active aspartic proteinase from Mucor circinelloides in Pichia pastoris

    OpenAIRE

    Gama Salgado, Jose Antonio; Kangwa, Martin; Fernandez-Lahore, Marcelo

    2013-01-01

    Background Extracellular aspartic proteinase (MCAP) produced by Mucor circinelloides in solid state fermentations has been shown to possess milk clotting activity and represents a potential replacement for bovine chymosin in cheese manufacturing. Despite its prospects in the dairy industry, the molecular characteristics of this enzyme remain unknown. This work focuses on MCAP cloning and optimization of heterologous expression in Pichia pastoris, and characterization of the enzyme. Results Th...

  1. Proteinase 3 contributes to transendothelial migration of NB1-positive neutrophils

    OpenAIRE

    Kuckleburg, Christopher J.; Tilkens, Sarah M.; Santoso, Sentot; Newman, Peter J.

    2012-01-01

    Neutrophil transmigration requires the localization of neutrophils to endothelial cell junctions, where receptor-ligand interactions and the action of serine proteases promote leukocyte diapedesis. NB1 (CD177) is a neutrophil-expressed surface molecule that has been reported to bind proteinase 3 (PR3), a serine protease released from activated neutrophils. PR3 has demonstrated proteolytic activity on a number of substrates, including extracellular matrix proteins, although its role in neutrop...

  2. Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis.

    Science.gov (United States)

    Kumar, Rohitashw; Saraswat, Darpan; Tati, Swetha; Edgerton, Mira

    2015-07-01

    Candida albicans, a commensal fungus of the oral microbiome, causes oral candidiasis in humans with localized or systemic immune deficiencies. Secreted aspartic proteinases (Saps) are a family of 10 related proteases and are virulence factors due to their proteolytic activity, as well as their roles in adherence and colonization of host tissues. We found that mice infected sublingually with C. albicans cells overexpressing Sap6 (SAP6 OE and a Δsap8 strain) had thicker fungal plaques and more severe oral infection, while infection with the Δsap6 strain was attenuated. These hypervirulent strains had highly aggregative colony structure in vitro and higher secreted proteinase activity; however, the levels of proteinase activity of C. albicans Saps did not uniformly match their abilities to damage cultured oral epithelial cells (SCC-15 cells). Hyphal induction in cells overexpressing Sap6 (SAP6 OE and Δsap8 cells) resulted in formation of large cell-cell aggregates. These aggregates could be produced in germinated wild-type cells by addition of native or heat-inactivated Sap6. Sap6 bound only to germinated cells and increased C. albicans adhesion to oral epithelial cells. The adhesion properties of Sap6 were lost upon deletion of its integrin-binding motif (RGD) and could be inhibited by addition of RGD peptide or anti-integrin antibodies. Thus, Sap6 (but not Sap5) has an alternative novel function in cell-cell aggregation, independent of its proteinase activity, to promote infection and virulence in oral candidiasis.

  3. Proteinases of the mammary gland: developmental regulation in vivo and vectorial secretion in culture

    OpenAIRE

    Talhouk, Rabih S.; CHIN, JENNIE R.; UNEMORI, ELAINE N.; Werb, Zena; Bissell, Mina J.

    1991-01-01

    The extracellular matrix (ECM) is an important regulator of mammary epithelial cell function both in vivo and in culture. Substantial remodeling of ECM accompanies the structural changes in the mammary gland during gestation, lactation and involution. However, little is known about the nature of the enzymes and the processes involved. We have characterized and studied the regulation of cell-associated and secreted mammary gland proteinases active at neutral pH that may be involved in degradat...

  4. Isolation and characterization of two forms of an acidic bromelain stem proteinase.

    Science.gov (United States)

    Harrach, T; Eckert, K; Maurer, H R; Machleidt, I; Machleidt, W; Nuck, R

    1998-05-01

    Two forms of an acidic bromelain proteinase isolated from crude bromelain, an extract from pineapple stem, were found by a two-step FPLC purification procedure. The basic main components were removed by cation exchange chromatography and the breakthrough fraction was further resolved by anion exchange chromatography into 15 protein fractions, only two of which, called SBA/a and SBA/b, were proteolytically active. These components were characterized by electrospray mass spectroscopy (ESMS), isoelectric focusing, N-terminal amino acid sequence analysis, monosaccharide analysis, and enzymatic parameters. The molecular masses of SBA/a and SBA/b were determined by ESMS to be 23,550 and 23,560, respectively. The isoelectric points (pI) of the two bands of SBA/a were 4.8 and 4.9; SBA/b focused as a single band at pI = 4.8. Partial N-terminal amino acid sequences (11 residues) were identical to SBA/a and SBA/b and identical with those of stem bromelain, the basic main proteinase of the pineapple stem, and fruit bromelain, the acidic main proteinase of the pineapple fruit. Both components are highly glycosylated; hydrolysis of SBA/a yielded about twofold more monosaccharide per protein than SBA/b. The comparison of the catalytic properties of SBA/a with those of SBA/b revealed no relevant differences in the hydrolysis of three peptidyl-NH-Mec substrates and in the inhibition profiles using chicken cystatin and E-64, indicating that these components can be considered as two forms of a single enzyme. Both forms are scarcely inhibited by chicken cystatin and slowly inactivated by E-64, hence are nontypical cysteine proteinases of the papain superfamily.

  5. Effect of acute ozone exposure on the proteinase-antiproteinase balance in the rat lung

    International Nuclear Information System (INIS)

    Lung disease may result from a persisting proteinase excess or a depletion of antiproteinase in pulmonary parenchyma. We investigated the in vivo effect of a 48-hr exposure to ozone at 0.5, 1.0, or 1.5 ppm on proteinase and antiproteinase activity of rat lungs. Elastase inhibitory capacities of serum, lung tissue, and airway washings were measured as indicators of antielastase activity. Trypsin inhibitory capacity was measured using an esterolytic procedure. Proteinase was measured as radioactive release from a 14C-globin substrate. The 48-hr exposures to O3 at levels up to 1 ppm produced concentration-dependent decreases of 35-80% of antiproteinase activities in serum and in lung tissue. However, exposure to 1.5 ppm O3 resulted in no decrease in antiproteinase activities. Acid proteinase activities (pH 4.2) were increased 65-120% by exposure to 1 or 1.5 ppm O3, which correlated with inflammatory cells noted histologically. At 1.5 ppm O3, pulmonary edema and hemorrhage were noted in histologic sections. These changes led to a flooding of the alveoli with up to 40 times normal protein levels and a greater than fivefold increase in airway antiproteinase. These data suggest that serum and soluble lung tissue antiproteinase activity decreased upon exposure to low levels of ozone. However, if O3 exposure is high enough to produce pulmonary hemorrhage, antiproteinase may increase following serum exudation. These changes may be important in the development of ozone-induced lung diseases, especially emphysema

  6. Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways

    OpenAIRE

    Lund, Leif R.; Rømer, John; Thomasset, Nicole; Solberg, Helene; Pyke, Charles; Bissell, Mina J.; Danø, Keld; Werb, Zena

    1996-01-01

    Postlactational involution of the mammary gland is characterized by two distinct physiological events: apoptosis of the secretory, epithelial cells undergoing programmed cell death, and proteolytic degradation of the mammary gland basement membrane. We examined the spatial and temporal patterns of apoptotic cells in relation to those of proteinases during involution of the BALB/c mouse mammary gland. Apoptosis was almost absent during lactation but became evident at day 2 of involution, when ...

  7. Comparison of self-processing of foot-and-mouth disease virus leader proteinase and porcine reproductive and respiratory syndrome virus leader proteinase nsp1α.

    Science.gov (United States)

    Steinberger, Jutta; Kontaxis, Georg; Rancan, Chiara; Skern, Tim

    2013-09-01

    The foot-and-mouth disease virus leader proteinase (Lb(pro)) cleaves itself off the nascent viral polyprotein. NMR studies on the monomeric variant Lb(pro) L200F provide structural evidence for intramolecular self-processing. (15)N-HSQC measurements of Lb(pro) L200F showed specifically shifted backbone signals in the active and substrate binding sites compared to the monomeric variant sLb(pro), lacking six C-terminal residues. This indicates transient intramolecular interactions between the C-terminal extension (CTE) of one molecule and its own active site. Contrastingly, the porcine reproductive and respiratory syndrome virus (PRRSV) leader proteinase nsp1α, with a papain-like fold like Lb(pro), stably binds its own CTE. Parts of the β-sheet domains but none of the α-helical domains of Lb(pro) and nsp1α superimpose; consequently, the α-helical domain of nsp1α is oriented differently relative to its β-sheet domain. This provides a large interaction surface for the CTE with the globular domain, stabilising the intramolecular complex. Consequently, self-processing inactivates nsp1α but not Lb(pro).

  8. Comparison of self-processing of foot-and-mouth disease virus leader proteinase and porcine reproductive and respiratory syndrome virus leader proteinase nsp1α

    International Nuclear Information System (INIS)

    The foot-and-mouth disease virus leader proteinase (Lbpro) cleaves itself off the nascent viral polyprotein. NMR studies on the monomeric variant Lbpro L200F provide structural evidence for intramolecular self-processing. 15N-HSQC measurements of Lbpro L200F showed specifically shifted backbone signals in the active and substrate binding sites compared to the monomeric variant sLbpro, lacking six C-terminal residues. This indicates transient intramolecular interactions between the C-terminal extension (CTE) of one molecule and its own active site. Contrastingly, the porcine reproductive and respiratory syndrome virus (PRRSV) leader proteinase nsp1α, with a papain-like fold like Lbpro, stably binds its own CTE. Parts of the β-sheet domains but none of the α-helical domains of Lbpro and nsp1α superimpose; consequently, the α-helical domain of nsp1α is oriented differently relative to its β-sheet domain. This provides a large interaction surface for the CTE with the globular domain, stabilising the intramolecular complex. Consequently, self-processing inactivates nsp1α but not Lbpro. - Highlights: • We examine self-processing of the leader protease of foot-and-mouth disease virus. • NMR analysis strongly supports intramolecular self-processing. • Self-processing is a dynamic process with no stable complex. • Structural comparison with nsp1α of PRRSV which forms stable intramolecular complex. • Subdomain orientation explains differences in stability of intramolecular complexes

  9. Characterization of certain proteinase isoenzymes produced by benign and virulent strains of Bacteroides nodosus.

    Science.gov (United States)

    Green, R S

    1985-11-01

    Three proteinase isoenzymes from one benign strain of Bacteroides nodosus and five proteinase isoenzymes from each of two virulent strains of B. nodosus were purified by horizontal slab polyacrylamide gel electrophoresis. The purified isoenzymes hydrolysed casein, collagen I, collagen III, elastin, alpha-elastin, fibrinogen, gelatin, haemoglobin and alpha-keratin. The pH optima of all the isoenzymes lay between 7.25 and 9.5, the range of 8.75-9.25 being common to all. The isoenzymes were inhibited by phenylmethylsulphonyl fluoride, diphenylcarbamyl chloride, L-(1-tosylamide-2-phenyl)ethyl chloromethyl ketone, EGTA and EDTA, indicating that they were chymotrypsin-like serine proteinases that require a metal ion for stability or activity. EDTA inhibition was not reversed by addition of Ca2+ or Mg2+. Some isoenzymes were activated by Mg2+, Ca2+, Cr3+ and Se4+ and all were inhibited by Fe2+, Co2+, Cu2+, Zn2+, Cd2+ and Hg2+. Isoenzymes from benign strains had a lower temperature stability, losing all activity at 55 degrees C, whereas those from virulent strains lost all activity at 60 degrees C.

  10. [Characteristics of proteinase digestive function in invertebrates--inhabitants of cold seas].

    Science.gov (United States)

    Mukhin, V A; Smirnova, E B; Novikov, V Iu

    2007-01-01

    Digestive proteinases of various taxa of invertebrates of the Northern seas have been studied: crustaceans Paralithodes camtchaticus, Pandalus borealis; molluscs Chlamys islandicus, Buccinum undatum, Serripes groenlandicus, and echinoderms Strongylocentrotus droebachiensis, Cucumaria frondosa, Asterias rubens, and Grossaster papposus. The presence of two proteolytic activity peaks in the acid (pH 2.5-3.5) and low alkaline zones (pH 7.5-8.5) and a similar proteinase spectrum have been revealed in digestive organs of the studied animals. The proteolytic activity in digestive organs of the Barents Sea invertebrates exceeds significantly that of terrestrial homoiothermal animals, which seems to be an extensive compensation for poor differentiation of the digestive system and for low substrate specificity of the enzymes as well as for cold conditions of the habitat. The principal qualitative difference between vertebrates and invertebrates consists in that the latter have no pepsin activity, but do have the cathepsin activity that is absent in vertebrate digestive organs. Contribution to the acid proteolysis is made by lysosomal cathepsins, rather than by pepsins. Activity in the alkaline and neutral pH zones is provided by serine proteinases. In digestive cavities of invertebrates, hydrolysis of proteins and mechanical processing of food occur only in the low alkaline zone, whereas acid proteolysis has intracellular lysosomal localization. PMID:18038635

  11. Cloning and characterization of an Eimeria acervulina sporozoite gene homologous to aspartyl proteinases.

    Science.gov (United States)

    Laurent, F; Bourdieu, C; Kaga, M; Chilmonczyk, S; Zgrzebski, G; Yvoré, P; Péry, P

    1993-12-01

    A lambda ZapII cDNA library was constructed using mRNA from Eimeria acervulina sporulated oocysts and screened with monoclonal antibodies raised against Eimeria tenella sporulated oocytes. Monoclonal antibody N3C8B12 identified a clone (6S2) potentially encoding an aspartyl proteinase since significant homology with cathepsin D, pepsin and renin proteinases was revealed by sequence comparisons. The 1500-bp cDNA fragment containing the coccidial gene was subcloned into pGEX-FA expression vector, leading to the production of an 80-kDa fusion protein (FA6S2) which was used to immunize rabbits. The anti-FA6S2 rabbit sera revealed a single 43-kDa protein present in Eimeria acervulina, Eimeria tenella, Eimeria maxima and Eimeria falciformis sporulated oocyst antigens. Indirect immunofluorescence and electron microscopy with mAb N3C8B12 localized the putative aspartyl proteinase in the refractile bodies of Eimeria tenella sporozoites.

  12. Rearch progress in the proteinase K%蛋白酶K的研究进展

    Institute of Scientific and Technical Information of China (English)

    吴彤; 王瑞明; 黄磊; 徐志南

    2013-01-01

    蛋白酶K是一种在生物科学研究和生物加工过程中具有多种应用的重要丝氨酸蛋白酶,并且在食品和饲料工业中有潜在的重要应用.本文对蛋白酶K的分子结构、催化特性、定向进化、重组表达和应用研究的最新进展进行了研究总结,并对蛋白酶K在未来的研究方向进行了展望.%Proteinase K is a kind of important serine protease which has a variety of application in biological science research and biological processing process and has an important potential application in the food and feed industry.In this paper,the molecular structure,catalytic properties of proteinase K,directed evolution,and the latest progress in the study of recombinant expression and application were summarized and the research direction of proteinase K in the future was prospected.

  13. Molecular cloning and characterization of cystatin, a cysteine protease inhibitor, from bufo melanostictus.

    Science.gov (United States)

    Liu, Wa; Ji, Senlin; Zhang, A-Mei; Han, Qinqin; Feng, Yue; Song, Yuzhu

    2013-01-01

    Cystatins are efficient inhibitors of papain-like cysteine proteinases, and they serve various important physiological functions. In this study, a novel cystatin, Cystatin-X, was cloned from a cDNA library of the skin of Bufo melanostictus. The single nonglycosylated polypeptide chain of Cystatin-X consisted of 102 amino acid residues, including seven cysteines. Evolutionary analysis indicated that Cystatin-X can be grouped with family 1 cystatins. It contains cystatin-conserved motifs known to interact with the active site of cysteine proteinases. Recombinant Cystatin-X expressed and purified from Escherichia coli exhibited obvious inhibitory activity against cathepsin B. rCystatin-X at a concentration of 8 µM inhibited nearly 80% of cathepsin B activity within 15 s, and about 90% of cathepsin B activity within 15 min. The Cystatin-X identified in this study can play an important role in host immunity and in the medical effect of B. melanostictus.

  14. Genetic analysis of regulatory mutants affecting synthesis of extracellular proteinases in the yeast Yarrowia lipolytica: identification of a RIM101/pacC homolog.

    OpenAIRE

    Lambert, M.; Blanchin-Roland, S; Le Louedec, F; Lepingle, A; Gaillardin, C.

    1997-01-01

    Depending on the pH of the growth medium, the yeast Yarrowia lipolytica secretes both an acidic proteinase and an alkaline proteinase, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins. Recessive mutations at four unlinked loci, named PAL1 to PAL4, were isolated which prevent alkaline proteinase derepression under conditions of carbon and nitrogen limitation at pH 6.8. These mutations markedly affect ma...

  15. Cloning and sequence analysis of serine proteinase of Gloydius ussuriensis venom gland

    International Nuclear Information System (INIS)

    Objective: To construct a cDNA library by using mRNA from Gloydius ussuriensis (G. Ussuriensis) venom gland, to clone and analyze serine proteinase gene from the cDNA library. Methods: Total RNA was isolated from venom gland of G. ussuriensis, mRNA was purified by using mRNA isolation Kit. The whole length cDNA was synthesized by means of smart cDNA synthesis strategy, and amplified by long distance PCR procedure, lately cDAN was cloned into vector pBluescrip-sk. The recombinant cDNA was transformed into E. coli DH5α. The cDNA of serine proteinase gene in the venom gland of G. ussuriensis was detected and amplified using the in situ hybridization. The cDNA fragment was inserted into pGEMT vector, cloned and its nucleotide sequence was determined. Results: The capacity of cDNA library of venom gland was above 2.3 x 106. Its open reading frame was composed of 702 nucleotides and coded a protein pre-zymogen of 234 amino acids. It contained 12 cysteine residues. The sequence analysis indicated that the deduced amino acid sequence of the cDNA fragment shared high identity with the thrombin-like enzyme genes of other snakes in the GenBank. the query sequence exhibited strong amino acid sequence homology of 85% to the serine proteas of T. gramineus, thrombin-like serine proteinase I of D. acutus and serine protease catroxase II of C. atrox respectively. Based on the amino acid sequences of other thrombin-like enzymes, the catalytic residues and disulfide bridges of this thrombin-like enzyme were deduced as follows: catalytic residues, His41, Asp86, Ser180; and six disulfide bridges Cys7-Cys139, Cys26-Cys42, Cys74-Cys232, Cys118-Cys186, Cys150-Cys165, Cys176-Cys201. Conclusion: The capacity of cDNA library of venom gland is above 2.3 x 106, overtop the level of 105 capicity. The constructed cDNA library of G. ussuriensis venom gland would be helpful platform to detect new target genes and further gene manipulate. The cloned serine proteinase gene exhibits strong amino

  16. Functional proteomic of Matrix Metallo-proteinases (MMP) dedicated to the detection of active forms of MMP in complex proteome

    International Nuclear Information System (INIS)

    The Matrix Metallo-proteinases (M.M.P.) represent a family of Zinc dependent extracellular proteinases able to cleave collectively all the proteins constituting the extracellular matrix. Currently, 23 human M.M.P. have been identified and are characterized by their sequence in amino-acids and their highly conserved 3 D structure. These enzymes are expressed constitutively during the tissue remodeling process. Their over-expression in various diseases tightly related to inflammatory processes (arthritis, emphysema, cancer) described M.M.P. as choice therapeutic targets. However, as the tissue remodeling implicates modification of cellular contacts, M.M.P. appear currently as proteins involved in signalling pathways. Recent works demonstrating that M.M.P. are able to cleave substrates, which are different than proteins constituting the extracellular matrix, reinforce this vision. In order to identify the individual role and the protein expression level of M.M.P. in pathological context, we developed a new technique of functional proteomics dedicated to the detection of active forms of M.M.P. in tumour samples. This technique relied on the development of a new photoaffinity probe, based on the structure of a potent phosphinic inhibitor of M.M.P., allowing targeting and isolating active forms of M.M.P. by photoaffinity labelling. Furthermore, as the new developed probe incorporated a radioactive element, photoaffinity labelling permitted to radiolabel the targeted proteins. This probe demonstrated in vitro its remarkable ability to covalently modify the h M.M.P.-12, with a singular cross-linking yield, determined at 42 %, displaying an extremely sensitive detection (2.5 fmoles of h M.M.P.-12). When added to complex proteome, the photoaffinity probe presents the same sensibility of detection for the h M.M.P.-12 (5 fmoles); importantly, in this case, h M.M.P.-12 represents only 0.001 % of the totality of the proteins present in the sample. Moreover, this technique allows

  17. Characterization of the acidic and basic limbs of a bell-shaped pH profile in the inhibitory activity of bromelain inhibitor VI.

    Science.gov (United States)

    Hatano, Ken-ichi; Sawano, Yoriko; Miyakawa, Takuya; Tanokura, Masaru

    2006-03-01

    Bromelain inhibitor VI (BI-VI) is a cysteine proteinase inhibitor from pineapple stem and a unique two-chain inhibitor composed of two distinct domains. BI-VI's inhibitory activity toward the target enzyme bromelain is maximal at pH 4 and shows a bell-shaped pH profile with pKa values of about 2.5 and 5.3. This pH profile is quite different from that of bromelain, which is optimally active around pH 7. In the present article, to characterize the acidic limb, we first expressed the recombinant inhibitors designed to lose two putative hydrogen bonds of Ser7(NH)-Asp28(beta-CO2H) and Lys38(NH)-Asp51(beta-CO2H) and confirmed the existence of the hydrogen bonds by two-dimensional nuclear magnetic resonance (NMR). Moreover, it was revealed that these hydrogen bonds are not the essential electrostatic factor and some ionizable groups would be responsible for the acidic limb in the pH-inhibition profile. On the other hand, to characterize the basic limb, we examined the pH-dependent inhibition using the cysteine proteinase papain, some of whose properties differ from those of bromelain, and compared the data with the corresponding data for bromelain. The result suggests that the basic limb would be affected by some electrostatic factors, probably some carboxyl groups in the target proteinase.

  18. Changes in blood levels of proteinase inhibitors, pregnancy zone protein, steroid carriers and complement factors induced by oral contraceptives

    DEFF Research Database (Denmark)

    Nielsen, Claus Henrik; Poulsen, Henning Kvist; Teisner, Børge;

    1993-01-01

    Three low-dose oral contraceptives Trinordiol, Gynatrol, and Marvelon, containing ethinylestradiol (EE) in combination with triphasic levonorgestrel (LNg), monophasic levonorgestrel, and monophasic desogestrel (DGS), respectively, were given to 65 healthy women, n = 21-22 in each group. Blood...

  19. Changes in blood levels of proteinase inhibitors, pregnancy zone protein, steroid carriers and complement factors induced by oral contraceptives

    DEFF Research Database (Denmark)

    Nielsen, C H; Poulsen, H K; Teisner, B;

    1993-01-01

    Three low-dose oral contraceptives Trinordiol, Gynatrol, and Marvelon, containing ethinylestradiol (EE) in combination with triphasic levonorgestrel (LNg), monophasic levonorgestrel, and monophasic desogestrel (DGS), respectively, were given to 65 healthy women, n = 21-22 in each group. Blood lev...

  20. Changes in blood levels of proteinase inhibitors, pregnancy zone protein, steroid carriers and complement factors induced by oral contraceptives

    DEFF Research Database (Denmark)

    Nielsen, C H; Poulsen, H K; Teisner, B;

    1993-01-01

    levels of antithrombin III (AT III), alpha 2-macroglobulin (alpha 2M) alpha 1-antitrypsin (alpha 1at), complement factors (factor B, C3, C4), pregnancy zone protein (PZP), corticosteroid binding globulin (CBG), sex hormone binding globulin (SHBG) and albumin were measured before treatment and during...... the first and third treatment cycles. AT III levels decreased and alpha 1at levels increased in all three groups during treatment. alpha 2M increased during cycle 3 in the Trinordiol and Gynatrol groups. CBG, PZP and SHBG levels increased in all 3 groups, the CBG and PZP increase being higher...... in the Marvelon group than in the Gynatrol group. Increases in SHBG levels were found in the order Marvelon > Trinordiol > Gynatrol. Plasma levels of complement factors B, C3 and C4 remained unchanged. It is concluded that the increase in alpha 1at partly compensates for the fall in AT III, that the rise in PZP...

  1. Comparison of self-processing of foot-and-mouth disease virus leader proteinase and porcine reproductive and respiratory syndrome virus leader proteinase nsp1α

    Energy Technology Data Exchange (ETDEWEB)

    Steinberger, Jutta [Max F. Perutz Laboratories, Medical University of Vienna, Department of Medical Biochemistry, Dr. Bohr-Gasse 9/3, A-1030 Vienna (Austria); Kontaxis, Georg [Max F. Perutz Laboratories, University of Vienna, Department of Structural and Computational Biology, Campus Vienna Biocenter 5, A-1030 Vienna (Austria); Rancan, Chiara [Helmholtz Zentrum München, Department of Gene Vectors, Haematologikum, Marchioninistrasse 25, D-81377 Munich (Germany); Skern, Tim, E-mail: timothy.skern@meduniwien.ac.at [Max F. Perutz Laboratories, Medical University of Vienna, Department of Medical Biochemistry, Dr. Bohr-Gasse 9/3, A-1030 Vienna (Austria)

    2013-09-01

    The foot-and-mouth disease virus leader proteinase (Lb{sup pro}) cleaves itself off the nascent viral polyprotein. NMR studies on the monomeric variant Lb{sup pro} L200F provide structural evidence for intramolecular self-processing. {sup 15}N-HSQC measurements of Lb{sup pro} L200F showed specifically shifted backbone signals in the active and substrate binding sites compared to the monomeric variant sLb{sup pro}, lacking six C-terminal residues. This indicates transient intramolecular interactions between the C-terminal extension (CTE) of one molecule and its own active site. Contrastingly, the porcine reproductive and respiratory syndrome virus (PRRSV) leader proteinase nsp1α, with a papain-like fold like Lb{sup pro}, stably binds its own CTE. Parts of the β-sheet domains but none of the α-helical domains of Lb{sup pro} and nsp1α superimpose; consequently, the α-helical domain of nsp1α is oriented differently relative to its β-sheet domain. This provides a large interaction surface for the CTE with the globular domain, stabilising the intramolecular complex. Consequently, self-processing inactivates nsp1α but not Lb{sup pro}. - Highlights: • We examine self-processing of the leader protease of foot-and-mouth disease virus. • NMR analysis strongly supports intramolecular self-processing. • Self-processing is a dynamic process with no stable complex. • Structural comparison with nsp1α of PRRSV which forms stable intramolecular complex. • Subdomain orientation explains differences in stability of intramolecular complexes.

  2. Purification, characterization, primary structure, crystallization and preliminary crystallographic study of a serine proteinase from Streptomyces fradiae ATCC 14544.

    Science.gov (United States)

    Kitadokoro, K; Tsuzuki, H; Nakamura, E; Sato, T; Teraoka, H

    1994-02-15

    A proteinase having wide substrate specificity was isolated from Streptomyces fradiae ATCC 14544. This proteinase, which we propose to call SFase-2, was purified from the culture filtrate by S-Sepharose chromatography. The purified enzyme showed an apparent molecular mass of 19 kDa on SDS/PAGE. When synthetic peptides were used as substrates, SFase-2 showed broad substrate specificity. It also hydrolyzed keratin, elastin and collagen as proteinaceous substrates. It was completely inhibited by diisopropylfluorophosphate and chymostatin, but not by tosylphenylalaninechloromethane, tosyllysinechloromethane or EDTA, indicating that it can be classified as a serine proteinase. The matured protein sequence of SFase-2 was determined by a combination of amino acid sequencing and the DNA sequencing of the gene. SFase-2, consisting of 191 amino acids, is a novel proteinase. It showed 76% similarity in the amino acid sequence with Streptomyces griseus proteinase A [Johnson P. and Smillie L. B. (1974) FEBS Lett. 47, 1-6]. For insight into the three-dimensional structure of SFase-2, we obtained single crystals by the vapor diffusion method using sodium phosphate as a precipitant. These crystals belonged to the orthorhombic, space group P2(1)2(1)2(1) with cell dimensions a = 6.92 nm, b = 7.28 nm, c = 2.99 nm; one molecule was present in the asymmetric unit.

  3. Heat shock induction of a 65 kDa ATP—binding proteinase in rat C6 glioma cells

    Institute of Scientific and Technical Information of China (English)

    XUCUNSHUAN; MARCOMEYER; 等

    1999-01-01

    The 45,55,65 and 100kDa ATP-binding proteinases(ATP-BPases) of the heat-shocked (44℃ for 30 min,recovery for 12h) rat C6 glioma cells were purified by DEAE-ionexchange and ATP-affinity chromatography.Their molecular masses,isoelectric points (pI),pH-optima and other properties were analyzed by native proteinase gels.It was shown that the 65 kDa ATP-BPase is specifically induced by heat shock and not detectable in control cells.Its N-terminal 1-9amino acid sequence was determined by Edman degradation,but no homologies to other proteins in the protein data bases were found.30 and 31kDa proteinases can be cleaved from the 45,55 and 65 kDa proteinases to which they are linked.A possible relationship of the heat-induced 65 kDa ATP-BPase with the ATP-dependent proteinases (ATP-DPases) in prokaryotes and eukaryotes is discussed.

  4. Insight derived from molecular dynamics simulation into substrate-induced changes in protein motions of proteinase K.

    Science.gov (United States)

    Tao, Yan; Rao, Zi-He; Liu, Shu-Qun

    2010-10-01

    Because of the significant industrial, agricultural and biotechnological importance of serine protease proteinase K, it has been extensively investigated using experimental approaches such as X-ray crystallography, site-directed mutagenesis and kinetic measurement. However, detailed aspects of enzymatic mechanism such as substrate binding, release and relevant regulation remain unstudied. Molecular dynamics (MD) simulations of the proteinase K alone and in complex with the peptide substrate AAPA were performed to investigate the effect of substrate binding on the dynamics/molecular motions of proteinase K. The results indicate that during simulations the substrate-complexed proteinase K adopt a more compact and stable conformation than the substrate-free form. Further essential dynamics (ED) analysis reveals that the major internal motions are confined within a subspace of very small dimension. Upon substrate binding, the overall flexibility of the protease is reduced; and the noticeable displacements are observed not only in substrate-binding regions but also in regions opposite the substrate-binding groove/pockets. The dynamic pockets caused by the large concerted motions are proposed to be linked to the substrate recognition, binding, orientation and product release; and the significant displacements in regions opposite the binding groove/pockets are considered to play a role in modulating the dynamics of enzyme-substrate interaction. Our simulation results complement the biochemical and structural studies, highlighting the dynamic mechanism of the functional properties of proteinase K.

  5. Protective role of purified cysteine proteinases against Fasciola gigantica infection in experimental animals.

    Science.gov (United States)

    El-Ahwany, Eman; Rabia, Ibrahim; Nagy, Faten; Zoheiry, Mona; Diab, Tarek; Zada, Suher

    2012-03-01

    Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freund's adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 F. gigantica metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, IgG(1), and IgG(2) (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-γ, and TNF-α, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-β, and IL-6, showed significant increases (P<0.05). In conclusion, it has been found that CP released by F. gigantica are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships. PMID:22451733

  6. Influence of immunoprotection on genetic variability of cysteine proteinases from Haemonchus contortus adult worms.

    Science.gov (United States)

    Martín, S; Molina, J M; Hernández, Y I; Ferrer, O; Muñoz, Ma C; López, A; Ortega, L; Ruiz, A

    2015-11-01

    The limitations associated with the use of anthelmintic drugs in the control of gastrotintestinal nematodosis, such as the emergence of anthelmintic resistance, have stimulated the study of the immunological control of many parasites. In the case of Haemonchus contortus, several vaccination trials using native and recombinant antigens have been conducted. A group of antigens with demonstrated immunoprotective value are cathepsin B - like proteolytic enzymes of the cysteine proteinase type. These enzymes, which have been observed in both excretory-secretory products and somatic extracts of H. contortus, may vary among different geographic isolates and on strains isolated from different hosts, or even from the same host, as has been demonstrated in some comparative studies of genetic variability. In the present study, we evaluated the genetic variability of the worms that fully developed their endogenous cycle in immunised sheep and goat in order to identify the alleles of most immunoprotective value. To address these objectives, groups of sheep and goats were immunised with PBS soluble fractions enriched for cysteine proteinases from adult worms of H. contortus from either a strain of H. contortus isolated from goats of Gran Canaria Island (SP) or a strain isolated from sheep of North America (NA). The results confirmed the immunoprophylactic value of this type of enzyme against haemonchosis in both sheep and goats in association with increased levels of specific IgG. The genetic analysis demonstrated that the immunisation had a genetic selection on proteinase-encoding genes. In all the immunised animals, allelic frequencies were statistically different from those observed in non-immunised control animals in the four analysed genes. The reduction in the allelic frequencies suggests that parasites expressing these proteases are selectively targeted by the vaccine, and hence they should be considered in any subunit vaccine approach to control haemonchosis in small

  7. In vitro assay for HCV serine proteinase expressed in insect cells

    Institute of Scientific and Technical Information of China (English)

    Li-Hua Hou; Gui-Xin Du; Rong-Bin Guan; Yi-Gang Tong; Hai-Tao Wang

    2003-01-01

    AIM: To produce the recombinant NS3 protease of hepatitis C virus with enzymatic activity in insect cells.METHODS: The gene of HCV serine proteinase domain which encodes 181 amino acids was inserted into pFastBacHTc and the recombinant plasmid pFBCNS3N was transformed into DH10Bac competent cells for transposition.After the recombinant bacmids had been determined to be correct by both blue-white colonies and PCR analysis, the isolated bacmid DNAs were transfected into Sf9 insect cells.The bacmids DNA was verified to replicate in insect cells and packaged into baculovirus particles via PCR and electronic microscopic analysis. The insect cells infected with recombinant baculovirus were determined by SDS-PAGE and Western-blot assays. The recombinant protein was soluted in N-lauryl sarcosine sodium (NLS) and purifed by metalchelated-affinity chromatography, then the antigenicity of recombinant protease was determined by enzyme-linked immunoabsorbant assay and its enzymatic activity was detected.RESULTS: The HCV NS3 protease domain was expressed in insect cells at high level and it was partially solved in NLS.Totally 0.2 mg recombinant serine proteinase domain with high purity was obtained by metal-chelated-affinity chromatography from 5×107 cells, and both antigenicity and specificity of the protein were evaluated to be high when used as antigen to detect hepatitis C patients′ sera in indirect ELISA format. In vitro cleavage assay corroborated its enzymatic activity.CONCLUSION: The recombinant HCV NS3 proteinase expressed by insect cells is a membrane-binding protein with good antigenicity and enzymatic activity.

  8. Morphological and molecular characterisation, and phylogenetic position of X. browni sp. n., X. penevi sp. n. and two known species of Xiphinema americanum-group (Nematoda, Longidoridae).

    Science.gov (United States)

    Lazarova, Stela; Peneva, Vlada; Kumari, Shesh

    2016-01-01

    Using ribosomal (18S, ITS1, ITS2, D2-D3 expansion segments of 28S rDNA) and mitochondrial (partial cox1 and nad4) DNA markers in a study of several populations of Xiphinema americanum-group from Europe and Morocco, two cryptic species Xiphinema browni sp. n. (formerly reported as Xiphinema pachtaicum) and Xiphinema penevi sp. n. were revealed. The species are described, illustrated and their phylogenetic relationships discussed. The first species is most similar to Xiphinema parasimile and is a member of Xiphinema simile species complex. The phylogenetic reconstructions inferred from three molecular markers (18S, D2-D3 28S rDNA and cox1) showed that Xiphinema penevi sp. n. is part of Xiphinema pachtaicum-subgroup and is closely related to Xiphinema incertum, Xiphinema pachtaicum, Xiphinema parapachydermum, Xiphinema plesiopachtaicum, Xiphinema astaregiense and Xiphinema pachydermum. Also, a separate "Xiphinema simile-subgroup", outside the Xiphinema pachtaicum-subgroup and so far consisting only of the parthenogenetic species Xiphinema simile, Xiphinema parasimile, Xiphinema browni sp. n. and probably Xiphinema vallense was formed. New primers for amplification and sequencing of part of the nad4 mitochondrial gene were designed and used.

  9. Morphological and molecular characterisation, and phylogenetic position of X. browni sp. n., X. penevi sp. n. and two known species of Xiphinema americanum-group (Nematoda, Longidoridae).

    Science.gov (United States)

    Lazarova, Stela; Peneva, Vlada; Kumari, Shesh

    2016-01-01

    Using ribosomal (18S, ITS1, ITS2, D2-D3 expansion segments of 28S rDNA) and mitochondrial (partial cox1 and nad4) DNA markers in a study of several populations of Xiphinema americanum-group from Europe and Morocco, two cryptic species Xiphinema browni sp. n. (formerly reported as Xiphinema pachtaicum) and Xiphinema penevi sp. n. were revealed. The species are described, illustrated and their phylogenetic relationships discussed. The first species is most similar to Xiphinema parasimile and is a member of Xiphinema simile species complex. The phylogenetic reconstructions inferred from three molecular markers (18S, D2-D3 28S rDNA and cox1) showed that Xiphinema penevi sp. n. is part of Xiphinema pachtaicum-subgroup and is closely related to Xiphinema incertum, Xiphinema pachtaicum, Xiphinema parapachydermum, Xiphinema plesiopachtaicum, Xiphinema astaregiense and Xiphinema pachydermum. Also, a separate "Xiphinema simile-subgroup", outside the Xiphinema pachtaicum-subgroup and so far consisting only of the parthenogenetic species Xiphinema simile, Xiphinema parasimile, Xiphinema browni sp. n. and probably Xiphinema vallense was formed. New primers for amplification and sequencing of part of the nad4 mitochondrial gene were designed and used. PMID:27110175

  10. Genetic changes after androgenesis in the millet Pennisetum americanum: Study of descendants from cultures of pollen of an F1 hybrid (Massue X Ligui)

    International Nuclear Information System (INIS)

    Self-fertilizing generations of double haploid plants obtained by androgenesis from an F1 hybrid (Massue X Ligui) of the millet Pennisetum americanum were investigated. Four enzyme systems were studied by electrophoresis (for four loci in particular: Adh1, esterase 1, peroxidase P5, catalase, which are heterozygous in F1), as well as 20 morphological and phenological characteristics (analysed by principal component analysis and by hierarchical variance analysis). The biochemical and biometric studies combined to show considerable distortions in segregation (generally in favour of characteristics of the Massue line) and great variability of genetic origin revealed during successive self-fertilizations of the double haploids. Part of this variability could be explained by partial non-reduction phenomena (never total non-reduction, since no plant produced by androgenesis has all the characteristics of the F1 generation). However, recourse must also be had to other phenomena analogous to genetic instabilities in order to account for the appearance of new functional states (detected by electrophoresis) in the S1 and S2 generations and the fact that some of the intra-family variations do not become weaker as the genealogy progresses. (author)

  11. Organic amendments impact the availability of heavy metal(loid)s in mine-impacted soil and their phytoremediation by Penisitum americanum and Sorghum bicolor.

    Science.gov (United States)

    Nawab, Javed; Khan, Sardar; Aamir, Muhammad; Shamshad, Isha; Qamar, Zahir; Din, Islamud; Huang, Qing

    2016-02-01

    The amendment of contaminated soil with organic materials is considered to be an environmentally friendly technique to immobilize heavy metal(loid)s and minimize their subsequent bioaccumulation in plants. This study focuses on the effects of different amendment techniques, such as the use of activated carbons (granulated or powder) and farmyard manure at various application rates (2 and 5 %). These techniques were applied on heavy metal(loid)s such as Ni, Cr, Cd, Pb, Mn, Cu, Zn, Fe, Co, and Al that were present in mine-impacted soil and caused bioaccumulation in cultivated plants. The results showed that, compared with the control, almost all the techniques significantly (P ≤ 0.01) reduced the bioavailability of heavy metal(loid)s in the amended soil. The bioaccumulation of heavy metal(loid)s in Penisitum americanum and Sorghum bicolor was significantly (P ≤ 0.01) reduced with all techniques, while Zn and Cd concentrations increased with the use of farmyard manure. Also compared with the control, plant growth was significantly decreased with the use of activated carbons, particularly with powder activated carbons, while farmyard manure (at 5 %) significantly (P ≤ 0.01) increased plant growth. Among the amendment techniques, powdered activated carbons (at 5 %) were best at reducing the bioavailability of heavy metal(loid)s in soil and plant accumulation. However, it negatively affected the growth of selected plant species.

  12. Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine beta-casein.

    OpenAIRE

    Rattray, F P; Fox, P. F.; Healy, A.

    1997-01-01

    The specificity of the extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine beta-casein was studied. Hydrolysis was monitored over time by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and urea-PAGE. The major pH 4.6-soluble peptides were isolated by high-performance liquid chromatography and identified by N-terminal amino acid sequencing and mass spectrometry. The major sites of hydrolysis were Ser-18-Ser-19, Glu-20-Glu-21, Gln-56-Ser-57, Gln-72-Asn-73, ...

  13. Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein.

    OpenAIRE

    Rattray, F P; Fox, P. F.; Healy, A.

    1996-01-01

    The specificity of the extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein was studied. Hydrolysis was monitored over time by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and urea-PAGE. The major pH 4.6-soluble peptides were isolated by high-performance liquid chromatography and identified by N-terminal amino acid sequencing and mass spectrometry. The time course of peptide formation indicated that His-8-Gln-9, Ser-161-Gly-162, and eithe...

  14. Biochemical and biological characterization of two serine proteinases from Colombian Crotalus durissus cumanensis snake venom.

    Science.gov (United States)

    Patiño, Arley Camilo; Pereañez, Jaime Andrés; Gutiérrez, José María; Rucavado, Alexandra

    2013-03-01

    Two clotting serine proteinases, named Cdc SI and Cdc SII, were isolated and characterized for the first time from Colombian Crotalus durissus cumanensis snake venom. The enzymes were purified using two chromatographic steps: molecular exclusion on Sephacryl S-200 and RP-HPLC on C8 Column. The molecular masses of the proteins, determined by MALDI-TOF mass spectrometry, were 28,561.4 and 28,799.2 Da for Cdc SI and Cdc SII, respectively. The aim of the present study was to evaluate enzymatic, coagulant and toxic properties of the two enzymes. The serine proteinases hydrolyzed specific chromogenic substrate (BaPNA) and exhibited a Michaelis-Menten behavior. Cdc SI had V(max) of 0.038 ± 0.003 nmol/min and K(M) of 0.034 ± 0.017 mM, while Cdc SII displayed values of V(max) of 0.267 ± 0.011 nmol/min and K(M) of 0.145 ± 0.023 mM. N-terminal sequences were VIGGDEXNIN and VIGGDICNINEHNFLVALYE for Cdc SI and Cdc SII, respectively. Molecular masses, N-terminal sequences, inhibition assays, and enzymatic profile suggest that Cdc SI and Cdc SII belong to the family of snake venom thrombin-like enzymes. These serine proteinases differed in their clotting activity on human plasma, showing a minimum coagulant dose of 25 μg and 0.571 μg for Cdc SI and Cdc SII, respectively. Enzymes also showed coagulant activity on bovine fibrinogen and degraded chain α of this protein. Toxins lack hemorrhagic and myotoxic activities, but are capable to induce defibrin(ogen)ation, moderate edema, and an increase in vascular permeability. These serine proteinases may contribute indirectly to the local hemorrhage induced by metalloproteinases, by causing blood clotting disturbances, and might also contribute to cardiovascular alterations characteristic of patients envenomed by C. d. cumanensis in Colombia.

  15. Comparison of ACE inhibitory activity in skimmed goat and cow milk hydrolyzed by alcalase, flavourzyme, neutral protease and proteinase K

    Directory of Open Access Journals (Sweden)

    Bao Chunju

    2016-06-01

    Full Text Available Angiotensin I converting enzyme (ACE inhibitory peptides derived from milk proteins have obvious effect of lowering blood pressure, safe and non-toxic side effects. This study compared four commercial proteases, namely alcalase, flavourzyme, neutral protease and proteinase K for their ACE inhibitory activity in skimmed goat and cow milk and identified the best one with higher ACE inhibitory activity. The degree of hydrolysis (DH of alcalase and proteinase K were much higher than flavourzyme, neutral protease for both skimmed goat and cow milk. Alcalase was the best enzyme to produce ACE inhibitory peptides from goat milk, with the ACE inhibitory activity 95.31%, while proteinase K was the optimal protease for hydrolyzing cow milk, with 81.28% ACE inhibitory activity. Furthermore, no correlation was obtained between the ACE inhibitory activity and DH for both goat and cow milk.

  16. Crystal structure of a putative aspartic proteinase domain of the Mycobacterium tuberculosis cell surface antigen PE_PGRS16☆

    Science.gov (United States)

    Barathy, Deivanayaga V.; Suguna, Kaza

    2013-01-01

    We report the crystal structure of the first prokaryotic aspartic proteinase-like domain identified in the genome of Mycobacterium tuberculosis. A search in the genomes of Mycobacterium species showed that the C-terminal domains of some of the PE family proteins contain two classic DT/SG motifs of aspartic proteinases with a low overall sequence similarity to HIV proteinase. The three-dimensional structure of one of them, Rv0977 (PE_PGRS16) of M. tuberculosis revealed the characteristic pepsin-fold and catalytic site architecture. However, the active site was completely blocked by the N-terminal His-tag. Surprisingly, the enzyme was found to be inactive even after the removal of the N-terminal His-tag. A comparison of the structure with pepsins showed significant differences in the critical substrate binding residues and in the flap tyrosine conformation that could contribute to the lack of proteolytic activity of Rv0977. PMID:23923105

  17. High-level expression of Proteinase K from Tritirachium album Limber in Pichia pastoris using multi-copy expression strains.

    Science.gov (United States)

    Yang, Hu; Zhai, Chao; Yu, Xianhong; Li, Zhezhe; Tang, Wei; Liu, Yunyun; Ma, Xiaojian; Zhong, Xing; Li, Guolong; Wu, Di; Ma, Lixin

    2016-06-01

    Proteinase K is widely used in scientific research and industries. This report was aimed to achieve high-level expression of proteinase K using Pichia pastoris GS115 as the host strain. The coding sequence of a variant of proteinase K that has higher activity than the wild type protein was chosen and optimized based on the codon usage preference of P. pastoris. The novel open reading frame was synthesized and a series of multi-copy expression vectors were constructed based on the pHBM905BDM plasmid, allowing for the tandem integration of multiple copies of the target gene into the genome of P. pastoris with a single recombination. These strains were used to study the correlation between the gene copy number and the expression level of proteinase K. The results of quantitative polymerase chain reaction (qPCR) indicated that the tandem expression cassettes were integrated into the host genome stably. Meanwhile, the results of qPCR and enzyme activity assays indicated that the mRNA and protein expression levels of the target gene increased as the gene copy number increased. Moreover, the effect of gene dosage on the expression level of the recombinant protein was more obvious using high-density fermentation. The maximum expression level and enzyme activity of proteinase K, which were obtained from the recombinant yeast strain bearing 5 copies of the target gene after an 84-h induction, were approximately 8.069 mg/mL and 108,295 U/mL, respectively. The recombinant proteinase was purified and characterized. The optimum pH and temperature for the activity of this protease were approximately pH 11 and 55 °C, respectively.

  18. Brewer's spent grain and corn steep liquor as alternative culture medium substrates for proteinase production by Streptomyces malaysiensis AMT-3

    Directory of Open Access Journals (Sweden)

    Rodrigo Pires do Nascimento

    2011-12-01

    Full Text Available Brewer's spent grain and corn steep liquor or yeast extract were used as the sole organic forms for proteinase production by Streptomyces malaysiensis in submerged fermentation. The influence of the C and N concentrations, as well as the incubation periods, were assessed. Eight proteolytic bands were detected through gelatin-gel-electrophoresis in the various extracts obtained from the different media and after different incubation periods, with apparent molecular masses of 20, 35, 43, 50, 70, 100, 116 and 212 kDa. The results obtained suggest an opportunity for exploring this alternative strategy for proteinases production by actinomycetes, using BSG and CSL as economically feasible substrates.

  19. Isolation and characterization of βA3-crystallin associated proteinase from α-crystallin fraction of human lenses

    OpenAIRE

    Srivastava, O.P.; Srivastava, K.; Chaves, J. M.

    2008-01-01

    Purpose The purpose was to characterize the properties of a proteinase activity associated with βA3-crystallin, which was isolated from the α-crystallin fraction of human lenses. Methods An inactive, Arg-bond hydrolyzing proteinase in the α-crystallin fraction, which was isolated from the water soluble (WS) protein fraction of 60- to 70-year-old human lenses, was activated by sodium deoxycholate treatment. The activated enzyme was purified by a three-step procedure that included a size-exclus...

  20. Production of proteinase A by Saccharomyces cerevisiae in a cell-recycling fermentation system: Experiments and computer simulations

    DEFF Research Database (Denmark)

    Grøn, S.; Biedermann, K.; Emborg, Claus

    1996-01-01

    Overproduction of proteinase A by recombinant Saccharomyces cerevisiae was investigated by cultivations in a cell-recycling bioreactor. Membrane filtration was used to separate cells from the broth. Recycling ratios and dilution rates were varied and the effect on enzyme production was studied both...... experimentally and by computer simulations. Experiments and simulations showed that cell mass and product concentration were enhanced by high ratios of recycling. Additional simulations showed that the proteinase A concentration decreased drastically at high dilution rates and the optimal volumetric...

  1. [Characteristics of the Effect of Cestodes Parasitizing the Fish Intestine on the Activity of the Host Proteinases].

    Science.gov (United States)

    Izvekova, G I; Solovyev, M M

    2016-01-01

    The activity and spectrum of proteinases in the intestines of host fishes change upon infestation with cestodes. Serine proteinases are found to make a greater contribution to the total proteolytic activity. The reduction of proteolytic activity is associated with adsorption of the enzymes of the host on the surface of cestodes, and the increase in the activity is caused by the injury of the intestinal mucosa by the attachment apparatuses of cestodes. The inhibition of proteainase activity indicates the possible participation of microbiota enzymes in protein hydrolyses.

  2. Cross-talk between toll-like receptor 4 (TLR4) and proteinase-activated receptor 2 (PAR2) is involved in vascular function

    Science.gov (United States)

    Bucci, M; Vellecco, V; Harrington, L; Brancaleone, V; Roviezzo, F; Mattace Raso, G; Ianaro, A; Lungarella, G; De Palma, R; Meli, R; Cirino, G

    2013-01-01

    Background and Purpose Proteinase-activated receptors (PARs) and toll-like receptors (TLRs) are involved in innate immune responses. The aim of this study was to evaluate the possible cross-talk between PAR2 and TLR4 in vessels in physiological condition and how it varies following stimulation of TLR4 by using in vivo and ex vivo models. Experimental Approach Thoracic aortas were harvested from both naïve and endotoxaemic rats for in vitro studies. Arterial blood pressure was monitored in anaesthetized rats in vivo. LPS was used as a TLR4 agonist while PAR2 activating peptide (AP) was used as a PAR2 agonist. Aortas harvested from TLR4–/– mice were also used to characterize the PAR2 response. Key Results PAR2, but not TLR4, expression was enhanced in aortas of endotoxaemic rats. PAR2AP-induced vasorelaxation was increased in aortic rings of LPS-treated rats. TLR4 inhibitors, curcumine and resveratrol, reduced PAR2AP-induced vasorelaxation and PAR2AP-induced hypotension in both naïve and endotoxaemic rats. Finally, in aortic rings from TLR4–/– mice, the expression of PAR2 was reduced and the PAR2AP-induced vasodilatation impaired compared with those from wild-type mice and both resveratrol and curcumine were ineffective. Conclusions and Implications Cross-talk between PAR2 and TLR4 contributes to vascular homeostasis. PMID:22957757

  3. Inhibition of invasion and metastasis of MHCC97H cells by expression of snake venom cystatin through reduction of proteinases activity and epithelial-mesenchymal transition.

    Science.gov (United States)

    Tang, Nanhong; Xie, Qun; Wang, Xiaoqian; Li, Xiujin; Chen, Yanlin; Lin, Xu; Lin, Jianyin

    2011-05-01

    Snake venom cystatin (sv-cystatin) is a member of the cystatin family of cysteine protease inhibitors. To further evaluate the possibility of sv-cystatin in cancer therapy, this study examined the effects of sv-cystatin on the invasion and metastasis of liver cancer cells (MHCC97H) in vitro and in vivo as well as the underlying mechanism. sv-cystatin cDNA was transfected into MHCC97H cells and the anti-invasion and antimetastasis effects of sv-cystatin were determined using migration and matrigel invasion assays and a lung-metastasis mice model. The results suggest that sv-cyst clone (sv-cystatin expression in MHCC97H cells) delayed the invasion and metastasis in vitro and in vivo compared to the parental, mock and si-sv-cyst clone cells (inhibited sv-cystatin expression by siRNA). The decreased activities of cathepsin B, MMP-2 and MMP-9 and EMT change index including higher E-cadherin, lower N-cadherin and decreased Twist activity were observed in the sv-cyst clone, which contributes to the change in invasion and metastasis ability of MHCC97H cells. This study provides evidence that expression of the sv-cystatin gene in MHCC97H cells inhibits tumor cell invasion and metastasis through the reduction of the proteinases activity and Epithelial-Mesenchymal Transition (EMT), which might contribute to the anticancer research of the sv-cystatin protein.

  4. INFLUENCE OF VACUUM-PULSE DRYING ON THE CONTENT OF FREE AMINO ACIDS, TRYPSINE INHIBITOR ACTIVITY AND COMPOSITION OF VOLATILE COMPONENTS OF MUSHROOMS

    OpenAIRE

    Shcheglova, I.; Vereshchagin, A.

    2015-01-01

    Wild-growing mushrooms traditionally are considered one of the sources of food fibers, vegetable proteins, macro and micronutrients, and also flavor components. However, the composition of mushrooms includes antinutritional substances capable to selectively reduce the absorption of certain nutrients. These are primarily antienzymes or proteinase inhibitors, which reduce the absorption of proteins. Previous studies have indicated applicability of vacuum-pulse drying to improve the nutritional ...

  5. Proteinase K-catalyzed synthesis of linear and star oligo(L-phenylalanine) conjugates.

    Science.gov (United States)

    Ageitos, Jose M; Baker, Peter J; Sugahara, Michihiro; Numata, Keiji

    2013-10-14

    Chemoenzymatic synthesis of peptides is a green and clean chemical reaction that offers high yields without using organic synthesis and serves as an alternative to traditional peptide synthesis methods. This report describes the chemoenzymatic synthesis of oligo(L-phenylalanine) mediated by proteinase K from Tritirachium album, which is one of the most widely used proteases in molecular biological studies. The synthesized linear oligo-phenylalanine showed a unique self-assembly in aqueous solutions. To further functionalize linear oligo(L-phenylalanine) as a low-molecular-weight gelator, it was cosynthesized with tris(2-aminoethyl)amine to obtain star-oligo(L-phenylalanine), which was bioconjugated to demonstrate its self-assembly into fluorescent fibers. The self-assembled fibers of star-oligo(L-phenylalanine) formed fibrous networks with various branching ratios, which depended on the molecular weights and molecular aspect ratios of star-oligo(L-phenylalanine). This is the first study to demonstrate that proteinase K is a suitable enzyme for chemoenzymatic cosynthesis of oligopeptides and star-shaped heteropeptides.

  6. Intracellular localization of Treponema denticola chymotrypsin-like proteinase in chronic periodontitis

    Directory of Open Access Journals (Sweden)

    Emilia Marttila

    2014-07-01

    Full Text Available Treponema denticola is an important periodontal pathogen capable of tissue invasion. Its chymotrypsin-like proteinase (CTLP can degrade a number of basement membrane components in vitro, thus suggesting a contribution to tissue invasion by the spirochete. The aim of this study was to analyze the localization of CTLP in chronic periodontitis tissues ex vivo. A polyclonal antibody specific to T. denticola cell-bound CTLP was used to detect the spirochetes in the gingival tissues of patients with moderate to severe chronic periodontitis (n=25 by immunohistochemistry and periodic acid-Schiff staining (PAS. The presence of T. denticola in the periodontal tissue samples was analyzed by PCR. Periodontal tissue samples of 12 of the 25 patients were found to be positive for T. denticola by PCR. Moreover, CTLP could be detected in the periodontal tissues of all these patients by immunohistochemistry. In the epithelium, the CTLP was mostly intracellular. Typically, the positive staining could be seen throughout the whole depth of the epithelium. When detected extracellularly, CTLP was localized mainly as granular deposits. The connective tissue stained diffusely positive in four cases. The positive staining co-localized with the PAS stain in nine cases. T. denticola and its CTLP could be detected in diseased human periodontium both intra- and extracellularly. The granular staining pattern was suggestive of the presence of T. denticola bacteria, whereas the more diffused staining pattern was indicative of the recent presence of the bacterium and shedding of the cell-bound proteinase.

  7. Morphological confocal microscopy in arthropods and the enhancement of autofluorescence after proteinase K extraction.

    Science.gov (United States)

    Valdecasas, Antonio G; Abad, Angela

    2011-02-01

    Procedures to study the molecular and morphological characteristics of microscopic organisms are often incompatible with each other. Therein, the realization of alternatives that make the characterization of these features compatible and simultaneously permit the deposition of the original material as a voucher sample into a reference collection is one of the foremost goals of biodiversity studies. In this study, we show that genomic extraction does not necessarily compromise the detailed study of the external morphology of microscopic organisms, and to do so, we used a group of aquatic mites (Acari, Hydrachnidia) as a test group. Hydrachnidia morphology is difficult to study when specimens have been stored in pure ethanol; however, proteinase K extraction leaves them flexible and easy to dissect, while, at the same time, maintaining all of their diagnostic features intact. Furthermore, autofluorescence is significantly enhanced after proteinase extraction. Our study was conducted with aquatic mites that were stored in absolute ethanol in the field and processed for DNA extraction using a Qiagen QIAamp minikit. Before and after molecular extraction, a laser scanning confocal microscopy morphological examination was carried out.

  8. Effect of the Solvent Temperatures on Dynamics of Serine Protease Proteinase K.

    Science.gov (United States)

    Sang, Peng; Yang, Qiong; Du, Xing; Yang, Nan; Yang, Li-Quan; Ji, Xing-Lai; Fu, Yun-Xin; Meng, Zhao-Hui; Liu, Shu-Qun

    2016-02-19

    To obtain detailed information about the effect of the solvent temperatures on protein dynamics, multiple long molecular dynamics (MD) simulations of serine protease proteinase K with the solute and solvent coupled to different temperatures (either 300 or 180 K) have been performed. Comparative analyses demonstrate that the internal flexibility and mobility of proteinase K are strongly dependent on the solvent temperatures but weakly on the protein temperatures. The constructed free energy landscapes (FELs) at the high solvent temperatures exhibit a more rugged surface, broader spanning range, and higher minimum free energy level than do those at the low solvent temperatures. Comparison between the dynamic hydrogen bond (HB) numbers reveals that the high solvent temperatures intensify the competitive HB interactions between water molecules and protein surface atoms, and this in turn exacerbates the competitive HB interactions between protein internal atoms, thus enhancing the conformational flexibility and facilitating the collective motions of the protein. A refined FEL model was proposed to explain the role of the solvent mobility in facilitating the cascade amplification of microscopic motions of atoms and atomic groups into the global collective motions of the protein.

  9. Characterization of a New Cell Envelope Proteinase PrtP from Lactobacillus rhamnosus CGMCC11055.

    Science.gov (United States)

    Guo, Tingting; Ouyang, Xudong; Xin, Yongping; Wang, Yue; Zhang, Susu; Kong, Jian

    2016-09-21

    Cell envelope proteinases (CEPs) play essential roles in lactic acid bacteria growth in milk and health-promoting properties of fermented dairy products. The genome of Lactobacillus rhamnosus CGMCC11055 possesses two putative CEP genes prtP and prtR2, and the PrtP displays the distinctive domain organization from PrtR2 reported. The PrtP was purified and biochemically characterized. The results showed that the optimal activity occurred at 44 °C, pH 6.5. p-Amidinophenylmethylsulfonyl fluoride obviously inhibited enzymatic activity, suggesting PrtP was a member of serine proteinases. Under the optimal conditions, β-casein was a favorite substrate over αS1- and κ-casein, and 35 oligopeptides were identified in the β-casein hydrolysate, including the phosphoserine peptide and bioactive isoleucine-proline-proline. By analysis of the amino acid sequences of those oligopeptides, proline was the preferred residue at the breakdown site. Therefore, we speculated that PrtP was a new type of CEPs from Lb. rhamnosus. PMID:27585760

  10. Diversity in proteinase specificity of thermophilic lactobacilli as revealed by hydrolysis of dairy and vegetable proteins.

    Science.gov (United States)

    Pescuma, Micaela; Espeche Turbay, María Beatriz; Mozzi, Fernanda; Font de Valdez, Graciela; Savoy de Giori, Graciela; Hebert, Elvira María

    2013-09-01

    Ability of industrially relevant species of thermophilic lactobacilli strains to hydrolyze proteins from animal (caseins and β-lactoglobulin) and vegetable (soybean and wheat) sources, as well as influence of peptide content of growth medium on cell envelope-associated proteinase (CEP) activity, was evaluated. Lactobacillus delbrueckii subsp. lactis (CRL 581 and 654), L. delbrueckii subsp. bulgaricus (CRL 454 and 656), Lactobacillus acidophilus (CRL 636 and 1063), and Lactobacillus helveticus (CRL 1062 and 1177) were grown in a chemically defined medium supplemented or not with 1 % Casitone. All strains hydrolyzed mainly β-casein, while degradation of αs-caseins was strain dependent. Contrariwise, κ-Casein was poorly degraded by the studied lactobacilli. β-Lactoglobulin was mainly hydrolyzed by CRL 656, CRL 636, and CRL 1062 strains. The L. delbrueckii subsp. lactis strains, L. delbrueckii subsp. bulgaricus CRL 656, and L. helveticus CRL 1177 degraded gliadins in high extent, while the L. acidophilus and L. helveticus strains highly hydrolyzed soy proteins. Proteinase production was inhibited by Casitone, the most affected being the L. delbrueckii subsp. lactis species. This study highlights the importance of proteolytic diversity of lactobacilli for rational strain selection when formulating hydrolyzed dairy or vegetable food products.

  11. HDAC Inhibitors.

    Science.gov (United States)

    Olzscha, Heidi; Bekheet, Mina E; Sheikh, Semira; La Thangue, Nicholas B

    2016-01-01

    Lysine acetylation in proteins is one of the most abundant posttranslational modifications in eukaryotic cells. The dynamic homeostasis of lysine acetylation and deacetylation is dictated by the action of histone acetyltransferases (HAT) and histone deacetylases (HDAC). Important substrates for HATs and HDACs are histones, where lysine acetylation generally leads to an open and transcriptionally active chromatin conformation. Histone deacetylation forces the compaction of the chromatin with subsequent inhibition of transcription and reduced gene expression. Unbalanced HAT and HDAC activity, and therefore aberrant histone acetylation, has been shown to be involved in tumorigenesis and progression of malignancy in different types of cancer. Therefore, the development of HDAC inhibitors (HDIs) as therapeutic agents against cancer is of great interest. However, treatment with HDIs can also affect the acetylation status of many other non-histone proteins which play a role in different pathways including angiogenesis, cell cycle progression, autophagy and apoptosis. These effects have led HDIs to become anticancer agents, which can initiate apoptosis in tumor cells. Hematological malignancies in particular are responsive to HDIs, and four HDIs have already been approved as anticancer agents. There is a strong interest in finding adequate biomarkers to predict the response to HDI treatment. This chapter provides information on how to assess HDAC activity in vitro and determine the potency of HDIs on different HDACs. It also gives information on how to analyze cellular markers following HDI treatment and to analyze tissue biopsies from HDI-treated patients. Finally, a protocol is provided on how to detect HDI sensitivity determinants in human cells, based on a pRetroSuper shRNA screen upon HDI treatment. PMID:27246222

  12. House dust mite Der p 1 downregulates defenses of the lung by inactivating elastase inhibitors.

    Science.gov (United States)

    Brown, Alan; Farmer, Kinley; MacDonald, Louise; Kalsheker, Noor; Pritchard, Dave; Haslett, Chris; Lamb, Jonathan; Sallenave, J-M

    2003-09-01

    House dust mites (HDM) are the most common source of aeroallergens and in genetic susceptible individuals can cause symptoms ranging from atopic dermatitis to bronchial asthma. Der p 1, a major target of the human immune responses to HDM, through its enzymatic properties can modulate the adaptive immune system by the cleavage of CD23 and CD25. The consequences of this would be to promote allergic inflammatory responses. Furthermore, by disrupting epithelial tight junctions Der p 1 facilitates the transport of allergen across the epithelium. Here, we report that Der p 1 has additional effects on the innate defense mechanisms of the lung, by inactivating in vitro and ex vivo the elastase inhibitors human (h) alpha1-proteinase inhibitor (h-A1-Pi), mouse (m-), (but not human [h])-SLPI and h-elafin. We confirm that Der p 1 contain both cysteine and serine proteinases, and extend this finding to demonstrate for the first time that h-elafin is particularly sensitive to the biological activity of the latter. Because these elastase inhibitors have antimicrobial, as well as antielastase activity, our results suggest that inactivation of these innate components of the lung defense system by Der p 1 may increase the susceptibility of patients with allergic inflammation to infection. PMID:12689923

  13. A comparison of the performance of regression models of Amblyomma americanum (L.) (Ixodidae) using life cycle or landscape data from administrative divisions.

    Science.gov (United States)

    Estrada-Peña, Agustín; de la Fuente, José; Cabezas-Cruz, Alejandro

    2016-06-01

    The distribution of Amblyomma americanum (L.) in the continental United States has been modelled using the reported distribution in counties as "established" (six or more ticks or two or more life stages were recorded during a specified time period), "reported" (fewer than six ticks of a single life stage were recorded), or "absent" (neither of the above categories were met) and a set of environmental and biotic explanatory variables. Categorical (vegetation, climate, and habitat suitability for the main host of the tick) or continuous variables (raw data on temperature, vegetation, and habitat fragmentation), as well as the processes of the tick's life cycle, were tested to build models using multiple logistic regression. The best results were obtained when the life cycle processes were used as descriptors of regressions. Better models derived from life cycle processes were obtained after inclusion of habitat suitability for the white tailed deer (the main host of the tick) and landscape fragmentation. Using this approach, 86% of "absent" and 83% of "established" counties were classified correctly, but all "reported" counties were erroneously classified. Modelling life cycle processes with descriptions of host abundance and habitat fragmentation produced the best outcome when coordinates were missing. When only standard categorical descriptors of climate or vegetation were included in models, results produced poor outcomes. Models were improved with remotely sensed information on temperature and vegetation but produced high rates of misclassification for 14% of "absent", 37% of "established", and 100% of "reported" counties. Modelling produced poor results in the absence of point distributions (coordinates). Therefore, "reported" counties cannot be handled adequately by modelling procedures, probably because this category does not reflect the true status of the tick distribution. We conclude that standard categorical classifications of the distribution of an

  14. Amblyomma americanum tick saliva insulin-like growth factor binding protein-related protein 1 binds insulin but not insulin-like growth factors.

    Science.gov (United States)

    Radulović, Ž M; Porter, L M; Kim, T K; Bakshi, M; Mulenga, A

    2015-10-01

    Silencing Amblyomma americanum insulin-like growth factor binding protein-related protein 1 (AamIGFBP-rP1) mRNA prevented ticks from feeding to repletion. In this study, we used recombinant (r)AamIGFBP-rP1 in a series of assays to obtain further insight into the role(s) of this protein in tick feeding regulation. Our results suggest that AamIGFBP-1 is an antigenic protein that is apparently exclusively expressed in salivary glands. We found that both males and females secrete AamIGFBP-rP1 into the host during feeding and confirmed that female ticks secrete this protein from within 24-48 h after attachment. Our data suggest that native AamIGFBP-rP1 is a functional insulin binding protein in that both yeast- and insect cell-expressed rAamIGFBP-rP1 bound insulin, but not insulin-like growth factors. When subjected to anti-blood clotting and platelet aggregation assays, rAamIGFBP-rP1 did not have any effect. Unlike human IGFBP-rP1, which is controlled by trypsinization, rAamIGFBP-rP1 is resistant to digestion, suggesting that the tick protein may not be under mammalian host control at the tick feeding site. The majority of tick-borne pathogens are transmitted 48 h after the tick has attached. Thus, the demonstrated antigenicity and secretion into the host within 24-48 h of the tick starting to feed makes AamIGFBP-rP1 an attractive target for antitick vaccine development.

  15. Effect of polysaccharide capsule of the microalgae Staurastrum iversenii var. americanum on diffusion of charged and uncharged molecules, using EPR technique

    International Nuclear Information System (INIS)

    The existence of a mucilaginous envelope, sheath or capsule is usual in many desmids, but few data concerning its function are available. Previous studies of the transport function and permeation of molecules through the algae capsules were done using the algae Spondylosium panduriforme and Nephrocytium lunatum, the Electron Paramagnetic Resonance (EPR) technique, and different spin labels. The results suggested that the capsule functions as a selective diffusion medium. In the present work charged and uncharged molecules (spin labels group A) and Staurastrum iversenii var. americanum (Desmids),whose alga presents a great mucilaginous capsule, were used. Charged nitroxide molecules similar to amino acids (spin labels group B) were also used allowing a better understanding of the electrostatic effect in the permeation process across the capsule. The role of the cell capsule in the solute diffusion was evaluated by determining the capsulated and decapsulated cell permeation times. The permeation times for all spin labels tested in the cells lacking capsules were always shorter than those containing this physical barrier. The decay times of spin labels group A observed for S. iversenii were compared to other studied algae. The results regarding the diffusion of charged spin labels group B suggested that the interaction of cell capsule occurs more strongly with negatively charged molecules than with positively charged ones. The results obtained in this work with spin labels group A confirm that the capsule is an essential structure for the cell, and that due to the polar interactions with the spin labels, it plays an important role in the selection of small molecules. Several parameters, mainly those of electrostatic nature, seem to control the permeation across the algal capsules of spin labels group B, showing that structures which are similar to amino acids could diffuse across the interior of the algal cell. (author)

  16. Temporal Gene Expression Analysis and RNA Silencing of Single and Multiple Members of Gene Family in the Lone Star Tick Amblyomma americanum

    Science.gov (United States)

    Karim, Shahid

    2016-01-01

    Saliva is an integral factor in the feeding success of veterinary and medically important ticks. Therefore, the characterization of the proteins present in tick saliva is an important area of tick research. Here, we confirmed previously generated sialotranscriptome data using quantitative real-time PCR. The information obtained in this in-depth study of gene expression was used to measure the effects of metalloprotease gene silencing on tick feeding. We analyzed the temporal expression of seven housekeeping genes and 44 differentially expressed salivary molecules selected from a previously published Amblyomma americanum sialotranscriptome. Separate reference genes were selected for the salivary glands and midgut from among the seven housekeeping genes, to normalize the transcriptional expression of differentially expressed genes. The salivary gland reference gene, ubiquitin, was used to normalize the expression of 44 salivary genes. Unsurprisingly, each gene family was expressed throughout the blood meal, but the expression of specific genes differed at each time point. To further clarify the complex nature of the many proteins found in the saliva, we disrupted the translation of several members of the metalloprotease family. Intriguingly, the nucleotide sequence similarity of the reprolysin metalloprotease gene family is so homologous that a single synthesized dsRNA sequence knocked down multiple members of the family. The use of multigene knockdown yielded a more significant picture of the role of metalloproteases in tick feeding success, and changes were observed in the female engorgement weight and larval hatching success. Interestingly, the depletion of metalloprotease transcripts also reduced the total number of bacteria present in the salivary glands. These data provide insight into the expression and functions of tick salivary proteins expressed while feeding on its host. PMID:26872360

  17. Kinetic modelling of enzyme inactivation Kinetics of heat inactivation of the extracellular proteinase from Pseudomonas fluorescens 22F.

    NARCIS (Netherlands)

    Schokker, E.P.

    1997-01-01

    The kinetics of heat inactivation of the extracellular proteinase from Pseudomonas fluorescens 22F was studied. It was established, by making use of kinetic modelling, that heat inactivation in the temperature range 35 - 70 °C was most likely caused by intermolecular autoproteolysis, where unfolded

  18. The epidermal growth factor precursor in the rat kidney seems to be processed by an aprotinin sensitive proteinase

    DEFF Research Database (Denmark)

    Nexø, Ebba; Poulsen, Steen Seier; Raaberg, Lasse

    1992-01-01

    Epidermal growth factor (EGF) is synthesized as a membrane bound precursor in the rat kidney. The precursor seems to be processed by an aprotinin sensitive proteinase. Intravenous infusion of aprotinin reduces the urinary excretion of EGF by 85% and increases the amount of renal EGF. Kidney...

  19. Molecular cloning and functional characterisation of a cathepsin L-like proteinases from the fish kinetoplastid parasite Trypanosoma carassii

    NARCIS (Netherlands)

    Ruszczyk, A.; Forlenza, M.; Savelkoul, H.F.J.; Wiegertjes, G.F.

    2008-01-01

    Trypanosoma carassii is a fish kinetoplastid parasite that belongs to the family Trypanosomatida. In the present study we cloned a cathepsin L-like proteinase from T. carassii. The nucleotide sequence of 1371 bp translated into a preproprotein of 456 amino acids. The preproprotein contained the oxya

  20. DNase I and proteinase K impair Listeria monocytogenes biofilm formation and induce dispersal of pre-existing biofilms.

    Science.gov (United States)

    Nguyen, Uyen T; Burrows, Lori L

    2014-09-18

    Current sanitation methods in the food industry are not always sufficient for prevention or dispersal of Listeria monocytogenes biofilms. Here, we determined if prevention of adherence or dispersal of existing biofilms could occur if biofilm matrix components were disrupted enzymatically. Addition of DNase during biofilm formation reduced attachment (biofilms with 100μg/ml of DNase for 24h induced incomplete biofilm dispersal, with biofilm remaining compared to control. In contrast, addition of proteinase K completely inhibited biofilm formation, and 72h biofilms-including those grown under stimulatory conditions-were completely dispersed with 100μg/ml proteinase K. Generally-regarded-as-safe proteases bromelain and papain were less effective dispersants than proteinase K. In a time course assay, complete dispersal of L. monocytogenes biofilms from both polystyrene and type 304H food-grade stainless steel occurred within 5min at proteinase K concentrations above 25μg/ml. These data confirm that both DNA and proteins are required for L. monocytogenes biofilm development and maintenance, and that these components of the biofilm matrix can be targeted for effective prevention and removal of biofilms.

  1. A distinct proteinase K resistant prion protein fragment in goats with no signs of disease in a classical scrapie outbreak

    NARCIS (Netherlands)

    Bouzalas, I.; Lörtscher, F.; Dovas, C.; Oevermann, A.; Langeveld, J.P.M.; Papanastassopoulou, M.; Papadopoulos, O.; Zurbriggen, A.; Seuberlich, T.

    2011-01-01

    Considerable efforts have been directed toward the identification of small-ruminant prion diseases, i.e., classical and atypical scrapie as well as bovine spongiform encephalopathy (BSE). Here we report the in-depth molecular analysis of the proteinase K-resistant prion protein core fragment (PrPres

  2. Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1.

    Science.gov (United States)

    Sakaguchi, Masayoshi; Osaku, Kanae; Maejima, Susumu; Ohno, Nao; Sugahara, Yasusato; Oyama, Fumitaka; Kawakita, Masao

    2014-01-01

    The proteinase K subfamily enzymes, thermophilic Aqualysin I (AQN) from Thermus aquaticus YT-1 and psychrophilic serine protease (VPR) from Vibrio sp. PA-44, have six and seven salt bridges, respectively. To understand the possible significance of salt bridges in the thermal stability of AQN, we prepared mutant proteins in which amino acid residues participating in salt bridges common to proteinase K subfamily members and intrinsic to AQN were replaced to disrupt the bridges one at a time. Disruption of a salt bridge common to proteinase K subfamily enzymes in the D183N mutant resulted in a significant reduction in thermal stability, and a massive change in the content of the secondary structure was observed, even at 70°C, in the circular dichroism (CD) analysis. These results indicate that the common salt bridge Asp183-Arg12 is important in maintaining the conformation of proteinase K subfamily enzymes and suggest the importance of proximity between the regions around Asp183 and the N-terminal region around Arg12. Of the three mutants that lack an AQN intrinsic salt bridge, D212N was more prone to unfolding at 80°C than the wild-type enzyme. Similarly, D17N and E237Q were less thermostable than the wild-type enzyme, although this may be partially due to increased autolysis. The AQN intrinsic salt bridges appear to confer additional thermal stability to this enzyme. These findings will further our understanding of the factors involved in stabilizing protein structure.

  3. Classification of Lactococcus lactis cell envelope proteinase based on gene sequencing, peptides formed after hydrolysis of milk, and computer modeling

    DEFF Research Database (Denmark)

    Børsting, Mette Winther; Qvist, K.B.; Brockmann, E.;

    2015-01-01

    Lactococcus lactis strains depend on a proteolytic system for growth in milk to release essential AA from casein. The cleavage specificities of the cell envelope proteinase (CEP) can vary between strains and environments and whether the enzyme is released or bound to the cell wall. Thirty-eight Lc...

  4. Divalent metals stabilize cellular prion proteins and alter the rate of proteinase-K dependent limited proteolysis

    Science.gov (United States)

    Background: The key biochemical event in the pathogenesis of prion diseases is the conversion of normal cellular prion proteins (PrP**c) to the proteinase K (PK) resistant, abnormal form (PrP**sc); however, the cellular mechanisms underlying the conversion remain enigmatic. Binding of divalent ca...

  5. Lipases and proteinases in milk : occurrence, heat inactivation, and their importance for the keeping quality of milk products

    NARCIS (Netherlands)

    Driessen, F.M.

    1983-01-01

    The occurrence and heat inactivation of native and bacterial lipases and proteinases in milk were studied.Production of these enzymes by Gram-negative psychrotrophic bacteria in milk was found to take place towards the end of exponential growth and in the stationary growth phase.Kinetics of heat ina

  6. Processing of predicted substrates of fungal Kex2 proteinases from Candida albicans, C. glabrata, Saccharomyces cerevisiae and Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Bader Oliver

    2008-07-01

    Full Text Available Abstract Background Kexin-like proteinases are a subfamily of the subtilisin-like serine proteinases with multiple regulatory functions in eukaryotes. In the yeast Saccharomyces cerevisiae the Kex2 protein is biochemically well investigated, however, with the exception of a few well known proteins such as the α-pheromone precursors, killer toxin precursors and aspartic proteinase propeptides, very few substrates are known. Fungal kex2 deletion mutants display pleiotropic phenotypes that are thought to result from the failure to proteolytically activate such substrates. Results In this study we have aimed at providing an improved assembly of Kex2 target proteins to explain the phenotypes observed in fungal kex2 deletion mutants by in vitro digestion of recombinant substrates from Candida albicans and C. glabrata. We identified CaEce1, CA0365, one member of the Pry protein family and CaOps4-homolog proteins as novel Kex2 substrates. Conclusion Statistical analysis of the cleavage sites revealed extended subsite recognition of negatively charged residues in the P1', P2' and P4' positions, which is also reflected in construction of the respective binding pockets in the ScKex2 enzyme. Additionally, we provide evidence for the existence of structural constrains in potential substrates prohibiting proteolysis. Furthermore, by using purified Kex2 proteinases from S. cerevisiae, P. pastoris, C. albicans and C. glabrata, we show that while the substrate specificity is generally conserved between organisms, the proteinases are still distinct from each other and are likely to have additional unique substrate recognition.

  7. Specificity of the collagenolytic serine proteinase from the pancreas of the catfish (Parasilurus asotus).

    Science.gov (United States)

    Yoshinaka, R; Sato, M; Yamashita, M; Itoko, M; Ikeda, S

    1987-01-01

    The collagenolytic serine proteinase from the pancreas of the catfish (Parasilus asotus) had a pH optimum of 7.5 for native, reconstituted calf skin collagen fibrils. The enzyme was most stable at pH 6-9. The enzyme hydrolyzed heat-denatured collagen (gelatin), casein, hemoglobin and elastin in addition to native collagen but not virtually Tos-Arg-OEe, Bz-Tyr-OEe and Suc-(Ala)3-NA. The enzyme cleaved Leu-Gly (or Gln-Gly), Gly-Ile and Ile-Ala bonds on DNP-Pro-Leu-Gly-Ile-Ala-Gly-Arg-NH2 and DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg. PMID:3480788

  8. Trichomonas vaginalis Cysteine Proteinases: Iron Response in Gene Expression and Proteolytic Activity.

    Science.gov (United States)

    Arroyo, Rossana; Cárdenas-Guerra, Rosa Elena; Figueroa-Angulo, Elisa Elvira; Puente-Rivera, Jonathan; Zamudio-Prieto, Olga; Ortega-López, Jaime

    2015-01-01

    We focus on the iron response of Trichomonas vaginalis to gene family products such as the cysteine proteinases (CPs) involved in virulence properties. In particular, we examined the effect of iron on the gene expression regulation and function of cathepsin L-like and asparaginyl endopeptidase-like CPs as virulence factors. We addressed some important aspects about CPs genomic organization and we offer possible explanations to the fact that only few members of this large gene family are expressed at the RNA and protein levels and the way to control their proteolytic activity. We also summarized all known iron regulations of CPs at transcriptional, posttranscriptional, and posttranslational levels along with new insights into the possible epigenetic and miRNA processes.

  9. The effect of proteinases (keratinases) in the pathogenesis of Dermatophyte infection using scanning electron microscope

    International Nuclear Information System (INIS)

    Objective: To study the inter-relationship between the stratum corneum of host and the fungal micro-organisms using scanning electron microscopy for a complete understanding of the host parasite relationship. Material and Methods: Skin surface biopsies were obtained two patients suffering from tinea cruris infection. One patient was infected with trichophyton rubrum and the other with epidermophytom floccosum strains. Results: The scanning electron microphotographs obtained from two patients showed a large number of villi in the infected area. The fungal hyphae were seen to placed intercellularly as well seem to be traversing through the corneocytes in many places. Conclusion: From the results observed in this study it could be suggested that the secretion of proteinases from the fungal hyphae together with the mechanical force of the invading organisms in vivo might be playing part in the invasion of the organisms. (author)

  10. Expression of Candida Albicans Secreted Aspartyl Proteinase in Acute Vaginal Candidiasis

    Institute of Scientific and Technical Information of China (English)

    LIN Nengxing; FENG Jing; TU Yating; FENG Aiping

    2007-01-01

    In order to analyze the in vivo expression of Candida albicans secreted aspartyl proteinases (SAP) in human vaginal infection, the vaginal secretion from 29 human subjects was collected by vaginal swab, and the expression of SAP1-SAP6 was detected by reverse-transcriptase polymerase chain reaction using specific primer sets. It was found that Sap2 and Sap5 were the most common genes expressed during infection; Sap3 and Sap4 were detected in all subjects and all 6 SAP genes were simultaneously expressed in some patients with vaginal candidiasis. It was suggested that the SAP family is expressed by Candida albicans during infection in human and that Candida albicans infection is associated with the differential expression of individual SAP genes which may be involved in the pathogenesis of vaginal candidiasis.

  11. Carcass characteristics, the calpain proteinase system, and aged tenderness of Angus and Brahman crossbred steers.

    Science.gov (United States)

    Pringle, T D; Williams, S E; Lamb, B S; Johnson, D D; West, R L

    1997-11-01

    We used 69 steers of varying percentage Brahman (B) breeding (0% B, n = 11; 25% B, n = 13; 37% B, n = 10; 50% B, n = 12; 75% B, n = 12; 100% B, n = 11) to study the relationship between carcass traits, the calpain proteinase system, and aged meat tenderness in intermediate B crosses. Calpains and calpastatin activities were determined on fresh longissimus muscle samples using anion-exchange chromatography. The USDA yield and quality grade data (24 h) were collected for each carcass. Longissimus steaks were removed and aged for 5 or 14 d for determination of shear force and 5 d for sensory panel evaluation. Even though some yield grade factors were affected by the percentage of B breeding, USDA yield grades did not differ (P > .15) between breed types. Marbling score and USDA quality grade decreased linearly (P Brahman crosses. PMID:9374310

  12. Carcass characteristics, the calpain proteinase system, and aged tenderness of Angus and Brahman crossbred steers.

    Science.gov (United States)

    Pringle, T D; Williams, S E; Lamb, B S; Johnson, D D; West, R L

    1997-11-01

    We used 69 steers of varying percentage Brahman (B) breeding (0% B, n = 11; 25% B, n = 13; 37% B, n = 10; 50% B, n = 12; 75% B, n = 12; 100% B, n = 11) to study the relationship between carcass traits, the calpain proteinase system, and aged meat tenderness in intermediate B crosses. Calpains and calpastatin activities were determined on fresh longissimus muscle samples using anion-exchange chromatography. The USDA yield and quality grade data (24 h) were collected for each carcass. Longissimus steaks were removed and aged for 5 or 14 d for determination of shear force and 5 d for sensory panel evaluation. Even though some yield grade factors were affected by the percentage of B breeding, USDA yield grades did not differ (P > .15) between breed types. Marbling score and USDA quality grade decreased linearly (P Brahman crosses.

  13. Quantification of the degree of biotinylation of proteins using proteinase K digestion and competition ELISA.

    Science.gov (United States)

    Rispens, Theo; Ooijevaar-de Heer, Pleuni

    2016-03-01

    Quantification of the degree of biotinylation of proteins is useful to achieve and maintain a high degree of consistency of reagents used in research and diagnostic setting. Unfortunately, existing protocols and commercial kits suffer from a number of shortcomings that limit their usefulness. Here, we describe a simple protocol that overcomes the limitations of current assays. A robust competition ELISA was developed that is easy to carry out, uses no specialized equipment other than a standard plate reader for absorbance measurements and only reagents that are commonly available. The protocol uses a proteinase K digestion step of a sample of biotinylated protein to eliminate multivalency issues and sterical hindrance from bulky proteins. Furthermore, the use of an anti-biotin antibody instead of streptavidin results in a convenient range of sensitivity, avoiding million-fold dilutions that may impair precision. The resulting assay typically consumes about 1 μg of biotinylated protein.

  14. Antioxidant activity of bovine casein hydrolysates produced by Ficus carica L.-derived proteinase.

    Science.gov (United States)

    Di Pierro, Giovanna; O'Keeffe, Martina B; Poyarkov, Alexey; Lomolino, Giovanna; FitzGerald, Richard J

    2014-08-01

    A Ficus carica L. latex proteinase preparation was investigated for its ability to produce antioxidant hydrolysates/peptides from bovine casein (CN). The Oxygen Radical Absorbance Capacity (ORAC) values for NaCN and β-CN hydrolysates ranged from 0.06 to 0.18, and from 0.51 to 1.19μmol Trolox equivalents/mg freeze-dried sample, respectively. Gel permeation HPLC showed that the β-CN hydrolysate with a degree of hydrolysis of 21% had 65% of peptide material with a molecular mass <500Da. The RP-UPLC profiles also indicated that β-CN was substantially hydrolysed during the early stages of hydrolysis. Analysis of the 4h β-CN hydrolysate by LC-ESI-MS/MS allowed identification of 8 peptide sequences with potential antioxidant properties.

  15. Trypsin inhibitor from tamarindus indica L. seeds reduces weight gain and food consumption and increases plasmatic cholecystokinin levels

    Directory of Open Access Journals (Sweden)

    Joycellane Alline do Nascimento Campos Ribeiro

    2015-02-01

    Full Text Available OBJECTIVES: Seeds are excellent sources of proteinase inhibitors, some of which may have satietogenic and slimming actions. We evaluated the effect of a trypsin inhibitor from Tamarindus indica L. seeds on weight gain, food consumption and cholecystokinin levels in Wistar rats. METHODS: A trypsin inhibitor from Tamarindus was isolated using ammonium sulfate (30-60% following precipitation with acetone and was further isolated with Trypsin-Sepharose affinity chromatography. Analyses were conducted to assess the in vivo digestibility, food intake, body weight evolution and cholecystokinin levels in Wistar rats. Histological analyses of organs and biochemical analyses of sera were performed. RESULTS: The trypsin inhibitor from Tamarindus reduced food consumption, thereby reducing weight gain. The in vivo true digestibility was not significantly different between the control and Tamarindus trypsin inhibitor-treated groups. The trypsin inhibitor from Tamarindus did not cause alterations in biochemical parameters or liver, stomach, intestine or pancreas histology. Rats treated with the trypsin inhibitor showed significantly elevated cholecystokinin levels compared with animals receiving casein or water. CONCLUSION: The results indicate that the isolated trypsin inhibitor from Tamarindus reduces weight gain by reducing food consumption, an effect that may be mediated by increased cholecystokinin. Thus, the potential use of this trypsin inhibitor in obesity prevention and/or treatment should be evaluated.

  16. Role of Candida albicans-Secreted Aspartyl Proteinases (Saps in Severe Early Childhood Caries

    Directory of Open Access Journals (Sweden)

    Wenqing Li

    2014-06-01

    Full Text Available Candida albicans is strongly associated with severe early childhood caries (S-ECC. However, the roles of secreted aspartyl proteinases (Saps, an important virulence factor of C. albicans, in the progress of S-ECC are not clear. In our study, the Saps activities were evaluated by the yeast nitrogen base–bovine serum albumi (YNB–BSA agar plate method and by the MTT method with bovine serum albumin (BSA as the substrate. Genotypes of C. albicans and gene expression of Sap1–5 were evaluated. The relationships of Saps activities and genotypes with S-ECC were analyzed. The results showed that enzyme activities of Saps in the S-ECC group were significantly higher than those in the caries free (CF group (p < 0.05. Genotypes A, B and C were detected in the S-ECC group, and genotypes A and C were detected in the CF group. In the genotype A group, Saps activity in the S-ECC group was significantly different from that in the CF group (p < 0.05. The gene expression level of Sap1 in the S-ECC group was significantly higher than that in the CF group (p = 0.001, while Sap4 expression was significantly lower than that in the CF group (p = 0.029. It can be concluded that Sap1–5 are the predominant proteinase genes expressed in C. albicans from dental biofilm and Sap1 may play an important role in the development of S-ECC.

  17. Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies.

    Science.gov (United States)

    Nada, R; Kumar, A; Kumar, V G; Gupta, K L; Joshi, K

    2016-01-01

    Renal biopsy interpretation requires histopathology, direct immunofluorescence (DIF) and electron microscopy. Formalin-fixed, paraffin-embedded tissue (FFPE) sent for light microscopy can be used for DIF after antigen retrieval. However, complement staining has not been satisfactory. We standardized DIF using proteinase-K for antigen retrieval in FFPE renal biopsies. A pilot study was conducted on known cases of membranous glomerulonephritis (MGN), membranoproliferative type-1 (MPGN-1), immunoglobulin A nephropathy (IgAN), and anti-glomerular basement disease (anti-GBM). Immunofluorescence panel included fluorescein isothiocyanate (FITC) conjugated IgG, IgA, IgM, complements (C3 and C1q), light chains (kappa, lambda) and fibrinogen antibodies. After standardization of the technique, 75 renal biopsies and 43 autopsies cases were stained. Out of 43 autopsy cases, immune-complex mediated glomerulonephritis (GN) was confirmed in 18 cases (Lupus nephritis-11, IgAN-6, MGN-1), complement-mediated dense deposit disease (DDD-1) and monoclonal diseases in 4 cases (amyloidosis-3, cast nephropathy-1). Immune-mediated injury was excluded in 17 cases (focal segmental glomerulosclerosis -3, crescentic GN-6 [pauci-immune-3, anti-GBM-3], thrombotic microangiopathy-5, atherosclerosis-3). Renal biopsies (n-75) where inadequate or no frozen sample was available; this technique classified 52 mesangiocapillary pattern as MPGN type-1-46, DDD-2 and (C3GN-4). Others were diagnosed as IgAN-3, lupus nephritis-2, MGN-4, diffuse proliferative glomerulonephritis (DPGN)-1, Non-IC crescentic GN-1, monoclonal diseases-3. In nine cases, DIF on FFPE tissue could not help in making diagnosis. Proteinase-K enzymatic digestion of FFPE renal biopsies can unmask complements (both C3 and C1q) in immune-complexes mediated and complement-mediated diseases. This method showed good results on autopsy tissues archived for as long as 15 years.

  18. Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies.

    Science.gov (United States)

    Nada, R; Kumar, A; Kumar, V G; Gupta, K L; Joshi, K

    2016-01-01

    Renal biopsy interpretation requires histopathology, direct immunofluorescence (DIF) and electron microscopy. Formalin-fixed, paraffin-embedded tissue (FFPE) sent for light microscopy can be used for DIF after antigen retrieval. However, complement staining has not been satisfactory. We standardized DIF using proteinase-K for antigen retrieval in FFPE renal biopsies. A pilot study was conducted on known cases of membranous glomerulonephritis (MGN), membranoproliferative type-1 (MPGN-1), immunoglobulin A nephropathy (IgAN), and anti-glomerular basement disease (anti-GBM). Immunofluorescence panel included fluorescein isothiocyanate (FITC) conjugated IgG, IgA, IgM, complements (C3 and C1q), light chains (kappa, lambda) and fibrinogen antibodies. After standardization of the technique, 75 renal biopsies and 43 autopsies cases were stained. Out of 43 autopsy cases, immune-complex mediated glomerulonephritis (GN) was confirmed in 18 cases (Lupus nephritis-11, IgAN-6, MGN-1), complement-mediated dense deposit disease (DDD-1) and monoclonal diseases in 4 cases (amyloidosis-3, cast nephropathy-1). Immune-mediated injury was excluded in 17 cases (focal segmental glomerulosclerosis -3, crescentic GN-6 [pauci-immune-3, anti-GBM-3], thrombotic microangiopathy-5, atherosclerosis-3). Renal biopsies (n-75) where inadequate or no frozen sample was available; this technique classified 52 mesangiocapillary pattern as MPGN type-1-46, DDD-2 and (C3GN-4). Others were diagnosed as IgAN-3, lupus nephritis-2, MGN-4, diffuse proliferative glomerulonephritis (DPGN)-1, Non-IC crescentic GN-1, monoclonal diseases-3. In nine cases, DIF on FFPE tissue could not help in making diagnosis. Proteinase-K enzymatic digestion of FFPE renal biopsies can unmask complements (both C3 and C1q) in immune-complexes mediated and complement-mediated diseases. This method showed good results on autopsy tissues archived for as long as 15 years. PMID:27194832

  19. A masquerade-like serine proteinase homologue is necessary for phenoloxidase activity in the coleopteran insect, Holotrichia diomphalia larvae.

    Science.gov (United States)

    Kwon, T H; Kim, M S; Choi, H W; Joo, C H; Cho, M Y; Lee, B L

    2000-10-01

    Previously, we reported the molecular cloning of cDNA for the prophenoloxidase activating factor-I (PPAF-I) that encoded a member of the serine proteinase group with a disulfide-knotted motif at the N-terminus and a trypsin-like catalytic domain at the C-terminus [Lee, S.Y., Cho, M.Y., Hyun, J.H., Lee, K.M., Homma, K.I., Natori, S. , Kawabata, S.I., Iwanaga, S. & Lee, B.L. (1998) Eur. J. Biochem. 257, 615-621]. PPAF-I is directly involved in the activation of pro-phenoloxidase (pro-PO) by limited proteolysis and the overall structure is highly similar to that of Drosophila easter serine protease, an essential serine protease zymogen for pattern formation in normal embryonic development. Here, we report purification and molecular cloning of cDNA for another 45-kDa novel PPAF from the hemocyte lysate of Holotrichia diomphalia larvae. The gene encodes a serine proteinase homologue consisting of 415 amino-acid residues with a molecular mass of 45 256 Da. The overall structure of the 45-kDa protein is similar to that of masquerade, a serine proteinase homologue expressed during embryogenesis, larval, and pupal development in Drosophila melanogaster. The 45-kDa protein contained a trypsin-like serine proteinase domain at the C-terminus, except for the substitution of Ser of the active site triad to Gly and had a disulfide-knotted domain at the N-terminus. A highly similar 45-kDa serine proteinase homologue was also cloned from the larval cDNA library of another coleopteran, Tenebrio molitor. By in vitro reconstitution experiments, we found that the purified 45-kDa serine proteinase homologue, the purified active PPAF-I and the purified pro-PO were necessary for expressing phenoloxidase activity in the Holotrichia pro-PO system. However, incubation of pro-PO with either PPAF-I or 45-kDa protein, no phenoloxidase activity was observed. Interestingly, when the 45-kDa protein was incubated with PPAF-I and pro-PO in the absence, but not in the presence of Ca2+, the 45-k

  20. Proton pump inhibitors

    Science.gov (United States)

    Proton pump inhibitors (PPIs) are medicines that work by reducing the amount of stomach acid made by ... Proton pump inhibitors are used to: Relieve symptoms of acid reflux, or gastroesophageal reflux disease (GERD). This ...

  1. Tissue factor pathway inhibitor-2 may interact with nuclear protein RASSF1C

    Institute of Scientific and Technical Information of China (English)

    Xudong Chen; Zhenwu Li; Jin Zhang; Zuohua Mao; Duan Ma; Huijun Wang

    2012-01-01

    Tissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa matrix-associated Kunitz-type serine proteinase inhibitor consisting of a short amino-terminal region,three tandem Kunitz-type domains,and a positively charged carboxyterminal tail.Human TFPI-2 (hTFPI-2) inhibits a broad spectrum of serine proteinases (including trypsin,plasmin,plasma kallikrein,XIa,and chymotrypsin) almost exclusively via its first Kunitz-type domain,and potentially plays an important role in the regulation of extracellular matrix digestion and remodeling [1].Reduced TFPI-2 synthesis has been related to numerous pathophysiological processes such as inflammation,angiogenesis,atherosclerosis [2,3],retinal degeneration,and tumor growth/metastasis [4-6].It has been suggested that TFPI-2 is a tumor suppressor gene in some cancers [7,8].However,the specific physiological functions of hTFPI-2 in humans are unclear,particularly its interactions with other proteins.To better understand the physiological function of hTFPI-2,we used yeast two-hybrid system screening and bioinformatics analysis to identify its interacting proteins and confirm its interactions with nuclear protein RASSF1C using confocal microscopy and co-immunoprecipitation.

  2. DNase I and proteinase K impair Listeria monocytogenes biofilm formation and induce dispersal of pre-existing biofilms.

    Science.gov (United States)

    Nguyen, Uyen T; Burrows, Lori L

    2014-09-18

    Current sanitation methods in the food industry are not always sufficient for prevention or dispersal of Listeria monocytogenes biofilms. Here, we determined if prevention of adherence or dispersal of existing biofilms could occur if biofilm matrix components were disrupted enzymatically. Addition of DNase during biofilm formation reduced attachment (bromelain and papain were less effective dispersants than proteinase K. In a time course assay, complete dispersal of L. monocytogenes biofilms from both polystyrene and type 304H food-grade stainless steel occurred within 5min at proteinase K concentrations above 25μg/ml. These data confirm that both DNA and proteins are required for L. monocytogenes biofilm development and maintenance, and that these components of the biofilm matrix can be targeted for effective prevention and removal of biofilms. PMID:25043896

  3. Proteinases from buckwheat (Fagopyrum esculentum moench seeds: Purification and properties of the 47 kDa enzyme

    Directory of Open Access Journals (Sweden)

    Timotijević Gordana S.

    2006-01-01

    Full Text Available Aspartic proteinases from buckwheat seeds are analyzed. Three forms of 47 kDa, 40 kDa and 28 kDa, were purified from mature buckwheat seeds, while two forms of 47 kDa and 28 kDa were detected in developing buckwheat seeds using pepstatin A affinity chromatography. A form of 47 kDa was selectively precipitated from other forms by ammonium sulfate precipitation. This enzyme resembles the chymosin-like pattern of proteolytic activity, as it was shown using BSA and k-casein as substrates, clarifying its ability for milk-clotting. The 47 kDa aspartic proteinase form is localized in the membrane fraction. .

  4. [Heterologous expression, purification, and properties of a chymotrypsin inhibitor isolated from potatoes].

    Science.gov (United States)

    2013-01-01

    The PKPIJ-B gene encoding a chymotrypsin inhibitor from a subfamily of potato Kunitz-type proteinase inhibitors (PKPI) in potatoes (Solanum tuberosum L. cv. Yubilei Zhukova) was cloned into a pET23a vector and then expressed in Escherichia coli. The recombinant PKPIJ-B protein obtained in the inclusion bodies was denatured, purified by high-performance liquid chromatography (HPLC) on Mono Q under denaturing conditions, and renaturated. The renaturated protein was additionally purified using HPLC on DEAE-ToyoPearl. The PKPIJ-B protein efficiently suppressed chymotrypsin activity, had a weaker effect on trypsin, and inhibited the growth and development of phytopathogenic microorganisms affecting potato plants. PMID:23662448

  5. Protective role of antimannan and anti-aspartyl proteinase antibodies in an experimental model of Candida albicans vaginitis in rats.

    OpenAIRE

    De Bernardis, F.; Boccanera, M; Adriani, D; Spreghini, E; G. Santoni; Cassone, A.

    1997-01-01

    The role of antibodies (Abs) in the resistance to vaginal infection by Candida albicans was investigated by using a rat vaginitis model. Animals receiving antimannoprotein (anti-MP) and anti-aspartyl proteinase (Sap) Ab-containing vaginal fluids from rats clearing a primary C. albicans infection showed a highly significant level of protection against vaginitis compared to animals given Ab-free vaginal fluid from noninfected rats. Preabsorption of the Ab-containing fluids with either one or bo...

  6. In vitro Candida albicans biofilm induced proteinase activity and SAP8 expression correlates with in vivo denture stomatitis severity.

    Science.gov (United States)

    Ramage, Gordon; Coco, Brent; Sherry, Leighann; Bagg, Jeremy; Lappin, David F

    2012-07-01

    Denture stomatitis is a common inflammatory disorder of the palatal mucosa amongst denture wearers. The pathological changes are induced by Candida albicans biofilm on the fitting surface of the upper denture, and different individuals experience different levels of disease. C. albicans is known to produce secreted aspartyl proteinases (SAPs) to aid adhesion, invasion and tissue destruction. We hypothesised that differential expression and activity of SAPs from denture stomatitis isolates results in different levels of disease amongst denture wearers. We selected C. albicans isolates from asymptomatic controls and three different severities of disease [Newton’s type (NT) 0, I, II and III]. We assessed biofilm formation and proteinase activity for each biofilm and investigated the transcriptional profile of SAPs 1, 2, 5, 6 and 8 from early (12 h) and mature (24 h) biofilms. There were no significant differences between isolates with respect to biofilm formation, whereas proteinase activity normalised to biofilm growth was significantly increased in the diseased groups (p < 0.0001). Proteinase activity correlated strongly with SAP expression (p < 0.0001). SAP8 expression was the greatest, followed by SAP5, 6, 2 and 1. The diseased groups showed the greatest levels of SAP expression, with significant differences also observed between the groups (p < 0.005). All SAPs except SAP5 were expressed in greater amounts in the mature biofilms compared to early biofilms. Overall, this study suggests that SAP activity in biofilms determined in vitro may help to explain differences in disease severity. SAP8 has been shown for the first time to play a prominent role in biofilms.

  7. Extensive expansion of A1 family aspartic proteinases in fungi revealed by evolutionary analyses of 107 complete eukaryotic proteomes

    OpenAIRE

    Revuelta, M.V.; Kan, van, J.; Kay, J; Have, ten, P.

    2014-01-01

    The A1 family of eukaryotic aspartic proteinases (APs) forms one of the 16 AP families. Although one of the best characterized families, the recent increase in genome sequence data has revealed many fungal AP homologs with novel sequence characteristics. This study was performed to explore the fungal AP sequence space and to obtain an in-depth understanding of fungal AP evolution. Using a comprehensive phylogeny of approximately 700 AP sequences from the complete proteomes of 87 fungi and 20 ...

  8. prtH2, Not prtH, Is the Ubiquitous Cell Wall Proteinase Gene in Lactobacillus helveticus▿

    OpenAIRE

    Genay, M.; Sadat, L.; Gagnaire, V.; Lortal, S

    2009-01-01

    Lactobacillus helveticus strains possess an efficient proteolytic system that releases peptides which are essential for lactobacillus growth in various fermented dairy products and also affect textural properties or biological activities. Cell envelope proteinases (CEPs) are bacterial enzymes that hydrolyze milk proteins. In the case of L. helveticus, two CEPs with low percentages of amino acid identity have been described, i.e., PrtH and PrtH2. However, the distribution of the genes that enc...

  9. Effects of systemic flunixin meglumine, topical oxytetracycline, and topical prednisolone acetate on tear film proteinases innormal horses

    OpenAIRE

    Rainbow, Marc E

    2004-01-01

    The purpose of this study was to determine the effects of three medical treatments, topical oxytetracycline, topical prednisolone acetate, and systemic flunixin meglumine, on the activity of two proteinases, matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), in equine tear film. The study design consisted of twelve ophthalmically normal horses separated into three groups of four in a cross-over study design. Each group was treated for 5 days with flunixin meglumine (...

  10. Proteinase-activated receptors 1 and 4 counter-regulate endostatin and VEGF release from human platelets

    OpenAIRE

    Ma, Li; Perini, Rafael; McKnight, Webb; Dicay, Michael; Klein, Andre; Hollenberg, Morley D.; Wallace, John L

    2004-01-01

    The roles of proteinase-activated receptors (PARs) in platelet functions other than aggregation are not well understood. Among these is the release of factors that regulate the process of angiogenesis, such as endostatin and VEGF, which, respectively, inhibit and promote angiogenesis. PAR1 and PAR4 are expressed on the surface of human platelets and can be activated by thrombin. In the present study, we have attempted to determine the roles of PAR1 and PAR4 in regulating release of endostatin...

  11. Phospholipase, proteinase and haemolytic activities of Candida albicans isolated from oral cavities of patients with type 2 diabetes mellitus

    OpenAIRE

    Tsang, CSP; Chu, FCS; Leung, WK; Jin, LJ; Samaranayake, LP; Siu, SC

    2007-01-01

    The aim of this study was to biotype and characterize phospholipase, proteinase and haemolytic activities of oral Candida albicans isolates from 210 Chinese patients with type 2 diabetes mellitus (DM) and 210 age- and sex-matched healthy controls. Seventy-six and 50 C. albicans isolates were obtained from type 2 DM patients and controls, respectively, using the oral rinse technique. The isolates were characterized with a biotyping system based on enzyme profiles, carbohydrate assimilation pat...

  12. Use of proteinase K in the excystation of Sarcocystis cruzi sporocysts for in vitro culture and DNA extraction.

    Science.gov (United States)

    Ndiritu, W; Cawthorn, R J; Kibenge, F S

    1994-03-01

    Proteinase K was used for the cleaning of Sarcocystis cruzi (Apicomplexa) sporocysts prior to excystation. Bovine pulmonary endothelial cell cultures inoculated with the excysted sporozoites remained free of bacterial contamination for the duration of the experiment and had high yields of merozoites. The excysted sporozoites also yielded genomic DNA that could be labelled efficiently with 32P dATP by the random priming method.

  13. The anthelmintic efficacy of plant-derived cysteine proteinases against the rodent gastrointestinal nematode, Heligmosomoides polygyrus, in vivo

    OpenAIRE

    Stepek, Gillian; Lowe, Ann; Buttle, David J.; Duce, I.R.; Behnke, Jerzy M.

    2007-01-01

    Gastrointestinal (GI) nematodes are important disease-causing organisms, controlled primarily through treatment with synthetic drugs, but the efficacy of these drugs has declined due to widespread resistance, and hence new drugs, with different modes of action, are required. Some medicinal plants, used traditionally for the treatment of worm infections, contain cysteine proteinases known to damage worms irreversibly in vitro. Here we (i) confirm that papaya latex has marked efficacy in vivo a...

  14. Modulation of enteroviral proteinase cleavage of poly(A)-binding protein (PABP) by conformation and PABP-associated factors

    OpenAIRE

    Rivera, Carlos I.; Lloyd, Richard E.

    2008-01-01

    Poliovirus (PV) causes a drastic inhibition of cellular cap-dependant protein synthesis due to the cleavage of translation factors eukaryotic initiation factor 4G (eIF4G) and poly (A) binding protein (PABP). Only about half of cellular PABP is cleaved by viral 2A and 3C proteinases during infection. We have investigated PABP cleavage determinants that regulate this partial cleavage. PABP cleavage kinetics analyses indicate that PABP exists in multiple conformations, some of which are resistan...

  15. The death enzyme CP14 is a unique papain-like cysteine proteinase with a pronounced S2 subsite selectivity.

    Science.gov (United States)

    Paireder, Melanie; Mehofer, Ulrich; Tholen, Stefan; Porodko, Andreas; Schähs, Philipp; Maresch, Daniel; Biniossek, Martin L; van der Hoorn, Renier A L; Lenarcic, Brigita; Novinec, Marko; Schilling, Oliver; Mach, Lukas

    2016-08-01

    The cysteine protease CP14 has been identified as a central component of a molecular module regulating programmed cell death in plant embryos. CP14 belongs to a distinct subfamily of papain-like cysteine proteinases of which no representative has been characterized thoroughly to date. However, it has been proposed that CP14 is a cathepsin H-like protease. We have now produced recombinant Nicotiana benthamiana CP14 (NbCP14) lacking the C-terminal granulin domain. As typical for papain-like cysteine proteinases, NbCP14 undergoes rapid autocatalytic activation when incubated at low pH. The mature protease is capable of hydrolysing several synthetic endopeptidase substrates, but cathepsin H-like aminopeptidase activity could not be detected. NbCP14 displays a strong preference for aliphatic over aromatic amino acids in the specificity-determining P2 position. This subsite selectivity was also observed upon digestion of proteome-derived peptide libraries. Notably, the specificity profile of NbCP14 differs from that of aleurain-like protease, the N. benthamiana orthologue of cathepsin H. We conclude that CP14 is a papain-like cysteine proteinase with unusual enzymatic properties which may prove of central importance for the execution of programmed cell death during plant development. PMID:27246477

  16. Resistance to Bacillus thuringiensis by the Indian meal moth, Plodia interpunctella: comparison of midgut proteinases from susceptible and resistant larvae.

    Science.gov (United States)

    Johnson, D E; Brookhart, G L; Kramer, K J; Barnett, B D; McGaughey, W H

    1990-03-01

    Midgut homogenates from susceptible and resistant strains of the Indian meal moth, Plodia interpunctella, were compared for their ability to activate the entomocidal parasporal crystal protein from Bacillus thuringiensis. The properties of midgut proteinases from both types of larvae were also examined. Electrophoretic patterns of crystal protein from B. thuringiensis subspecies kurstaki (HD-1) and aizawai (HD-133 and HD-144) were virtually unchanged following digestion by either type of midgut homogenate. Changes in pH (9.5 to 11.5) or midgut homogenate concentration during digestion failed to substantially alter protein electrophoretic patterns of B. thuringiensis HD-1 crystal toxin. In vitro toxicity of crystal protein activated by either type of midgut preparation was equal toward cultured insect cells from either Manduca sexta or Choristoneura fumiferana. Electrophoresis of midgut extracts in polyacrylamide gels containing gelatin as substrate also yielded matching mobility patterns of proteinases from both types of midguts. Quantitation of midgut proteolytic activity using tritiated casein as a substrate revealed variation between midgut preparations, but no statistically significant differences between proteolytic activities from susceptible and resistant Indian meal moth larvae. Inhibition studies indicated that a trypsin-like proteinase with maximal activity at pH 10 is a major constituent of Indian meal moth midguts. The results demonstrated that midguts from susceptible and resistant strains of P. interpunctella are similar both in their ability to activate B. thuringiensis protoxin and in their proteolytic activity.

  17. Isolation of a putative receptor for KDEL-tailed cysteine proteinase (SH-EP) from cotyledons of Vigna mungo seedlings.

    Science.gov (United States)

    Tsuru-Furuno, A; Okamoto, T; Minamikawa, T

    2001-10-01

    SH-EP is the major papain-type proteinase expressed in cotyledons of germinated Vigna mungo seeds. The proteinase possesses a KDEL sequence at the C-terminus although the mature form of SH-EP is localized in vacuoles. It has also been shown that the proform of SH-EP is accumulated at the edge or middle region of the endoplasmic reticulum, and the accumulated proSH-EP is directly transported to vacuoles via the KDEL-tailed cysteine proteinase-accumulating vesicle, KV. In this study, to address the transport machinery of proSH-EP through KV, putative receptor for proSH-EP was isolated from membrane proteins of cotyledons of V. mungo seedlings using a proSH-EP-immobilized column. The deduced amino acid sequence from cDNA to the protein revealed that the putative receptor for proSH-EP is a member of vacuolar sorting receptor, VSR, that is known to be localized in the Golgi-complex and/or clathrin coated vesicle. We carried out subcellular fractionation of cotyledon cells and subsequently conducted SDS-PAGE/immunoblotting and immunocytochemistry with anti-V. mungo VSR (VmVSR) or SH-EP antibody. The results showed that VmVSR is co-localized in the fraction of the gradient in which KV existed.

  18. A thermolabile aspartic proteinase from Mucor mucedo DSM 809: gene identification, cloning, and functional expression in Pichia pastoris.

    Science.gov (United States)

    Yegin, Sirma; Fernandez-Lahore, Marcelo

    2013-06-01

    In this study, the cDNA encoding the aspartic proteinase of Mucor mucedo DSM 809 has been identified by RNA ligased-mediated and oligo-capping rapid amplification of cDNA ends (RACE) technique. The gene contained an open reading frame of 1,200 bp and encoded for a signal peptide of 21 amino acid residues. Two N-glycosylation sites were observed within the identified sequence. The proteinase gene was cloned into the vector pGAPZαA and expressed in Pichia pastoris X-33 for the first time. The protein has been secreted in functionally active form into the culture medium. The expression system does not require any acid activation process. The factors affecting the expression level were optimized in shaking flask cultures. Maximum enzyme production was observed with an initial medium pH of 3.5 at 20 °C and 220 rpm shaking speed utilizing 4 % glucose as a carbon and energy source. The enzyme was purified with cation exchange chromatography and further studies revealed that the enzyme was secreted in glycosylated form. The purified enzyme exhibited remarkable sensitivity to thermal treatment and became completely inactivated after incubation at 55 °C for 10 min. These results indicated that the recombinant proteinase could be considered as a potential rennet candidate for the cheese-making industry.

  19. Phospholipase, proteinase and haemolytic activities of Candida albicans isolated from oral cavities of patients with type 2 diabetes mellitus.

    Science.gov (United States)

    Tsang, C S P; Chu, F C S; Leung, W K; Jin, L J; Samaranayake, L P; Siu, S C

    2007-10-01

    The aim of this study was to biotype and characterize phospholipase, proteinase and haemolytic activities of oral Candida albicans isolates from 210 Chinese patients with type 2 diabetes mellitus (DM) and 210 age- and sex-matched healthy controls. Seventy-six and 50 C. albicans isolates were obtained from type 2 DM patients and controls, respectively, using the oral rinse technique. The isolates were characterized with a biotyping system based on enzyme profiles, carbohydrate assimilation patterns and boric acid resistance of the yeasts, and the isolates were further tested for in vitro phospholipase, proteinase and haemolytic activities. The major biotypes of C. albicans isolates from the type 2 DM and control groups were A1R (42.1 %) and J1R (36.0 %), respectively. Significantly higher proteinase and haemolytic activities were found in the isolates from the type 2 DM group (P or =10 years of DM history than those with DM patients than in those from male counterparts (PCandida isolates taken from DM patients.

  20. Characterization of proteinases from the midgut of Rhipicephalus (Boophilus microplus involved in the generation of antimicrobial peptides

    Directory of Open Access Journals (Sweden)

    Craik Charles S

    2010-07-01

    Full Text Available Abstract Background Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins. A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins. Results An aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1'. Conclusions BmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora.

  1. Impact of Mercury(II) on proteinase K catalytic center: investigations via classical and Born-Oppenheimer molecular dynamics.

    Science.gov (United States)

    Panek, Jarosław J; Mazzarello, Riccardo; Novič, Marjana; Jezierska-Mazzarello, Aneta

    2011-02-01

    Mercury(II) has a strong affinity for the thiol groups in proteins often resulting in the disruption of their biological functions. In this study we present classical and first-principles, DFT-based molecular dynamics (MD) simulations of a complex of Hg(II) and proteinase K, a well-known serine protease with a very broad and diverse enzymatic activity. It contains a catalytic triad formed by Asp39, His69, and Ser224, which is responsible for its biological activity. It was found previously by X-ray diffraction experiments that the presence of Hg(II) inhibits the enzymatic action of proteinase K by affecting the stereochemistry of the triad. Our simulations predict that (i) the overall structure as well as the protein backbone dynamics are only slightly affected by the mercury cation, (ii) depending on the occupied mercury site, the hydrogen bonds of the catalytic triad are either severely disrupted (both bonds for mercury at site 1, and the His69-Ser224 contact for mercury at site 2) or slightly strengthened (the Asp39-His69 bond when mercury is at site 2), (iii) the network of hydrogen bonds of the catalytic triad is not static but undergoes constant fluctuations, which are significantly modified by the presence of the Hg(II) cation, influencing in turn the triad's ability to carry out the enzymatic function--these facts explain the experimental findings on the inhibition of proteinase K by Hg(II).

  2. Functional properties of a cysteine proteinase from pineapple fruit with improved resistance to fungal pathogens in Arabidopsis thaliana.

    Science.gov (United States)

    Wang, Wei; Zhang, Lu; Guo, Ning; Zhang, Xiumei; Zhang, Chen; Sun, Guangming; Xie, Jianghui

    2014-01-01

    In plant cells, many cysteine proteinases (CPs) are synthesized as precursors in the endoplasmic reticulum, and then are subject to post-translational modifications to form the active mature proteinases. They participate in various cellular and physiological functions. Here, AcCP2, a CP from pineapple fruit (Ananas comosus L.) belonging to the C1A subfamily is analyzed based on the molecular modeling and homology alignment. Transcripts of AcCP2 can be detected in the different parts of fruits (particularly outer sarcocarps), and gradually increased during fruit development until maturity. To analyze the substrate specificity of AcCP2, the recombinant protein was overexpressed and purified from Pichia pastoris. The precursor of purified AcCP2 can be processed to a 25 kDa active form after acid treatment (pH 4.3). Its optimum proteolytic activity to Bz-Phe-Val-Arg-NH-Mec is at neutral pH. In addition, the overexpression of AcCP2 gene in Arabidopsis thaliana can improve the resistance to fungal pathogen of Botrytis cinerea. These data indicate that AcCP2 is a multifunctional proteinase, and its expression could cause fruit developmental characteristics of pineapple and resistance responses in transgenic Arabidopsis plants.

  3. Functional Properties of a Cysteine Proteinase from Pineapple Fruit with Improved Resistance to Fungal Pathogens in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Wei Wang

    2014-02-01

    Full Text Available In plant cells, many cysteine proteinases (CPs are synthesized as precursors in the endoplasmic reticulum, and then are subject to post-translational modifications to form the active mature proteinases. They participate in various cellular and physiological functions. Here, AcCP2, a CP from pineapple fruit (Ananas comosus L. belonging to the C1A subfamily is analyzed based on the molecular modeling and homology alignment. Transcripts of AcCP2 can be detected in the different parts of fruits (particularly outer sarcocarps, and gradually increased during fruit development until maturity. To analyze the substrate specificity of AcCP2, the recombinant protein was overexpressed and purified from Pichia pastoris. The precursor of purified AcCP2 can be processed to a 25 kDa active form after acid treatment (pH 4.3. Its optimum proteolytic activity to Bz-Phe-Val-Arg-NH-Mec is at neutral pH. In addition, the overexpression of AcCP2 gene in Arabidopsis thaliana can improve the resistance to fungal pathogen of Botrytis cinerea. These data indicate that AcCP2 is a multifunctional proteinase, and its expression could cause fruit developmental characteristics of pineapple and resistance responses in transgenic Arabidopsis plants.

  4. Efeito da cobertura morta de milheto (Pennisetum americanum sobre a eficácia do herbicida metribuzin no controle de Ipomoea grandifolia e Sida rhombifolia Effect of Pennisetum americanum mulch in the metribuzin efficacy on the control of Ipomoea grandifolia and Sida rhombifolia

    Directory of Open Access Journals (Sweden)

    M.C. Godoy

    2007-03-01

    Full Text Available Com o objetivo de avaliar a eficácia do metribuzin (480 g i.a. ha-1 associado à palha de milheto no controle de Ipomoea grandifolia e Sida rhombifolia, foram realizados dois experimentos em casa de vegetação. No primeiro, os tratamentos constituíram-se de diferentes posicionamentos do herbicida, aplicado sobre e sob a palha em diferentes condições de umidade. No segundo, foram estudados diferentes períodos de permanência (0, 7, 14 e 21 dias do herbicida sobre a palha de milheto antes da ocorrência da primeira chuva. Após o preenchimento dos vasos com solo, as plantas daninhas (I. grandifolia e S. rhombifolia foram semeadas superficialmente e, em seguida, cobertas com palha de milheto (8 t ha-1. O delineamento experimental utilizado em ambos os experimentos foi o inteiramente casualizado, com quatro repetições. Foram realizadas avaliações visuais de controle (0 a 100%, contagem das plantas daninhas aos 7, 14, 21, 28 e 35 dias após a aplicação (DAA e biomassa seca ao final. Verificou-se controle excelente das duas espécies nos diferentes posicionamentos do herbicida, exceto para I. grandifolia na condição de aplicação em palha úmida, seguido de período seco. Observou-se, ainda, que o herbicida promoveu controle eficaz em pós-emergência e em pré-emergência, mesmo sem ocorrência de chuva após a aplicação. No segundo experimento, constatou-se controle excelente (>96% de I. grandifolia nos os períodos sem chuva de até 7 DAA; nos demais períodos, tal controle foi insatisfatório. Para S. rhombifolia, observou-se controle excelente para os períodos até 14 dias sem ocorrência de chuvas. Para o período de 28 dias, não se obteve controle satisfatório.Two experiments were carried out under greenhouse conditions to evaluate the efficacy of the metribuzin herbicide associated to Pennisetum americanum mulch in the control of the weeds Ipomoea grandifolia and Sida rhombifolia. The treatments in the first experiment were

  5. Stable and Simple Immobilization of Proteinase K Inside Glass Tubes and Microfluidic Channels.

    Science.gov (United States)

    Küchler, Andreas; Bleich, Julian N; Sebastian, Bernhard; Dittrich, Petra S; Walde, Peter

    2015-11-25

    Engyodontium album proteinase K (proK) is widely used for degrading proteinaceous impurities during the isolation of nucleic acids from biological samples, or in proteomics and prion research. Toward applications of proK in flow reactors, a simple method for the stable immobilization of proK inside glass micropipette tubes was developed. The immobilization of the enzyme was achieved by adsorption of a dendronized polymer-enzyme conjugate from aqueous solution. This conjugate was first synthesized from a polycationic dendronized polymer (denpol) and proK and consisted, on average, of 2000 denpol repeating units and 140 proK molecules, which were attached along the denpol chain via stable bis-aryl hydrazone bonds. Although the immobilization of proK inside the tube was based on nonspecific, noncovalent interactions only, the immobilized proK did not leak from the tube and remained active during prolonged storage at 4 °C and during continuous operation at 25 °C and pH = 7.0. The procedure developed was successfully applied for the immobilization of proK on a glass/PDMS (polydimethylsiloxane) microchip, which is a requirement for applications in the field of proK-based protein analysis with such type of microfluidic devices. PMID:26536248

  6. Original features of cell-envelope proteinases of Lactobacillus helveticus. A review.

    Science.gov (United States)

    Sadat-Mekmene, Leila; Genay, Magali; Atlan, Danièle; Lortal, Sylvie; Gagnaire, Valérie

    2011-03-15

    Lactobacillus helveticus is a lactic acid bacterium very used in fermented milks and cheese. The rapid growth of L. helveticus in milk is supported by an efficient cell envelope proteinase (CEP) activity, due to subtilisin-like serine proteases. These enzymes play also crucial roles in texture and flavor formation in dairy products as well as in generating in situ bioactive peptides. In L. helveticus, several genes encoding putative CEPs were detected and characterized by a large intraspecific diversity; little is known about regulation of expression of CEP-encoding genes. Anchored at the bacterial surface, CEPs are large-sized enzymes (> 150 kDa) hydrolyzing β- and α(s1)-casein as well. Substrate cleavages occur after almost all types of amino acids residues, but mass spectrometry analysis revealed L. helveticus strains with specific profiles of substrate hydrolysis, which could explain identification of strains associated with interesting technological properties. In this review, the most recent data regarding CEP-encoding genes, CEP activities toward caseins and L. helveticus strain diversity are discussed. PMID:21354644

  7. The relative anthelmintic efficacy of plant-derived cysteine proteinases on intestinal nematodes.

    Science.gov (United States)

    Luoga, W; Mansur, F; Buttle, D J; Duce, I R; Garnett, M C; Lowe, A; Behnke, J M

    2015-03-01

    We examined the in vitro and in vivo efficacy of plant cysteine proteinases (CPs) derived from pineapple (Ananas comosus) and kiwi fruit (Actinidia deliciosa), and compared their efficacy as anthelmintics to the known effects of CPs from the latex of papaya (Carica papaya) against the rodent intestinal nematode, Heligmosomoides bakeri. Both fruit bromelain and stem bromelain had significant in vitro detrimental effects on H. bakeri but in comparison, actinidain from kiwi fruit had very little effect. However, in vivo trials indicated far less efficacy of stem bromelain and fruit bromelain than that expected from the in vitro experiments (24.5% and 22.4% reduction in worm burdens, respectively) against H. bakeri. Scanning electron microscopy revealed signs of cuticular damage on worms incubated in fruit bromelain, stem bromelain and actinidain, but this was far less extensive than on those incubated in papaya latex supernatant. We conclude that, on the basis of presently available data, CPs derived from pineapples and kiwi fruits are not suitable for development as novel anthelmintics for intestinal nematode infections. PMID:24176056

  8. The pep4 gene encoding proteinase A is involved in dimorphism and pathogenesis of Ustilago maydis.

    Science.gov (United States)

    Soberanes-Gutiérrez, Cinthia V; Juárez-Montiel, Margarita; Olguín-Rodríguez, Omar; Hernández-Rodríguez, César; Ruiz-Herrera, José; Villa-Tanaca, Lourdes

    2015-10-01

    Vacuole proteases have important functions in different physiological processes in fungi. Taking this aspect into consideration, and as a continuation of our studies on the analysis of the proteolytic system of Ustilago maydis, a phytopathogenic member of the Basidiomycota, we have analysed the role of the pep4 gene encoding the vacuolar acid proteinase PrA in the pathogenesis and morphogenesis of the fungus. After confirmation of the location of the protease in the vacuole using fluorescent probes, we obtained deletion mutants of the gene in sexually compatible strains of U. maydis (FB1 and FB2), and analysed their phenotypes. It was observed that the yeast to mycelium dimorphic transition induced by a pH change in the medium, or the use of a fatty acid as sole carbon source, was severely reduced in Δpep4 mutants. In addition, the virulence of the mutants in maize seedlings was reduced, as revealed by the lower proportion of plants infected and the reduction in size of the tumours induced by the pathogen, when compared with wild-type strains. All of these phenotypic alterations were reversed by complementation of the mutant strains with the wild-type gene. These results provide evidence of the importance of the pep4 gene for the morphogenesis and virulence of U. maydis.

  9. Proteinase K activity determination with β-galactosidase as sensitive macromolecular substrate.

    Science.gov (United States)

    Ghéczy, Nicolas; Küchler, Andreas; Walde, Peter

    2016-11-15

    Proteinase K from Engyodontium album (proK) is a relatively unspecific serine endopeptidase which is known to attack proteins yet in their native states. If the attacked protein is an enzyme, even a partial hydrolysis by proK may lead to an inactivation of the enzyme, which can be monitored by measuring the loss of catalytic activity of the attacked enzyme. E. coli β-galactosidase (β-Gal) was used in this work as such enzyme. It was found to be a convenient and sensitive macromolecular model substrate for comparing the "native protein-attacking ability" of free and immobilized proK at pH = 7.0 and 23 °C. The β-Gal activity was measured spectrophotometrically with o-nitrophenyl-β-galactopyranoside. Reproducible proK determinations were possible for as little as 4.3 ng proK by using a proK analyte solution of 10 nM. Compared to free proK, immobilized proK was much less efficient in inactivating β-Gal, most likely due to a decreased mobility of immobilized proK and a restricted accessibility of β-Gal to the active site of proK. Worth noting is, that under conditions at which β-Gal was completely inactivated by proK, the activity of hen egg lysozyme, horseradish peroxidase, or Aspergillus sp. glucose oxidase remained unaltered. PMID:27594349

  10. Thrombin-Mediated Direct Activation of Proteinase-Activated Receptor-2: Another Target for Thrombin Signaling.

    Science.gov (United States)

    Mihara, Koichiro; Ramachandran, Rithwik; Saifeddine, Mahmoud; Hansen, Kristina K; Renaux, Bernard; Polley, Danny; Gibson, Stacy; Vanderboor, Christina; Hollenberg, Morley D

    2016-05-01

    Thrombin is known to signal to cells by cleaving/activating a G-protein-coupled family of proteinase-activated receptors (PARs). The signaling mechanism involves the proteolytic unmasking of an N-terminal receptor sequence that acts as a tethered receptor-activating ligand. To date, the recognized targets of thrombin cleavage and activation for signaling are PAR1 and PAR4, in which thrombin cleaves at a conserved target arginine to reveal a tethered ligand. PAR2, which like PAR1 is also cleaved at an N-terminal arginine to unmask its tethered ligand, is generally regarded as a target for trypsin but not for thrombin signaling. We now show that thrombin, at concentrations that can be achieved at sites of acute injury or in a tumor microenvironment, can directly activate PAR2 vasorelaxation and signaling, stimulating calcium and mitogen-activated protein kinase responses along with triggeringβ-arrestin recruitment. Thus, PAR2 can be added alongside PAR1 and PAR4 to the targets, whereby thrombin can affect tissue function.

  11. Proteinase K and the structure of PrPSc: The good, the bad and the ugly.

    Science.gov (United States)

    Silva, Christopher J; Vázquez-Fernández, Ester; Onisko, Bruce; Requena, Jesús R

    2015-09-01

    Infectious proteins (prions) are, ironically, defined by their resistance to proteolytic digestion. A defining characteristic of the transmissible isoform of the prion protein (PrP(Sc)) is its partial resistance to proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunodetection of PK-digested PrP(Sc) by Western blot, ELISA or immunohistochemical detection. PK digestion has also been used to detect differences in prion strains. Thus, PK has been a crucial tool to detect and, thereby, control the spread of prions. PK has also been used as a tool to probe the structure of PrP(Sc). Mass spectrometry and antibodies have been used to identify PK cleavage sites in PrP(Sc). These results have been used to identify the more accessible, flexible stretches connecting the β-strand components in PrP(Sc). These data, combined with physical constraints imposed by spectroscopic results, were used to propose a qualitative model for the structure of PrP(Sc). Assuming that PrP(Sc) is a four rung β-solenoid, we have threaded the PrP sequence to satisfy the PK proteolysis data and other experimental constraints.

  12. Proteinase K activity determination with β-galactosidase as sensitive macromolecular substrate.

    Science.gov (United States)

    Ghéczy, Nicolas; Küchler, Andreas; Walde, Peter

    2016-11-15

    Proteinase K from Engyodontium album (proK) is a relatively unspecific serine endopeptidase which is known to attack proteins yet in their native states. If the attacked protein is an enzyme, even a partial hydrolysis by proK may lead to an inactivation of the enzyme, which can be monitored by measuring the loss of catalytic activity of the attacked enzyme. E. coli β-galactosidase (β-Gal) was used in this work as such enzyme. It was found to be a convenient and sensitive macromolecular model substrate for comparing the "native protein-attacking ability" of free and immobilized proK at pH = 7.0 and 23 °C. The β-Gal activity was measured spectrophotometrically with o-nitrophenyl-β-galactopyranoside. Reproducible proK determinations were possible for as little as 4.3 ng proK by using a proK analyte solution of 10 nM. Compared to free proK, immobilized proK was much less efficient in inactivating β-Gal, most likely due to a decreased mobility of immobilized proK and a restricted accessibility of β-Gal to the active site of proK. Worth noting is, that under conditions at which β-Gal was completely inactivated by proK, the activity of hen egg lysozyme, horseradish peroxidase, or Aspergillus sp. glucose oxidase remained unaltered.

  13. Stable and Simple Immobilization of Proteinase K Inside Glass Tubes and Microfluidic Channels.

    Science.gov (United States)

    Küchler, Andreas; Bleich, Julian N; Sebastian, Bernhard; Dittrich, Petra S; Walde, Peter

    2015-11-25

    Engyodontium album proteinase K (proK) is widely used for degrading proteinaceous impurities during the isolation of nucleic acids from biological samples, or in proteomics and prion research. Toward applications of proK in flow reactors, a simple method for the stable immobilization of proK inside glass micropipette tubes was developed. The immobilization of the enzyme was achieved by adsorption of a dendronized polymer-enzyme conjugate from aqueous solution. This conjugate was first synthesized from a polycationic dendronized polymer (denpol) and proK and consisted, on average, of 2000 denpol repeating units and 140 proK molecules, which were attached along the denpol chain via stable bis-aryl hydrazone bonds. Although the immobilization of proK inside the tube was based on nonspecific, noncovalent interactions only, the immobilized proK did not leak from the tube and remained active during prolonged storage at 4 °C and during continuous operation at 25 °C and pH = 7.0. The procedure developed was successfully applied for the immobilization of proK on a glass/PDMS (polydimethylsiloxane) microchip, which is a requirement for applications in the field of proK-based protein analysis with such type of microfluidic devices.

  14. Purification and characterization of a proteinase from pineapple fruit, fruit bromelain FA2.

    Science.gov (United States)

    Yamada, F; Takahashi, N; Murachi, T

    1976-06-01

    Fruit bromelain FA2, the main proteinase component of the juice of pineapple fruit, has been purified and characterized. 1. Efficient extraction of this enzyme from the crude material was possible using "Cellulosin AP," a microbial polysaccharidase preparation containing cellulase, hemicellulase, and pectinase. The enzyme was purified mainly by successive applications of anion-exchange chromatography, yielding an apparently homogeneous protein as judged by several physical, chemical, and immunochemical criteria. Properties of FA2 include: molecular weight, 31,000; isoelectric point, pH 4.6; absorbance at 280 nm of a 1% solution at pH 7.0 per cm, 19.2. 2. FA2 gave only alanine phenylthiohydantoin upon amino-terminal group analysis by the Edman procedure. Stepwise degradation yielded the amino-terminal sequence Ala-Val-Pro-Gln-Ser-Ile-Asp-Trp-Arg-Asp-Tyr-Gly-Ala. The amino acid composition of FA2 was not markedly different from that of stem bromelain, except for a much smaller lysine content and a smaller alanine content relative to glycine in FA2. FA2 contained neither amino sugars nor neutral carbohydrates as determined by several methods, so FA2 is not a glycoprotein. 3. By labeling the reactive cysteine residue (CYS) with [14C]iodoacetate, the following partial amino acid sequence has been determined. Asn-Glx-Asn-Pro-Cys-Gly-Ala-CYS.

  15. A subset of ulcerative colitis with positive proteinase-3antineutrophil cytoplasmic antibody

    Institute of Scientific and Technical Information of China (English)

    Jin Xu; Chuan-Hua Yang; Xiao-Yu Chen; Xu-Hang Li; Min Dai; Shu-Dong Xiao

    2008-01-01

    A small subset of patients with active ulcerative colitis is non-responsive to major known non-biological therapies.We reported 5 patients with positive serum proteinase-3 antineutrophil cytoplasmic antibody (PR3-ANCA) and tried to (1) identify the common clinical features of these patients; (2) investigate the efficacy of a novel therapy using a Chinese medicine compound; and (3) attract more gastroenterologists to be engaged in further study of this subset of patients. The common manifestations of disease in these 5 patients included recurrent bloody diarrhea and inflammatory lesions involving the entire colorectal mucosa. Initial treatment with intravenous methylprednisolone successfully induced remission.Four of these 5 patients were steroid-dependence,and immunosuppressants, such as azathioprine and cyclophosphamide, were ineffective. In 3 patients,only the particular Chinese medicine compound could induce and maintain remission. One patient underwent colectomy. No vascular inflammatory lesions were found by histopathological examination. Although more cases are needed for confirmation, our study indicates that ulcerative colitis with positive PR3-ANCA may belong to a subtype of refractory ulcerative colitis. The particular Chinese medicine compound used in our study is by far the most effective in the management of these patients,with additional advantages of having no noticeable sideeffects and less financial burden.

  16. Accumulation of helper component/proteinase and coat protein of turnip mosaic virus in intact plants.

    Science.gov (United States)

    Ohshima, K

    1999-02-01

    The helper component/proteinase (HC/Pro) protein of turnip mosaic virus (TuMV) was fused with glutathione S-transferase (GST) and expressed as a fusion protein in Escherichia coli. The quality of antiserum raised against the GST-HC/Pro fusion protein was compared to that of antiserum raised against coat protein (CP) by image analyser. The result showed that these antisera were of similar quality. Then the both antisera were used to follow the time course of accumulation of HC/Pro protein and CP in intact TuMV-infected leaves. CP appeared first at day 3 post inoculation (p.i.) and gradually accumulated in uninoculated upper leaves, whereas HC/Pro protein appeared first at day 4 p.i., accumulated up to day 7 p.i. and then gradually decreased. Potyvirus proteins are encoded by a single translation unit spanning most of the genome and are presumably synthesized in equimolar ratios. Therefore, the reduced accumulation of HC/Pro protein in relation to CP at one month p.i. in infected plants is presumed to be the result of its degradation. PMID:10672341

  17. Purification and characterization of a salt-activated and organic solvent-stable heterotrimer proteinase from Virgibacillus sp. SK33 isolated from Thai fish sauce.

    Science.gov (United States)

    Sinsuwan, Sornchai; Rodtong, Sureelak; Yongsawatdigul, Jirawat

    2010-01-13

    A NaCl-activated proteinase produced by Virgibacillus sp. SK33 was purified to homogeneity using phenyl-Sepharose and Sephadex G-75 with a yield of 12% and purification of 2.6-fold. A single protein was detected at approximately 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, three subunits with molecular weights of 27,858, 33,918, and 35,368 Da were obtained from MALDI-TOF mass spectra, implying that the enzyme was a heterotrimer. The isoelectric point of the proteinase was 5.4. Optimum catalytic activity was at 55 degrees C and pH 7.5. The enzyme showed serine characteristics as it was completely inhibited by phenylmethanesulfonyl fluoride. The purified proteinase showed broad specificity toward oxidized insulin B including Gln4, Cys7, Glu13, Ala14, Leu15,17, Tyr16,26, Arg22, Phe24,25, and Lys29. Dominant cleavage sites of the enzyme were Tyr16-Leu17 and Phe25-Tyr26, indicating that it preferably hydrolyzed aromatic amino acids located on the P1 site. Among various substrates studied, the enzyme hydrolyzed anchovy protein to the greatest extent at 4 M NaCl. Activity increased with either CaCl2 or NaCl concentration with the maximum 2-fold increase at either 50 mM CaCl2 or 4 M NaCl. The enzyme was also highly stable up to 500 mM CaCl2 or 4 M NaCl. The proteinase showed high stability in various organic solvents (25%, v/v) including dimethylsulfoxide, methanol, acetonitrile, and ethanol. Results of peptide mass fingerprint and de novo peptide sequencing showed that the purified proteinase is a novel proteinase. The proteinase from Virgibacillus sp. SK33 could have a potential application in high ionic strength environments and aqueous-organic solvent systems. PMID:19938835

  18. 分泌型天冬氨酸蛋白酶—抗真菌药物作用的新靶点%Secreted aspartyl proteinase:a novel antifungal target

    Institute of Scientific and Technical Information of China (English)

    刘杨; 盛春泉; 张万年

    2011-01-01

    近年来,随着临床上致病真菌耐药性的日趋严重,寻找具全新作用机制的新型先导化合物成为抗真菌药物研究的重要方向.大量实验证明,分泌型天冬氨酸蛋白酶(Sap)是白色念珠菌致病的重要因素,将成为一个极具前景的抗真菌药物作用新靶点.该文综述了Sap的生物学性质与功能、催化反应机制、三维结构及抑制剂等方面的研究进展.%In recent years,the incidence of drug-resistant Candia is increasing dramatically in clinic,and it is of great importance to develop novel antifungal agents with new mechanism of action. Secreted aspartyl pro-teinase( Sap) was demonstrated to be the important virulence factor of Candida albicans, which represents a promising new target for antifungal agents. Of all the Saps,Sap2 is supposed to be the most potential drug target. Most of the Sap inhibitors are peptides and peptidomimetics, which generally have poor in vivo antifungal efficacy. There is an urgent need to discover small molecule inhibitors with high potency and selectivity. Although there are no drug candidates in clinical now, the determination of the crystal structures of various Saps will facilitate the process of rational drug design and improve the efficiency of drug discovery. This review summarizes the biological properties,structure and function,catalytic mechanism,and inhibitors of Sap.

  19. MMpI: A WideRange of Available Compounds of Matrix Metalloproteinase Inhibitors.

    Science.gov (United States)

    Muvva, Charuvaka; Patra, Sanjukta; Venkatesan, Subramanian

    2016-01-01

    Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases involved in the regulation of the extracellular signaling and structural matrix environment of cells and tissues. MMPs are considered as promising targets for the treatment of many diseases. Therefore, creation of database on the inhibitors of MMP would definitely accelerate the research activities in this area due to its implication in above-mentioned diseases and associated limitations in the first and second generation inhibitors. In this communication, we report the development of a new MMpI database which provides resourceful information for all researchers working in this field. It is a web-accessible, unique resource that contains detailed information on the inhibitors of MMP including small molecules, peptides and MMP Drug Leads. The database contains entries of ~3000 inhibitors including ~72 MMP Drug Leads and ~73 peptide based inhibitors. This database provides the detailed molecular and structural details which are necessary for the drug discovery and development. The MMpI database contains physical properties, 2D and 3D structures (mol2 and pdb format files) of inhibitors of MMP. Other data fields are hyperlinked to PubChem, ChEMBL, BindingDB, DrugBank, PDB, MEROPS and PubMed. The database has extensive searching facility with MMpI ID, IUPAC name, chemical structure and with the title of research article. The MMP inhibitors provided in MMpI database are optimized using Python-based Hierarchical Environment for Integrated Xtallography (Phenix) software. MMpI Database is unique and it is the only public database that contains and provides the complete information on the inhibitors of MMP. Database URL: http://clri.res.in/subramanian/databases/mmpi/index.php. PMID:27509041

  20. Differential expression of chemokines, chemokine receptors and proteinases by foreign body giant cells (FBGCs) and osteoclasts.

    Science.gov (United States)

    Khan, Usman A; Hashimi, Saeed M; Khan, Shershah; Quan, Jingjing; Bakr, Mahmoud M; Forwood, Mark R; Morrison, Nigel M

    2014-07-01

    Osteoclasts and foreign body giant cells (FBGCs) are both derived from the fusion of macropahges. These cells are seen in close proximity during foreign body reactions, therefore it was assumed that they might interact with each other. The aim was to identify important genes that are expressed by osteoclasts and FBGCs which can be used to understand peri-implantitis and predict the relationship of these cells during foreign body reactions. Bone marrow macrophages (BMM) were treated with receptor activator of nuclear factor kappa B ligand (RANKL) to produce osteoclasts. Quantitative PCR (qPCR) was used to identify the genes that were expressed by osteoclasts and FBGCs compared to macrophage controls. TRAP staining was used to visualise the cells while gelatine zymography and western blots were used for protein expression. Tartrate-resistant acid phosphatase (TRAP), matrix metallo proteinase 9 (MMP9), nuclear factor of activated T cells 1 (NFATc1), cathepsin K (CTSK) and RANK were significantly lower in FBGCs compared to osteoclasts. Inflammation specific chemokines such as monocyte chemotactic protein (MCP1 also called CCL2), macrophage inflammatory protein 1 alpha (MIP1α), MIP1β and MIP1γ, and their receptors CCR1, CCR3 and CCR5, were highly expressed by FBGCs. FBGCs were negative for osteoclast specific markers (RANK, NFATc1, CTSK). FBGCs expressed chemokines such as CCL2, 3, 5 and 9 while osteoclasts expressed the receptors for these chemokines i.e. CCR1, 2 and 3. Our findings show that osteoclast specific genes are not expressed by FBGCs and that FBGCs interact with osteoclasts during foreign body reaction through chemokines.

  1. Membrane lipid peroxidation in neurodegeneration: Role of thrombin and proteinase-activated receptor-1.

    Science.gov (United States)

    Citron, Bruce A; Ameenuddin, Syed; Uchida, K; Suo, William Z; SantaCruz, Karen; Festoff, Barry W

    2016-07-15

    Thrombin and membrane lipid peroxidation (MLP) have been implicated in various central nervous system (CNS) disorders from CNS trauma to stroke, Alzheimer's (AD) and Parkinson's (PD) diseases. Because thrombin also induces MLP in platelets and its involvement in neurodegenerative diseases we hypothesized that its deleterious effects might, in part, involve formation of MLP in neuronal cells. We previously showed that thrombin induced caspase-3 mediated apoptosis in motor neurons, via a proteinase-activated receptor (PAR1). We have now investigated thrombin's influence on the oxidative state of neurons leading to induction of MLP-protein adducts. Translational relevance of thrombin-induced MLP is supported by increased levels of 4-hydroxynonenal-protein adducts (HNEPA) in AD and PD brains. We now report for the first time that thrombin dose-dependently induces formation of HNEPA in NSC34 mouse motor neuron cells using anti-HNE and anti-acrolein monoclonal antibodies. The most prominent immunoreactive band, in SDS-PAGE, was at ∼54kDa. Membrane fractions displayed higher amounts of the protein-adduct than cytosolic fractions. Thrombin induced MLP was mediated, at least in part, through PAR1 since a PAR1 active peptide, PAR1AP, also elevated HNEPA levels. Of interest, glutamate and Fe2SO4 also increased the ∼54kDa HNEPA band in these cells but to a lesser extent. Taken together our results implicate the involvement of thrombin and MLP in neuronal cell loss observed in various CNS degenerative and traumatic pathologies. PMID:27138068

  2. Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis

    Science.gov (United States)

    Millet, Arnaud; Martin, Katherine R.; Bonnefoy, Francis; Saas, Philippe; Mocek, Julie; Alkan, Manal; Terrier, Benjamin; Kerstein, Anja; Tamassia, Nicola; Satyanarayanan, Senthil Kumaran; Ariel, Amiram; Ribeil, Jean-Antoine; Guillevin, Loïc; Cassatella, Marco A.; Mueller, Antje; Thieblemont, Nathalie; Lamprecht, Peter; Mouthon, Luc; Perruche, Sylvain; Witko-Sarsat, Véronique

    2015-01-01

    Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating CD4+ T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology. PMID:26436651

  3. Mandatory role of proteinase-activated receptor 1 in experimental bladder inflammation

    Directory of Open Access Journals (Sweden)

    Davis Carole A

    2007-03-01

    Full Text Available Abstract Background In general, inflammation plays a role in most bladder pathologies and represents a defense reaction to injury that often times is two edged. In particular, bladder neurogenic inflammation involves the participation of mast cells and sensory nerves. Increased mast cell numbers and tryptase release represent one of the prevalent etiologic theories for interstitial cystitis and other urinary bladder inflammatory conditions. The activity of mast cell-derived tryptase as well as thrombin is significantly increased during inflammation. Those enzymes activate specific G-protein coupled proteinase-activated receptors (PARs. Four PARs have been cloned so far, and not only are all four receptors highly expressed in different cell types of the mouse urinary bladder, but their expression is altered during experimental bladder inflammation. We hypothesize that PARs may link mast cell-derived proteases to bladder inflammation and, therefore, play a fundamental role in the pathogenesis of cystitis. Results Here, we demonstrate that in addition to the mouse urinary bladder, all four PA receptors are also expressed in the J82 human urothelial cell line. Intravesical administration of PAR-activating peptides in mice leads to an inflammatory reaction characterized by edema and granulocyte infiltration. Moreover, the inflammatory response to intravesical instillation of known pro-inflammatory stimuli such as E. coli lipopolysaccharide (LPS, substance P, and antigen was strongly attenuated by PAR1-, and to a lesser extent, by PAR2-deficiency. Conclusion Our results reveal an overriding participation of PAR1 in bladder inflammation, provide a working model for the involvement of downstream signaling, and evoke testable hypotheses regarding the role of PARs in bladder inflammation. It remains to be determined whether or not mechanisms targeting PAR1 gene silencing or PAR1 blockade will ameliorate the clinical manifestations of cystitis.

  4. A thermo-halo-tolerant and proteinase-resistant endoxylanase from Bacillus sp. HJ14.

    Science.gov (United States)

    Zhou, Junpei; Wu, Qian; Zhang, Rui; Mo, Minghe; Tang, Xianghua; Li, Junjun; Xu, Bo; Ding, Junmei; Lu, Qian; Huang, Zunxi

    2014-09-01

    A glycosyl hydrolase family 10 endoxylanase from Bacillus sp. HJ14 was grouped in a separated cluster with another six Bacillus endoxylanases which have not been characterized. These Bacillus endoxylanases showed less than 52% amino acid sequence identity with other endoxylanases and far distance with endoxylanases from most microorganisms. Signal peptide was not detected in the endoxylanase. The endoxylanase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant enzyme (rXynAHJ14) was characterized. rXynAHJ14 was apparent optimal at 62.5 °C and pH 6.5 and retained more than 55% of the maximum activity when assayed at 40-75 °C, 23% at 20 °C, 16% at 85 °C, and even 8% at 0 °C. Half-lives of the enzyme were more than 60 min, approximately 25 and 4 min at 70, 75, and 80 °C, respectively. The enzyme exhibited more than 62% xylanase activity and stability at the concentration of 3-30% (w/v) NaCl. No xylanase activity was lost after incubation of the purified rXynAHJ14 with trypsin and proteinase K at 37 °C for 60 min. Different components of oligosaccharides were detected in the time-course hydrolysis of beechwood xylan by the enzyme. During the simulated intestinal digestion phase in vitro, 11.5-19.0, 15.3-19.0, 21.9-27.7, and 28.2-31.2 μmol/mL reducing sugar were released by the purified rXynAHJ14 from soybean meal, wheat bran, beechwood xylan, and rapeseed meal, respectively. The endoxylanase might be an alternative for potential applications in the processing of sea food and saline food and in aquaculture as agastric fish feed additive.

  5. Use of proteinase K for RT-PCR of cytokine mRNA in formalin fixed tissue

    DEFF Research Database (Denmark)

    Davies, G N; Bevan, I S; Banner, Jytte;

    1996-01-01

    Fresh tissue from cases of sudden infant death syndrome is becoming increasingly scarce and therefore researchers interesting in studying the aetiology of this syndrome have had to resort to archival tissue, usually in the form of paraffin wax sections. A simple method for isolating mRNA from...... formalin fixed, paraffin wax embedded material of sufficient purity for reverse transcription (RT)-PCR is described. Proteinase K treatment of formalin fixed, wax embedded tissue followed by RNA STAT-60 extraction was successful in isolating mRNA suitable for RT-PCR. Interleukin (IL)-1alpha, IL-6...

  6. Proteinase-activated receptor-2 is required for normal osteoblast and osteoclast differentiation during skeletal growth and repair.

    Science.gov (United States)

    Georgy, S R; Pagel, C N; Ghasem-Zadeh, A; Zebaze, R M D; Pike, R N; Sims, N A; Mackie, E J

    2012-03-01

    Proteinase-activated receptor-2 (PAR(2)) is a G-protein coupled receptor expressed by osteoblasts and monocytes. PAR(2) is activated by a number of proteinases including coagulation factors and proteinases released by inflammatory cells. The aim of the current study was to investigate the role of PAR(2) in skeletal growth and repair using wild type (WT) and PAR(2) knockout (KO) mice. Micro computed tomography and histomorphometry were used to examine the structure of tibias isolated from uninjured mice at 50 and 90 days of age, and from 98-day-old mice in a bone repair model in which a hole had been drilled through the tibias. Bone marrow was cultured and investigated for the presence of osteoblast precursors (alkaline phosphatase-positive fibroblastic colonies), and osteoclasts were counted in cultures treated with M-CSF and RANKL. Polymerase chain reaction (PCR) was used to determine which proteinases that activate PAR(2) are expressed in bone marrow. Regulation of PAR(2) expression in primary calvarial osteoblasts from WT mice was investigated by quantitative PCR. Cortical and trabecular bone volumes were significantly greater in the tibias of PAR(2) KO mice than in those of WT mice at 50 days of age. In trabecular bone, osteoclast surface, osteoblast surface and osteoid volume were significantly lower in KO than in WT mice. Bone marrow cultures from KO mice showed significantly fewer alkaline phosphatase-positive colony-forming units and osteoclasts compared to cultures from WT mice. Significantly less new bone and significantly fewer osteoclasts were observed in the drill sites of PAR(2) KO mice compared to WT mice 7 days post-surgery. A number of activators of PAR(2), including matriptase and kallikrein 4, were found to be expressed by normal bone marrow. Parathyroid hormone, 1,25 dihydroxyvitamin D(3), or interleukin-6 in combination with its soluble receptor down-regulated PAR(2) mRNA expression, and fibroblast growth factor-2 or thrombin stimulated PAR(2

  7. The leader proteinase of foot-and-mouth disease virus: structure-function relationships in a proteolytic virulence factor

    Science.gov (United States)

    Steinberger, Jutta; Skern, Tim

    2016-01-01

    The leader proteinase (Lpro) of foot-and-mouth disease virus, inhibits the host innate immune response by at least three different mechanisms. The most well characterised is the prevention of the synthesis of cytokines such as interferons immediately after infection, brought about by specific proteolytic cleavage of the eukaryotic initiation factor 4G. This prevents the recruitment of capped cellular mRNA; the viral RNA can however be translated under these conditions. The two other mechanisms are the induction of NF-κB cleavage and the deubiquitination of immune signalling molecules. This review focuses on the structure-function relationships in Lpro responsible for these widely divergent activities. PMID:24670358

  8. Flexibility of cold- and heat-adapted subtilisin-like serine proteinases evaluated with fluorescence quenching and molecular dynamics

    DEFF Research Database (Denmark)

    Sigtryggsdóttir, Asta Rós; Papaleo, Elena; Thorbjarnardóttir, Sigríður H.;

    2014-01-01

    activity of cold adapted enzymes when compared to homologues from thermophiles, reflects their higher molecular flexibility. To assess a potential difference in molecular flexibility between the two homologous proteinases, we have measured their Trp fluorescence quenching by acrylamide at different...... have similar flexibility profiles, the cold adapted VPR displays higher flexibility in most regions of the protein structure. Some of these regions contain or are in proximity to some of the Trp residues (Trp6, Trp114 and Trp208) in the proteins. Thus, we observe an overall agreement between...

  9. Use of proteinase K nonspecific digestion for selective and comprehensive identification of interpeptide cross-links: application to prion proteins.

    Science.gov (United States)

    Petrotchenko, Evgeniy V; Serpa, Jason J; Hardie, Darryl B; Berjanskii, Mark; Suriyamongkol, Bow P; Wishart, David S; Borchers, Christoph H

    2012-07-01

    Chemical cross-linking combined with mass spectrometry is a rapidly developing technique for structural proteomics. Cross-linked proteins are usually digested with trypsin to generate cross-linked peptides, which are then analyzed by mass spectrometry. The most informative cross-links, the interpeptide cross-links, are often large in size, because they consist of two peptides that are connected by a cross-linker. In addition, trypsin targets the same residues as amino-reactive cross-linkers, and cleavage will not occur at these cross-linker-modified residues. This produces high molecular weight cross-linked peptides, which complicates their mass spectrometric analysis and identification. In this paper, we examine a nonspecific protease, proteinase K, as an alternative to trypsin for cross-linking studies. Initial tests on a model peptide that was digested by proteinase K resulted in a "family" of related cross-linked peptides, all of which contained the same cross-linking sites, thus providing additional verification of the cross-linking results, as was previously noted for other post-translational modification studies. The procedure was next applied to the native (PrP(C)) and oligomeric form of prion protein (PrPβ). Using proteinase K, the affinity-purifiable CID-cleavable and isotopically coded cross-linker cyanurbiotindipropionylsuccinimide and MALDI-MS cross-links were found for all of the possible cross-linking sites. After digestion with proteinase K, we obtained a mass distribution of the cross-linked peptides that is very suitable for MALDI-MS analysis. Using this new method, we were able to detect over 60 interpeptide cross-links in the native PrP(C) and PrPβ prion protein. The set of cross-links for the native form was used as distance constraints in developing a model of the native prion protein structure, which includes the 90-124-amino acid N-terminal portion of the protein. Several cross-links were unique to each form of the prion protein, including

  10. Protease inhibitors from marine actinobacteria as a potential source for antimalarial compound.

    Directory of Open Access Journals (Sweden)

    L Karthik

    Full Text Available The study was planned to screen the marine actinobacterial extract for the protease inhibitor activity and its anti- Pf activity under in vitro and in vivo conditions. Out of 100 isolates, only 3 isolates exhibited moderate to high protease inhibitor activities on trypsin, chymotrypsin and proteinase K. Based on protease inhibitor activity 3 isolates were chosen for further studies. The potential isolate was characterized by polyphasic approach and identified as Streptomyces sp LK3 (JF710608. The lead compound was identified as peptide from Streptomyces sp LK3. The double-reciprocal plot displayed inhibition mode is non-competitive and it confirms the irreversible nature of protease inhibitor. The peptide from Streptomyces sp LK3 extract showed significant anti plasmodial activity (IC50: 25.78 µg/ml. In in vivo model, the highest level of parasitemia suppression (≈ 45% was observed in 600 mg/kg of the peptide. These analyses revealed no significant changes were observed in the spleen and liver tissue during 8 dpi. The results confirmed up-regulation of TGF-β and down regulation of TNF-α in tissue and serum level in PbA infected peptide treated mice compared to PbA infection. The results obtained infer that the peptide possesses anti- Pf activity activity. It suggests that the extracts have novel metabolites and could be considered as a potential source for drug development.

  11. Effects of proteinase A on cultivation and viability characteristics of industrial Saccharomyces cerevisiae WZ65

    Institute of Scientific and Technical Information of China (English)

    Hong-bo ZHANG; Hai-feng ZHANG; Qi-he CHEN; Hui RUAN; Ming-liang FU; Guo-qing HE

    2009-01-01

    Proteinase A (PrA), encoded by PEP4 gene, is a key enzyme in the vacuoles of Saccharomyces cerevisiae. We characterized the effects of PrA on cell growth and glucose metabolism in the industrial S. cerevisiae WZ65. It was observed that the lag phase of cell growth of partial PEP4 gene deletion mutant (36 h) and PrA-negative mutant (48 h) was significantly ex-tended, compared with the wild type strain (24 h) (P<0.05), but PrA had no effect on glucose metabolism either under shaking or steady state cultivations. The logistic model was chosen to evaluate the effect of PrA on S. cerevisiae cell growth, and PrA was found to promote cell growth against insufficient oxygen condition in steady state cultivation, but had no effect in shaking culti-vation. The effects of glucose starvation on cell growth of partial PEP4 gene deletion strain and PrA-negative mutant were also evaluated. The results show that PrA partial deficiency increased the adaption ofS. cerevisiae to unfavorable nutrient environment, but had no effect on glucose metabolism under the stress of low glucose. During heat shock test, at 60 ℃ the reduced cell viability rate (RCVR) was 10% for the wild type S. cerevisiae and 90% for both mutant strains (P<0.01), suggesting that PrA was a negative factor for S. cerevisiae cells to survive under heat shock. As temperatures rose from 60 ℃ to 70 ℃, the wild type S. cerevisiae had significantly lower relative glucose consumption rate (RGCR) (61.0% and 80.0%) than the partial mutant (78.0% and 98.5%) and the complete mutant (80.0% and 98.0%) (P<0.05), suggesting that, in coping with heat shock, cells of the PrA mutants increased their glucose consumption to survive. The present study may provide meaningful information for brewing industry; however, the role of PrA in industrial S. cerevisiae physiology is complex and needs to be further investigated.

  12. Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper

    Directory of Open Access Journals (Sweden)

    A.V Pérez

    2008-01-01

    Full Text Available A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg and fibrinogen (minimum coagulant dose = 4.2 µg in vitro, and promotes defibrin(ogenation in vivo (minimum defibrin(ogenating dose = 1.0 µg. In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

  13. Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

    Directory of Open Access Journals (Sweden)

    CB Toaldo

    2001-01-01

    Full Text Available The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of ß-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70 and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of ß-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain.

  14. Assessing proteinase K resistance of fish prion proteins in a scrapie-infected mouse neuroblastoma cell line.

    Science.gov (United States)

    Salta, Evgenia; Kanata, Eirini; Ouzounis, Christos A; Gilch, Sabine; Schätzl, Hermann; Sklaviadis, Theodoros

    2014-11-13

    The key event in prion pathogenesis is the structural conversion of the normal cellular protein, PrP(C), into an aberrant and partially proteinase K resistant isoform, PrP(Sc). Since the minimum requirement for a prion disease phenotype is the expression of endogenous PrP in the host, species carrying orthologue prion genes, such as fish, could in theory support prion pathogenesis. Our previous work has demonstrated the development of abnormal protein deposition in sea bream brain, following oral challenge of the fish with natural prion infectious material. In this study, we used a prion-infected mouse neuroblastoma cell line for the expression of three different mature fish PrP proteins and the evaluation of the resistance of the exogenously expressed proteins to proteinase K treatment (PK), as an indicator of a possible prion conversion. No evidence of resistance to PK was detected for any of the studied recombinant proteins. Although not indicative of an absolute inability of the fish PrPs to structurally convert to pathogenic isoforms, the absence of PK-resistance may be due to supramolecular and conformational differences between the mammalian and piscine PrPs.

  15. Crystallization of proteinase K complexed with substrate analogue peptides on US space missions STS-91 and STS-95

    Science.gov (United States)

    Eschenburg, Susanne; Degenhardt, Michael; Moore, Karen; DeLucas, Lawrence J.; Peters, Klaus; Fittkau, Siegfried; Weber, Wolfgang; Betzel, Christian

    2000-01-01

    Crystals of proteinase K in complex with synthetic substrate analogues have been grown under microgravity on the US space shuttle missions STS-91 and STS-95 using the vapor diffusion apparatus (c-VDA) supplied by the Center for Macromolecular Crystallography at the University of Alabama at Birmingham. The crystals obtained under microgravity are compared with those grown simultanously on ground in identical c-VDA reactors and in conventional hanging-drop set-ups. The diffraction quality of space- and ground-grown crystals has been assessed by collecting complete data sets with a conventional X-ray source and with synchrotron radiation. Crystals grown in microgravity are clearly superior to those grown in the identical hardware on earth in terms of crystal habit and diffraction power. In comparison to best terrestrial crystals obtained in conventional hanging-drop set-ups the differences in crystal size and diffraction quality are less, but still confirm the benefit of microgravity for the crystallization of proteinase K-substrate analogue complexes.

  16. Production and purification of antioxidant peptides from a mungbean meal hydrolysate by Virgibacillus sp. SK37 proteinase.

    Science.gov (United States)

    Lapsongphon, Nawaporn; Yongsawatdigul, Jirawat

    2013-11-15

    Antioxidant peptides of mungbean meal hydrolysed by Virgibacillus sp. SK37 proteinases (VH), Alcalase (AH) and Neutrase (NH) were investigated. The antioxidant activities based on 2,2'-azinobis (3-ethyl-benzothiazoline-6-sulphonate) (ABTS) radical-scavenging, ferric-reducing antioxidant power (FRAP) and metal chelation of VH were comparable to those of NH. VH was purified using ultrafiltration, ion exchange and gel filtration chromatography. The purified peptides (F37) from VH, which had the highest specific antioxidant activity, consisted of four peptides containing an arginine residue at their C-termini. In addition, the ABTS radical-scavenging activity of the purified peptides (F42) at 0.148mg/ml was comparable to that of 1mM of butylated hydroxytoluene (BHT). These two fractions were stable over a wide pH (4-10) and temperature (25-121°C) range. Virgibacillus sp. SK37 proteinase is a potential processing-aid for the production of a mungbean meal hydrolyzate with antioxidant properties. PMID:23790878

  17. Detergent-Stable Salt-Activated Proteinases from Virgibacillus halodenitrificans SK1-3-7 Isolated from Fish Sauce Fermentation.

    Science.gov (United States)

    Montriwong, Aungkawipa; Rodtong, Sureelak; Yongsawatdigul, Jirawat

    2015-05-01

    The NaCl-activated and detergent-stable proteinases from Virgibacillus halodenitrificans SK1-3-7 isolated from fish sauce fermentation were purified and characterized. The enzymes with molecular masses of 20 and 36 kDa showed caseinolytic activity on a zymogram. Optimum azocaseinolytic activity was at 60 °C and pH 9. The proteolytic activity increased in the presence of 10 mM CaCl2 and 0.5 M NaCl and showed high stability at 0-2 M NaCl. The enzymes were stable at pH 4-10 and 10-50 °C. The enzymes preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-pNA and were completely inhibited by phenylmethanesulfonyl fluoride (PMSF), showing subtilisin-like characteristics. Activity and stability remained high in the presence of H2O2 and various surfactants. The enzymes exhibited high stability (>95%) in various organic solvents (DMSO, butanol, ethanol, 2-propanol, and acetonitrile) at concentration of 50%. The V. halodenitrificans SK1-3-7 proteinases showed potential as a biocatalyst in aqueous-organic solvent systems and as an additive in laundry detergent. PMID:25820449

  18. Proteinase activity in latex of three plants of the family Euphorbiaceae

    Directory of Open Access Journals (Sweden)

    Andréa Michel Sobottka

    2014-09-01

    Full Text Available In the family of Euphorbiaceae,the genera Euphorbia and Sapium are known to contain essentially latex-bearing species. In the present study, the latex of Euphorbia selloi(Klotzsch & Garcke Boiss., Euphorbia papillosa A.St.-Hil., and Sapium glandulosum (L. Morong, plants native from Brazil, were examined concerning proteolytic activity. All studied species have proteins with significant proteolytic activity and E. papillosa has the greatest specific activity. Aiming to verify the type of protease present, an assay with different inhibitors was performed. In the three tested plants, the proteolytic activity was significantly inhibited by a serine protease inhibitor 4-(2-aminoethyl-benzenesulfonyl fluoride hydrochloride (AEBSF. Using techniques of electrophoresis with polyacrylamide gels (SDS-PAGE, the subunits of proteins were separated according to their molecular masses, and the protein activity was visually detected by zymography.

  19. 萌发绿豆种子中的一种大豆胰蛋白酶抑制剂钝化酶%A Proteinase from Mung Bean Sprouts That Inactivaties Soybean Trypsin Inhib itor

    Institute of Scientific and Technical Information of China (English)

    陈中; 杨晓泉; 赵谋明

    2001-01-01

    By 30%-60% (NH4)2SO4 fractional precipitation, anion-exchange chromatogr aphy o n DEAE-Sepharose CL-6B, gel filtration on Sephacryl S-200 and anion-exchange chr omatography on Waters AP-1 column (ProteinPM-Pak DEAE 15HR), a proteinase which can inactivate soybean trypsin inhibitor (STI) was purified from mun g bean (Vigna rabiata (L.) Wilczek) sprouts. Its molecular weight wa s estimated to be 29.8 kD by SDS-PAGE, and its Km and V max for STI were 769.2 N-α-benzoyl-L-arginine ethyl ester BAEE/mL and 115.3 BAEE· mL-1·min-1 respectively. This proteinase was stable at temperatures lower than 50 ℃ and pH 6.5-8.5, and 90.91% STI activity of defatted soybean powder was inact ivated by this preparation, with proteolytic activity 5 000 BAEE/mL at 50 ℃ and pH 8.0 in 4 h.%通过30%~60% (NH4)2SO4分级沉淀、DEAE-Sepharose CL-6B离子交换层析、Sepha c ryl S-200凝胶过滤层析和Waters AP-1离子交换层析,从萌发的绿豆(Vigna rabiata ( L.) Wilczek)种子中分离纯化出一种可降解大豆胰蛋白酶抑制剂(STI)的蛋白酶.SDS-PAG E测定该酶的分子量为29.8 kD.该酶催化降解STI的Km值为769.2 BAEE/mL,V max为115.3 BAEE·mL-1·min-1.该酶在50 ℃、pH 8.0、相对酶活力5 0 00 BAEE/mL和4 h的反应时间时可将脱脂大豆粉中的STI 活性钝化90.91%.该酶在温度低于5 0 ℃及pH 6.5~8.5时能保持其活性.

  20. Elastase, but not proteinase 3 (PR3), induces proteinuria associated with loss of glomerular basement membrane heparan sulphate after in vivo renal perfusion in rats

    NARCIS (Netherlands)

    Heeringa, P; VanDenBorn, J; Brouwer, E; Dolman, KM; Klok, PA; Huitema, MG; Limburg, PC; Bakker, MAH; Berden, JHM; Daha, MR; Kallenberg, CGM

    1996-01-01

    Elastase, but not PR3, induces proteinuria associated with loss of glomerular basement membrane (GEM) heparan sulphate after in vivo renal perfusion in rats. PR3 and elastase are cationic neutral serine proteinases present in the azurophilic granules of polymorphonuclear leucocytes. Release of these

  1. Characterisation of cysteine proteinases responsible for digestive proteolysis in guts of larval Western corn rootworm (Diabrotica virgifera) by expression in the yeast Pichia pastoris

    NARCIS (Netherlands)

    Bown, D.P.; Wilkinson, H.S.; Jongsma, M.A.; Gatehouse, J.A.

    2004-01-01

    Cysteine proteinases are the major class of enzymes responsible for digestive proteolysis in western corn rootworm (Diabrotica virgifera), a serious pest of maize. A larval gut extract hydrolysed typical cathepsin substrates, such as Z-phe-arg-AMC and Z-arg-arg-AMC, and hydrolysis was inhibited by Z

  2. Correction: Short-term variability of biomarkers of proteinase activity in patients with emphysema associated with type Z alpha-1-antitrypsin deficiency.

    NARCIS (Netherlands)

    Stolk, J.; Veldhuisen, B.; Annovazzi, L.; Zanone, C.; Versteeg, E.M.M.; Kuppevelt, A.H.M.S.M. van; Nieuwenhuizen, W.; Iadarola, P.; Berden, J.H.M.; Luisetti, M.

    2006-01-01

    BACKGROUND: The burden of proteinases from inflammatory cells in the lung of subjects with type Pi ZZ of alpha-1-antitrypsin deficiency is higher than in those without the deficiency. Cross-sectional studies have shown increased levels of biomarkers of extracellular matrix degradation in vivo. Longi

  3. Short-term variability of biomarkers of proteinase activity in patients with emphysema associated with type Z alpha-1-antitrypsin deficiency

    NARCIS (Netherlands)

    Stolk, J; Veldhuisen, B; Annovazzi, L; Zanone, C; Versteeg, EM; van Kuppevelt, TH; Nieuwenhuizen, W; Iadarola, P; Luisetti, M

    2005-01-01

    Background: The burden of proteinases from inflammatory cells in the lung of subjects with type Pi ZZ of alpha-1-antitrypsin deficiency is higher than in those without the deficiency. Cross-sectional studies have shown increased levels of biomarkers of extracellular matrix degradation in vivo. Longi

  4. Short-term variability of biomarkers of proteinase activity in patients with emphysema associated with type Z alpha-1-antitrypsin deficiency.

    NARCIS (Netherlands)

    Stolk, J.; Veldhuisen, B.; Annovazzi, L.; Zanone, C.; Versteeg, E.M.M.; Kuppevelt, A.H.M.S.M. van; Nieuwenhuizen, W.; Iadarola, P.; Luisetti, M.

    2005-01-01

    BACKGROUND: The burden of proteinases from inflammatory cells in the lung of subjects with type Pi ZZ of alpha-1-antitrypsin deficiency is higher than in those without the deficiency. Cross-sectional studies have shown increased levels of biomarkers of extracellular matrix degradation in vivo. Longi

  5. A Nonhost Peptidase Inhibitor of ~14 kDa from Butea monosperma (Lam. Taub. Seeds Affects Negatively the Growth and Developmental Physiology of Helicoverpa armigera

    Directory of Open Access Journals (Sweden)

    Prabhash K. Pandey

    2014-01-01

    Full Text Available Helicoverpa armigera is one of the major devastating pests of crop plants. In this context a serine peptidase inhibitor purified from the seeds of Butea monosperma was evaluated for its effect on developmental physiology of H. armigera larvae. B. monosperma peptidase inhibitor on 12% denaturing polyacrylamide gel electrophoresis exhibited a single protein band of ~14 kDa with or without reduction. In vitro studies towards total gut proteolytic enzymes of H. armigera and bovine trypsin indicated measurable inhibitory activity. B. monosperma peptidase inhibitor dose for 50% mortality and weight reduction by 50% were 0.5% w/w and 0.10% w/w, respectively. The IC50 of B. monosperma peptidase inhibitor against total H. armigera gut proteinases activity was 2.0 µg/mL. The larval feeding assays suggested B. monosperma peptidase inhibitor to be toxic as reflected by its retarded growth and development, consequently affecting fertility and fecundity of pest and prolonging the larval-pupal duration of the insect life cycle of H. armigera. Supplementing B. monosperma peptidase inhibitor in artificial diet at 0.1% w/w, both the efficiencies of conversion of ingested as well as digested food were downregulated, whereas approximate digestibility and metabolic cost were enhanced. The efficacy of Butea monosperma peptidase inhibitor against progressive growth and development of H. armigera suggest its usefulness in insect pest management of food crops.

  6. The Research of Aspartic Protease Inhibitors from Soy Protein Hydrolyzed by Proteases%酶解大豆蛋白产天冬氨酸蛋白酶抑制剂的研究

    Institute of Scientific and Technical Information of China (English)

    张国强; 田亚平

    2012-01-01

    In this paper, we reported a kind of aspartie protease inhibitor from soy protein ( soy aspartie proteinase inhibitor,SAPI) after controlled enzymatic hydrolysis. The final inhibitory activity was 254.2 IU /mg and the purification fold was 62 after DEAE-52 ion-exchange chromatography,Superdex Peptide 10/300 GL gel chromatography and SOURCE 15RPC reversed-phase chromatography. The IC50, value of SAPI was 25. 67 μg/mL to pepsin(3000 U) , SAPI showed good thermostability and belonged to a kind of non-competitive inhibitor.%本文通过采用可控酶解的方式酶解大豆蛋白获得了一种源自大豆的天冬氨酸蛋白酶抑制剂SAPI( soy aspartic proteinase inhibitor).酶解液经过DEAE-52阴离子交换层析、Superdex Peptide 10/300 GL凝胶层析、SOURCE 15RPC反相层析分离后最终比抑制活力为254.2 IU/mg,纯化倍数为62.SAPI对胃蛋白酶(3000U)半数抑制浓度IC50为25.67 μg/mL,有良好的热稳定性,属于一种非竞争性抑制剂.

  7. Cholinesterase inhibitors from botanicals

    Directory of Open Access Journals (Sweden)

    Faiyaz Ahmed

    2013-01-01

    Full Text Available Alzheimer′s disease (AD is a progressive neurodegenerative disease, wherein a progressive loss of cholinergic synapses occurs in hippocampus and neocortex. Decreased concentration of the neurotransmitter, acetylcholine (ACh, appears to be critical element in the development of dementia, and the most appropriate therapeutic approach to treat AD and other form of dementia is to restore acetylcholine levels by inhibiting both major form of cholinesterase: Acetylcholinesterase (AChE and butyrylcholinesterase (BChE. Consequently, researches have focused their attention towards finding cholinesterase inhibitors from natural products. A large number of such inhibitors have been isolated from medicinal plants. This review presents a comprehensive account of the advances in field of cholinesterase inhibitor phytoconstituents. The structures of some important phytoconstituents (collected through www.Chemspider.com are also presented and the scope for future research is discussed.

  8. Correlation of Inhibitor Proteinase in Varieties and Lines of Cotton (Gossypium hirsutum L.) to Different Geographic Population of Verticillium dathliae Klebahn

    Institute of Scientific and Technical Information of China (English)

    KIM Robert; AMANTURDIEV Alisher; MEJLUMYAN Larisa; BABAYEV Yashen; KIM Michael

    2008-01-01

    @@ Breeding for wilt resistance and its theoretical basis are primarily responsible for increases in cotton yield and fiber quality.Breeding for immunity is the most efficient method in our struggle with infectious diseases.

  9. Correlation of Inhibitor Proteinase in Varieties and Lines of Cotton(Gossypium hirsutum L.) to Different Geographic Population of Verticillium dathliae Klebahn

    Institute of Scientific and Technical Information of China (English)

    KIM; Robert; AMANTURDIEV; Alisher; MEJLUMYAN; Larisa; BABAYEV; Yashen; KIM; Michael

    2008-01-01

    Breeding for wilt resistance and its theoretical basis are primarily responsible for increases in cotton yield and fiber quality. Breeding for immunity is the most efficient method in our struggle with infectious diseases.

  10. DECOMPOSTION OF GENETICALLY ENGINEERED TOBACCO UNDER FIELD CONDITIONS: PERSISTENCE OF THE PROTEINASE INHIBITOR I PRODUCT AND EFFECTS OF SOIL MICROBIAL RESPIRATION AND PROTOZOA, NEMATODE AND MICROARTHR

    Science.gov (United States)

    1. To evaluate the potential effects of genetically engineered (transgenic) plants on soil ecosystems, litterbags containing leaves of non-engineered (parental) and transgenic tobacco plants were buried in field plots. The transgenic tobacco plants were genetically engineered to ...

  11. Development, characterization and diagnostic application of a monoclonal antibody specific for a proteinase K resistant Lawsonia intracellularis antigen

    DEFF Research Database (Denmark)

    Boesen, Henriette T.; Jensen, Tim Kåre; Jungersen, Gregers;

    2005-01-01

    (mAb), Law1-DK, isotyped as IgG2b was selected by indirect immunofluorescence antibody test (IFAT). Histological sections of the intestines from pigs affected by proliferative enteropathy and in vitro grown bacteria in cell culture were tested positive for the presence of L. intracellularis...... and was resistant to proteinase K digestion, suggesting it to be non-protein, e.g., lipopolysaccharide (LPS). This suggestion was supported by its presence in the aqueous phase of a phenol-water extract. The inhibitory effect of periodate oxidation on the antigen-antibody binding confirmed the participation...... of a carbohydrate epitope. The new mAb was tested highly specific for L. intracellularis by applying in situ hybridization with a L. intracellularis specific probe targeting 16S ribosomal RNA simultaneously with the IFAT....

  12. Proteinases aspárticas em flores de Cynara Humilis : Estudos de expressão durante o desenvolvimento floral

    OpenAIRE

    Sousa, Américo José da Silva Martins

    2002-01-01

    Dissertação de Mestrado em Biologia do Desenvolvimento e Reprodução Vegetal apresentada à Faculdade de Ciências da Universidade do Porto As proteinases aspárticas (EC 3.4.23) são uma sub-subclasse de enzimas que se encontram amplamente distribuídas na natureza. Os estudos realizados descrevem-nas em animais, plantas, bactérias, fungos, retrovírus e vírus; onde desempenham diversas funções, desde a digestão de proteínas no sistema digestivo dos vertebrados até à clivagem de poliproteínas vi...

  13. Proteinase production in Pseudomonas fluorescens ON2 is affected by carbon sources and allows surface-attached but not planktonic cells to utilize protein for growth in lake water

    DEFF Research Database (Denmark)

    Nicolaisen, Mette Haubjerg; Worm, Jakob; Jørgensen, Niels O. G.;

    2012-01-01

    Proteins may be an important carbon and nitrogen source to bacteria in aquatic habitats, yet knowledge on the actual utilization of this substrate by proteolytic bacteria is scarce. In the present study, Pseudomonas fluorescens ON2 produced an alkaline proteinase (AprX) during growth...... and there was no evidence for cell density-regulated or starvation-induced proteinase production. Proteinase was produced in the absence of an organic nitrogen source, and citrate had a negative while glucose had a positive effect on the production. Hence P. fluorescens ON2 seems to exploit protein sources by expressing...

  14. N-Terminal Domain of Feline Calicivirus (FCV) Proteinase-Polymerase Contributes to the Inhibition of Host Cell Transcription.

    Science.gov (United States)

    Wu, Hongxia; Zu, Shaopo; Sun, Xue; Liu, Yongxiang; Tian, Jin; Qu, Liandong

    2016-01-01

    Feline Calicivirus (FCV) infection results in the inhibition of host protein synthesis, known as "shut-off". However, the precise mechanism of shut-off remains unknown. Here, we found that the FCV strain 2280 proteinase-polymerase (PP) protein can suppress luciferase reporter gene expression driven by endogenous and exogenous promoters. Furthermore, we found that the N-terminal 263 aa of PP (PPN-263) determined its shut-off activity using the expression of truncated proteins. However, the same domain of the FCV strain F9 PP protein failed to inhibit gene expression. A comparison between strains 2280 and F9 indicated that Val27, Ala96 and Ala98 were key sites for the inhibition of host gene expression by strain 2280 PPN-263, and PPN-263 exhibited the ability to shut off host gene expression as long as it contained any two of the three amino acids. Because the N-terminus of the PP protein is required for its proteinase and shut-off activities, we investigated the ability of norovirus 3C-like proteins (3CLP) from the GII.4-1987 and -2012 isolates to interfere with host gene expression. The results showed that 3CLP from both isolates was able to shut off host gene expression, but 3CLP from GII.4-2012 had a stronger inhibitory activity than that from GII.4-1987. Finally, we found that 2280 PP and 3CLP significantly repressed reporter gene transcription but did not affect mRNA translation. Our results provide new insight into the mechanism of the FCV-mediated inhibition of host gene expression. PMID:27447663

  15. Vegetative Storage Protein with Trypsin Inhibitor Activity Occurs in Sapindus mukorassi,a Sapindaceae Deciduous Tree

    Institute of Scientific and Technical Information of China (English)

    Shi-Biao Liu; Xu-Chu Wang; Min-Jing Shi; Yue-Yi Chen; Zheng-Hai Hu; Wei-Min Tian

    2009-01-01

    A vegetative storage protein (VSP) with trypsin inhibitor activity in a deciduous tree,Sapindus mukorassi,was characterized by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis,Western-blot,immuno-histochemical localization,light- and electro-microscopy,together with analysis of proteinase inhibitor activity of the purified VSP in vitro.There were two proteins with molecular masses of about 23 and 27 kDa in a relatively high content in the bark tissues of terminal branches of S.mukorassi in leafless periods.The proteins decreased markedly during young shoot development,indicating their role in seasonal nitrogen storage.Immuno-histochemical localization with the polyclonal antibodies raised against the 23 kDa protein demonstrated that the 23 kDa protein was the major component of protein inclusions in protein-storing cells.The protein inclusions were identified by protein-specific staining and should correspond to the electron-dense materials in different forms in the vacuoles of phloem parenchyma cells and phloem ray parenchyma cells under an electron microscope.So,the 23 kDa protein was a typical VSP in S.mukorassi.The 23 and 27 kDa proteins shared no immuno-relatedness,whereas the 23 kDa protein was immuno-related with the 22 kDa VSP in lychee and possessed trypsin inhibitor activity.The 23 kDa protein may confer dual functions:nitrogen storage and defense.

  16. Sunflower trypsin inhibitor-1.

    Science.gov (United States)

    Korsinczky, Michael L J; Schirra, Horst Joachim; Craik, David J

    2004-10-01

    SFTI-1 is a bicyclic 14 amino acid peptide that was originally isolated from the seeds of the sunflower Helianthus annuus. It is a potent inhibitor of trypsin, with a sub-nanomolar K(i) value and is homologous to the active site region of the well-known family of serine protease inhibitors known as the Bowman-Birk trypsin inhibitors. It has a cyclic backbone that is cross-braced by a single disulfide bridge and a network of hydrogen bonds that result in a well-defined structure. SFTI-1 is amenable to chemical synthesis, allowing for the creation of synthetic variants. Alterations to the structure such as linearising the backbone or removing the disulfide bridge do not reduce the potency of SFTI-1 significantly, and minimising the peptide to as few as nine residues results in only a small decrease in reactivity. The creation of linear variants of SFTI-1 also provides a tool for investigating putative linear precursor peptides. The mechanism of biosynthesis of SFTI-1 is not yet known but it seems likely that it is a gene-coded product that has arisen from a precursor protein that may be evolutionarily related to classic Bowman-Birk inhibitors. PMID:15544530

  17. Inhibitors of histone demethylases

    DEFF Research Database (Denmark)

    Lohse, Brian; Kristensen, Jesper L; Kristensen, Line H;

    2011-01-01

    Methylated lysines are important epigenetic marks. The enzymes involved in demethylation have recently been discovered and found to be involved in cancer development and progression. Despite the relative recent discovery of these enzymes a number of inhibitors have already appeared. Most of the i...

  18. Inhibitors of histone deacetylase

    DEFF Research Database (Denmark)

    2015-01-01

    The present invention relates to compounds of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, wherein X1, X2, X3, X4, X5, W1, W2, W3, and W4 are as described. The present invention relates generally to inhibitors of histone deacetylase and to methods...

  19. Suppression of Tumor Growth in Mice by Rationally Designed Pseudopeptide Inhibitors of Fibroblast Activation Protein and Prolyl Oligopeptidase

    Directory of Open Access Journals (Sweden)

    Kenneth W. Jackson

    2015-01-01

    Full Text Available Tumor microenvironments (TMEs are composed of cancer cells, fibroblasts, extracellular matrix, microvessels, and endothelial cells. Two prolyl endopeptidases, fibroblast activation protein (FAP and prolyl oligopeptidase (POP, are commonly overexpressed by epithelial-derived malignancies, with the specificity of FAP expression by cancer stromal fibroblasts suggesting FAP as a possible therapeutic target. Despite overexpression in most cancers and having a role in angiogenesis, inhibition of POP activity has received little attention as an approach to quench tumor growth. We developed two specific and highly effective pseudopeptide inhibitors, M83, which inhibits FAP and POP proteinase activities, and J94, which inhibits only POP. Both suppressed human colon cancer xenograft growth >90% in mice. By immunohistochemical stains, M83- and J94-treated tumors had fewer microvessels, and apoptotic areas were apparent in both. In response to M83, but not J94, disordered collagen accumulations were observed. Neither M83- nor J94-treated mice manifested changes in behavior, weight, or gastrointestinal function. Tumor growth suppression was more extensive than noted with recently reported efforts by others to inhibit FAP proteinase function or reduce FAP expression. Diminished angiogenesis and the accompanying profound reduction in tumor growth suggest that inhibition of either FAP or POP may offer new therapeutic approaches that directly target TMEs.

  20. [JAK2 inhibitors].

    Science.gov (United States)

    Hernández Boluda, Juan Carlos; Gómez, Montse; Pérez, Ariadna

    2016-07-15

    Pharmacological inhibition of the kinase activity of JAK proteins can interfere with the signaling of immunomodulatory cytokines and block the constitutive activation of the JAK-STAT pathway that characterizes certain malignancies, including chronic myeloproliferative neoplasms. JAK inhibitors may, therefore, be useful to treat malignancies as well as inflammatory or immune disorders. Currently, the most significant advances have been made in the treatment of myelofibrosis, where these drugs may lead to a remarkable improvement in the control of hyperproliferative manifestations. However, available data suggest that this treatment is not curative of myelofibrosis. In general, JAK2 inhibition induces cytopaenias, with this being considered a class side-effect. By contrast, the extrahaematologic toxicity profile varies significantly among the different JAK inhibitors. At present, there are several clinical trials evaluating the combination of ruxolitinib with other drugs, in order to improve its therapeutic activity as well as reducing haematologic toxicity. PMID:27033437