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Sample records for alpha proteinase inhibitor

  1. [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

  2. Identification and characterization of alpha-I-proteinase inhibitor from common carp sarcoplasmic proteins.

    Science.gov (United States)

    Siriangkanakun, Siriphon; Li-Chan, Eunice C Y; Yongsawadigul, Jirawat

    2016-02-01

    Purification of proteinase inhibitor from common carp (Cyprinus carpio) sarcoplasmic proteins resulted in 2.8% yield with purification fold of 111. Two inhibitors, namely inhibitor I and II, exhibited molecular mass of 47 and 52 kDa, respectively, based on non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both inhibitors I and II were identified to be alpha-1-proteinase inhibitor (α1-PI) based on LC-MS/MS. They were glycoproteins and molecular mass after peptide-N-glycosidase F treatment was 38 and 45 kDa, respectively. The N-glycosylation sites of both inhibitors were determined to be at N214 and N226. The inhibitors specifically inhibited trypsin. The common carp α1-PI showed high thermal stability with denaturation temperatures of 65.43 and 73.31 °C, which were slightly less than those of ovomucoid. High stability toward NaCl was also evident up to 3M. The common carp α1-PI effectively reduced autolytic degradation of bigeye snapper surimi at the concentration as low as 0.025%. PMID:26304452

  3. [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

  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. Intravenous administration of alpha-1-proteinase inhibitor in patients of PiZ and PiM phenotype. Preliminary report

    International Nuclear Information System (INIS)

    Nine patients with moderate pulmonary emphysema, six of PiZ phenotype and three of PiM phenotype, have received a single intravenous infusion of alpha-1-proteinase inhibitor (human) (A1PI), in a dose of 60 mg/kg over a 30-minute period. They also received a tracer dose (300 microCi) of 131I-labeled A1PI. No active or passive immunization against hepatitis was given. No acute toxicity was observed. Compared with baseline data, significant elevations of serum A1PI (measured both antigenically and as anti-elastase activity) occurred, with a serum half-life approximating 110 hours. Bronchoalveolar lavage fluid, obtained 48 hours after infusion, reflected a significant increase in A1PI concentration versus baseline bronchoalveolar lavage fluid values. Serial gamma camera images of the lungs confirmed persistence of enhanced lung radioactivity for several days. Urinary desmosine excretion did not change following A1PI infusion. During the period of follow-up thus far, no patient has had chronic toxicity, results of liver function tests have been stable, and there has been no development of hepatitis B antigen or antibodies to hepatitis B surface or core antigens

  6. Alpha-1 proteinase inhibitors for the treatment of alpha-1 antitrypsin deficiency: safety, tolerability, and patient outcomes

    Directory of Open Access Journals (Sweden)

    Chotirmall SH

    2015-01-01

    Full Text Available Sanjay H Chotirmall,1 Mazen Al-Alawi,2 Thomas McEnery,2 Noel G McElvaney2 1Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore; 2Department of Respiratory Medicine, Beaumont Hospital, Dublin, Republic of Ireland Abstract: Alpha-1 antitrypsin (AAT deficiency remains an underrecognized genetic disease with predominantly pulmonary and hepatic manifestations. AAT is derived primarily from hepatocytes; however, macrophages and neutrophils are secondary sources. As the natural physiological inhibitor of several proteases, most importantly neutrophil elastase (NE, it plays a key role in maintaining pulmonary protease–antiprotease balance. In deficient states, unrestrained NE activity promotes damage to the lung matrix, causing structural defects and impairing host defenses. The commonest form of AAT deficiency results in a mutated Z AAT that is abnormally folded, polymerized, and aggregated in the liver. Consequently, systemic levels are lower, resulting in diminished pulmonary concentrations. Hepatic disease occurs due to liver aggregation of the protein, while lung destruction ensues from unopposed protease-mediated damage. In this review, we will discuss AAT deficiency, its clinical manifestations, and augmentation therapy. We will address the safety and tolerability profiles of AAT replacement in the context of patient outcomes and cost-effectiveness and outline future directions for work in this field. Keywords: alpha-1, augmentation, deficiency, replacement, emphysema

  7. Alpha-1 proteinase inhibitors for the treatment of alpha-1 antitrypsin deficiency: safety, tolerability, and patient outcomes

    OpenAIRE

    Chotirmall, Sanjay

    2015-01-01

    Sanjay H Chotirmall,1 Mazen Al-Alawi,2 Thomas McEnery,2 Noel G McElvaney2 1Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore; 2Department of Respiratory Medicine, Beaumont Hospital, Dublin, Republic of Ireland Abstract: Alpha-1 antitrypsin (AAT) deficiency remains an underrecognized genetic disease with predominantly pulmonary and hepatic manifestations. AAT is derived primarily from hepatocytes; however, macrophages and neutrophils are secondary sources. As the...

  8. [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

  9. 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

  10. Retroviral proteinases and their inhibitors

    Czech Academy of Sciences Publication Activity Database

    Sedláček, Juraj

    2000-01-01

    Roč. 3, 3,4 (2000), s. 23-24. [Proteolytic enzymes and their inhibitors in physiology and pathogenesis.. 14.09.2000, Plzen] Institutional research plan: CEZ:AV0Z5052915 Subject RIV: EB - Genetics ; Molecular Biology

  11. 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

  12. Action of plant proteinase inhibitors on enzymes of physiopathological importance

    OpenAIRE

    Oliva, Maria Luiza V.; Sampaio, Misako U.

    2009-01-01

    Obtained from leguminous seeds, various plant proteins inhibit animal proteinases, including human, and can be considered for the development of compounds with biological activity. Inhibitors from the Bowman-Birk and plant Kunitz-type family have been characterized by proteinase specificity, primary structure and reactive site. Our group mostly studies the genus Bauhinia, mainly the species bauhinioides, rufa, ungulata and variegata. In some species, more than one inhibitor was characterized,...

  13. Action of plant proteinase inhibitors on enzymes of physiopathological importance.

    Science.gov (United States)

    Oliva, Maria Luiza V; Sampaio, Misako U

    2009-09-01

    Obtained from leguminous seeds, various plant proteins inhibit animal proteinases, including human, and can be considered for the development of compounds with biological activity. Inhibitors from the Bowman-Birk and plant Kunitz-type family have been characterized by proteinase specificity, primary structure and reactive site. Our group mostly studies the genus Bauhinia, mainly the species bauhinioides, rufa, ungulata and variegata. In some species, more than one inhibitor was characterized, exhibiting different properties. Although proteins from this group share high structural similarity, they present differences in proteinase inhibition, explored in studies using diverse biological models. PMID:19722028

  14. Action of plant proteinase inhibitors on enzymes of physiopathological importance

    Directory of Open Access Journals (Sweden)

    Maria Luiza V. Oliva

    2009-09-01

    Full Text Available Obtained from leguminous seeds, various plant proteins inhibit animal proteinases, including human, and can be considered for the development of compounds with biological activity. Inhibitors from the Bowman-Birk and plant Kunitz-type family have been characterized by proteinase specificity, primary structure and reactive site. Our group mostly studies the genus Bauhinia, mainly the species bauhinioides, rufa, ungulata and variegata. In some species, more than one inhibitor was characterized, exhibiting different properties. Although proteins from this group share high structural similarity, they present differences in proteinase inhibition, explored in studies using diverse biological models.Obtidas de sementes leguminosas, várias proteínas inibem proteinases de origem animal, incluindo humanas, e podem ser consideradas para o desenvolvimento de compostos com atividade biológica. Inibidores da família Bowman-Birk e da família Kunitz vegetal tem sido caracterizados em relação a especificidade para proteinase, estrutura primária e sitio reativo. O nosso grupo majoritariamente vem estudando o gênero Bauhinia, principalmente as espécies bauhinioides, rufa, ungulatae variegata. Em algumas espécies, mais de um inibidor com propriedades diferentes foi caracterizado. Embora tais proteínas apresentem alta similaridade estrutural, diferem quanto à inibição de proteinases, e foram exploradas em estudos utilizando diversos modelos biológicos.

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

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    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

  16. Characterization of peptide proteinase inhibitors isolated from boar seminal plasma

    Czech Academy of Sciences Publication Activity Database

    Jelínková, Petra; Tichá, M.; Jonáková, Věra

    Praha : UOCHB AV ČR, 2003 - (Slaninová, J.; Collinsová, M.; Klasová, L.), s. 1-57 [Biologicky aktivní peptidy /8./. Praha (CZ), 23.04.2003-25.04.2003] R&D Projects: GA ČR GA303/99/0357; GA ČR GP303/02/P069; GA MZd NJ7463 Institutional research plan: CEZ:MSM 113100001 Keywords : boar seminal plasma proteins * proteinase inhibitors Subject RIV: CE - Biochemistry

  17. 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.

  18. Characterization of a novel Kazal-type serine proteinase inhibitor of Arabidopsis thaliana.

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    Pariani, Sebastián; Contreras, Marisol; Rossi, Franco R; Sander, Valeria; Corigliano, Mariana G; Simón, Francisco; Busi, María V; Gomez-Casati, Diego F; Pieckenstain, Fernando L; Duschak, Vilma G; Clemente, Marina

    2016-04-01

    Many different types of serine proteinase inhibitors have been involved in several kinds of plant physiological processes, including defense mechanisms against phytopathogens. Kazal-type serine proteinase inhibitors, which are included in the serine proteinase inhibitor family, are present in several organisms. These proteins play a regulatory role in processes that involve serine proteinases like trypsin, chymotrypsin, thrombin, elastase and/or subtilisin. In the present work, we characterized two putative Kazal-type serine proteinase inhibitors from Arabidopsis thaliana, which have a single putative Kazal-type domain. The expression of these inhibitors is transiently induced in response to leaf infection by Botrytis cinerea, suggesting that they play some role in defense against pathogens. We also evaluated the inhibitory specificity of one of the Kazal-type serine proteinase inhibitors, which resulted to be induced during the local response to B. cinerea infection. The recombinant Kazal-type serine proteinase inhibitor displayed high specificity for elastase and subtilisin, but low specificity for trypsin, suggesting differences in its selectivity. In addition, this inhibitor exhibited a strong antifungal activity inhibiting the germination rate of B. cinerea conidia in vitro. Due to the important role of proteinase inhibitors in plant protection against pathogens and pests, the information about Kazal-type proteinase inhibitors described in the present work could contribute to improving current methods for plant protection against pathogens. PMID:26853817

  19. 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. PMID:11545387

  20. 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

  1. Structure and function of invertebrate Kazal-type serine proteinase inhibitors.

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    Rimphanitchayakit, Vichien; Tassanakajon, Anchalee

    2010-04-01

    Proteinases and proteinase inhibitors are involved in several biological and physiological processes in all multicellular organisms. The proteinase inhibitors function as modulators for controlling the extent of deleterious proteinase activity. The Kazal-type proteinase inhibitors (KPIs) in family I1 are among the well-known families of proteinase inhibitors, widely found in mammals, avian and a variety of invertebrates. Like those classical KPIs, the invertebrate KPIs can be single or multiple domain proteins containing one or more Kazal inhibitory domains linked together by peptide spacers of variable length. All invertebrate Kazal domains of about 40-60 amino acids in length share a common structure which is dictated by six conserved cysteine residues forming three intra-domain disulfide cross-links despite the variability of amino acid sequences between the half-cystines. Invertebrate KPIs are strong inhibitors as shown by their extremely high association constant of 10(7)-10(13)M(-1). The inhibitory specificity of a Kazal domain varies widely with a different reactive P(1) amino acid. Different invertebrate KPI domains may arise from gene duplication but several KPI proteins can also be derived from alternative splicing. The invertebrate KPIs function as anticoagulants in blood-sucking animals such as leech, mosquitoes and ticks. Several KPIs are likely involved in protecting host from microbial proteinases while some from the parasitic protozoa help protecting the parasites from the host digestive proteinase enzymes. Silk moths produce KPIs to protect their cocoon from predators and microbial destruction. PMID:19995574

  2. Serine proteinase inhibitors from nematodes and the arms race between host and pathogen

    OpenAIRE

    Zang, Xingxing; Maizels, Rick

    2001-01-01

    Parasite nematode genomics is a relatively new field9, but already two of the most interesting gene families to be found encode serine proteinase inhibitors. This article describes a family of nematode proteinase inhibitors with homology to mammalian serpins, as well as a distinct set of lower-molecularweight inhibitors first discovered by biochemical analysis of the human roundworm Ascaris10.Taking these two examples into account, it thus appears that parasitic nem...

  3. Alpha glucosidase inhibitors.

    Science.gov (United States)

    Kalra, Sanjay

    2014-04-01

    Alpha glucosidase inhibitors (AGIs) are a unique class of anti-diabetic drugs. Derived from bacteria, these oral drugs are enzyme inhibitors which do not have a pancreato -centred mechanism of action. Working to delay carbohydrate absorption in the gastrointestinal tract, they control postprandial hyperglycaemia and provide unquestioned cardiovascular benefit. Specially suited for a traditional Pakistani carbohydrate-rich diet, AGIs have been termed the 'untapped diamonds' of diabetology. The use of these oral antidiabetic drugs (OADs) that target pathophysiology in the early stages of type 2 diabetes, notably to reduce postprandial hyperglycaemia and hyperinsulinaemia will inevitably increase with time. This review describes the history of their development, mechanism of action, basic and clinical pharmacology, and suggests practical, evidence-based guidance for their optimal use. PMID:24864650

  4. 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.

  5. Thiol proteinase inhibitor - Oryzacystatin. Molecular cloning and expression in E. coli

    International Nuclear Information System (INIS)

    Insect depredation is a major reason for the reduction in crop yields world-wide. Promising results have already been achieved with transgenic plants expression cowpea trypsin inhibitor (CpTI) genes and modified delta-endotoxin genes. Insects, in general, hydrolyse ingested proteins with a variety of proteinase. The effect of the serine proteinase inhibitor against Lepidopteran insects is probably caused by the preponderance of serine type gut proteinase and a luminal pH in the neutral to alkaline range. On the other hand, the insect orders Coleoptera and Hemiptera have gut pHs in the mildly acidic range and commonly have thiol type gut proteinases. Plant transformation with a gene coding for a thiol proteinase inhibitor has been suggested as a strategy for interfering with the digestive physiology of Coleopteran and Hemipteran insects. Co-transformation of both the serine proteinase inhibitor and the thiol proteinase inhibitor genes might result in a broader spectrum of activity and increased durability of protection

  6. 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

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

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    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

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

    Directory of Open Access Journals (Sweden)

    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.

  9. The aspartic proteinase from Saccharomyces cerevisiae folds its own inhibitor into a helix

    DEFF Research Database (Denmark)

    Li, M; Phylip, L H; Lees, W E; Winther, Jakob R.; Dunn, B M; Wlodawer, A; Kay, J; Gustchina, A

    2000-01-01

    Aspartic proteinase A from yeast is specifically and potently inhibited by a small protein called IA3 from Saccharomyces cerevisiae. Although this inhibitor consists of 68 residues, we show that the inhibitory activity resides within the N-terminal half of the molecule. Structures solved at 2.2 a...

  10. 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.

  11. The Characterization of SaPIN2b, a Plant Trichome-Localized Proteinase Inhibitor from Solanum americanum

    Directory of Open Access Journals (Sweden)

    Zeng-Fu Xu

    2012-11-01

    Full Text Available Proteinase inhibitors play an important role in plant resistance of insects and pathogens. In this study, we characterized the serine proteinase inhibitor SaPIN2b, which is constitutively expressed in Solanum americanum trichomes and contains two conserved motifs of the proteinase inhibitor II (PIN2 family. The recombinant SaPIN2b (rSaPIN2b, which was expressed in Escherichia coli, was demonstrated to be a potent proteinase inhibitor against a panel of serine proteinases, including subtilisin A, chymotrypsin and trypsin. Moreover, rSaPIN2b also effectively inhibited the proteinase activities of midgut trypsin-like proteinases that were extracted from the devastating pest Helicoverpa armigera. Furthermore, the overexpression of SaPIN2b in transgenic tobacco plants resulted in enhanced resistance against H. armigera. Taken together, our results demonstrated that SaPIN2b is a potent serine proteinase inhibitor that may act as a protective protein in plant defense against insect attacks.

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

    Science.gov (United States)

    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

  13. 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.

  14. Proteinase inhibitory activities of two two-domain Kazal proteinase inhibitors from the freshwater crayfish Pacifastacus leniusculus and the importance of the P(2) position in proteinase inhibitory activity.

    Science.gov (United States)

    Donpudsa, Suchao; Söderhäll, Irene; Rimphanitchayakit, Vichien; Cerenius, Lage; Tassanakajon, Anchalee; Söderhäll, Kenneth

    2010-11-01

    Serine proteinase inhibitors are found ubiquitously in living organisms and involved in homeostasis of processes using proteinases as well as innate immune defense. Two two-domain Kazal-type serine proteinase inhibitors (KPIs), KPI2 and KPI8, have been identified from the hemocyte cDNA library of the crayfish Pacifastacus leniusculus. Unlike other KPIs from P. leniusculus, they are found specific to the hemocytes and contain an uncommon P(2) amino acid residue, Gly. To unveil their inhibitory activities, the two KPIs and their domains were over-expressed. By testing against subtilisin, trypsin, chymotrypsin and elastase, the KPI2 was found to inhibit strongly against subtilisin and weakly against trypsin, while the KPI8 was strongly active against only trypsin. With their P(1) Ser and Lys residues, the KPI2_domain2 and KPI8_domain2 were responsible for strong inhibition against subtilisin and trypsin, respectively. Mutagenesis of KPI8_domain1 at P(2) amino acid residue from Gly to Pro, mimicking the P(2) residue of KPI8_domain2, rendered the KPI8_domain1 strongly active against trypsin, indicating the important role of P(2) residue in inhibitory activities of the Kazal-type serine proteinase inhibitors. Only the KPI2 was found to inhibit against the extracellular serine proteinases from the pathogenic oomycete of the freshwater crayfish, Aphanomyces astaci. PMID:20621193

  15. Development and bioassay of transgenic Chinese cabbage expressing potato proteinase inhibitor II gene

    OpenAIRE

    Zhang, Junjie; Liu, Fan; Yao, Lei; Luo, Chen; Yin, Yue; Wang, Guixiang; Huang, Yubi

    2012-01-01

    Lepidopteran larvae are the most injurious pests of Chinese cabbage production. We attempted the development of transgenic Chinese cabbage expressing the potato proteinase inhibitor II gene (pinII) and bioassayed the pest-repelling ability of these transgenic plants. Cotyledons with petioles from aseptic seedlings were used as explants for Agrobacterium-mediated in vitro transformation. Agrobacterium tumefaciens C58 contained the binary vector pBBBasta-pinII-bar comprising pinII and bar genes...

  16. Sulfonate salts of amino acids: novel inhibitors of the serine proteinases.

    Science.gov (United States)

    Groutas, W C; Brubaker, M J; Zandler, M E; Stanga, M A; Huang, T L; Castrisos, J C; Crowley, J P

    1985-04-16

    A series of amino acid-derived sulfonate salts have been synthesized. They were found to inactivate efficiently and selectively human leukocyte elastase. The sulfonate salts of the methyl esters of L-norleucine, L-norvaline and L-valine were the most potent. The enzyme is inactivated irreversibly with concomitant release of bisulfite ion. The results demonstrate for the first time that ionic compounds can indeed function as novel inhibitors for the serine proteinases. PMID:3885950

  17. 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.

  18. 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...

  19. On the structure, function and biosynthesis of human inter-. alpha. inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Swaim, M.W.

    1989-01-01

    Human inter-{alpha} inhibitor (I{alpha}I) is a {approx}200-kD serum glycoprotein with serine proteinase-inhibitory activity whose physiologic role remains unclear. I{alpha}I is related to smaller inhibitors found in physiologic fluids and is a complex of {approx}40-kD light and {approx}90-kD heavy chains. I{alpha}I proteinase-inhibitory activity resides exclusively in the light chain, which has tandem Kunitz inhibitory domains with methionine and arginine residues, respectively, at position P{sub 1}. The inhibitory activity of the reactive centers was heretofore uncharacterized. Cis-dichlorodiammineplatinum (II) (cis-DDP) reacts with sulfur containing residues in a limited and selective fashion. In preliminary studies, cis-DDP was evaluated as a reagent to modify the methionine reactive centers of two other plasma proteinase inhibitors, {alpha}{sub 1}-antitrypsin and {alpha}{sub 2}-antiplasmin. Cis-DDP readily abolished the proteinase-inhibitory activity of both proteins. Methionine oxidation, papain digestion, and platinum binding assays showed that cis-DDP inactivates {alpha}-antitrypsin by binding exclusively to its reactive-center methionine. Cis-DDP partially eliminated I{alpha}I inhibitory activity against cathepsin G and neutrophil elastase but did not affect inhibition of trypsin or chymotrypsin. Conversely, reaction with the arginine-modifying reagent 2,3-butanedione afforded complete loss of activity against trypsin and chymotrypsin but partial loss of activity against cathepsin G and elastase. Employment of both reagents eliminated inhibition of cathepsin G and elastase. Thus eathepsin G and elastase are apparently inhibited at either reactive center. Trypsin and chymotrypsin are inhibited exclusively at the arginine reactive center.

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

    Science.gov (United States)

    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

  1. Kazal-type proteinase inhibitor from disk abalone (Haliotis discus discus): molecular characterization and transcriptional response upon immune stimulation.

    Science.gov (United States)

    Wickramaarachchi, W D Niroshana; De Zoysa, Mahanama; Whang, Ilson; Wan, Qiang; Lee, Jehee

    2013-09-01

    Proteinases and proteinase inhibitors are involved in several biological and physiological processes in all multicellular organisms. Proteinase inhibitors play a key role in regulating the activity of the respective proteinases. Among serine proteinase inhibitors, kazal-type proteinase inhibitors (KPIs) are widely found in mammals, avians, and a variety of invertebrates. In this study, we describe the identification of a kazal-type serine proteinase inhibitor (Ab-KPI) from the disk abalone, Haliotis discus discus, which is presumably involved in innate immunity. The full-length cDNA of Ab-KPI includes 600 bp nucleotides with an open reading frame (ORF) encoding a polypeptide of 143 amino acids. The deduced amino acid sequence of Ab-KPI contains a putative 17-amino acid signal peptide and two tandem kazal domains with high similarity to other kazal-type SPIs. Each kazal domain consists of reactive site (P1) residue containing a leucine (L), and a threonine (T) located in the second amino acid position after the second conserved cysteine of each domain. Temporal expression of Ab-KPI was assessed by real time quantitative PCR in hemocytes and mantle tissue following bacterial and viral hemorrhagic septicemia virus (VHSV) challenge, and tissue injury. At 6 h post-bacterial and -VHSV challenge, Ab-KPI expression in hemocytes was increased 14-fold and 4-fold, respectively, compared to control samples. The highest up-regulations upon tissue injury were shown at 9 h and 12 h in hemocytes and mantle, respectively. The transcriptional modulation of Ab-KPI following bacterial and viral challenges and tissue injury indicates that it might be involved in immune defense as well as wound healing process in abalone. PMID:23859879

  2. Cloning eleven midgut trypsin cDNAs and evaluating the interaction of proteinase inhibitors with Cry1Ac against the tobacco budworm Heliothis virescens (F.) (Lepidoptera: Noctuidae)

    Science.gov (United States)

    Midgut trypsins are associated with Bt protoxin activation and toxin degradation. Proteinase inhibitors have potential insecticidal toxicity against a wide range of insect species. Proactive action to examine trypsin gene profiles and proteinase inhibitors for interaction with Bt toxin is necessary ...

  3. Coexpression of potato type I and II proteinase inhibitors gives cotton plants protection against insect damage in the field

    OpenAIRE

    Dunse, K. M.; Stevens, J. A.; Lay, F. T.; Gaspar, Y. M.; Heath, R. L.; Anderson, M. A.

    2010-01-01

    Potato type I and II serine protease inhibitors are produced by solanaceous plants as a defense mechanism against insects and microbes. Nicotiana alata proteinase inhibitor (NaPI) is a multidomain potato type II inhibitor (pin II) that is produced at high levels in the female reproductive tissues of the ornamental tobacco, Nicotiana alata. The individual inhibitory domains of NaPI target the major classes of digestive enzymes, trypsin and chymotrypsin, in the gut of lepidopteran larval pests....

  4. 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

  5. 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.

  6. High sequence variability among hemocyte-specific Kazal-type proteinase inhibitors in decapod crustaceans.

    Science.gov (United States)

    Cerenius, Lage; Liu, Haipeng; Zhang, Yanjiao; Rimphanitchayakit, Vichien; Tassanakajon, Anchalee; Gunnar Andersson, M; Söderhäll, Kenneth; Söderhäll, Irene

    2010-01-01

    Crustacean hemocytes were found to produce a large number of transcripts coding for Kazal-type proteinase inhibitors (KPIs). A detailed study performed with the crayfish Pacifastacus leniusculus and the shrimp Penaeus monodon revealed the presence of at least 26 and 20 different Kazal domains from the hemocyte KPIs, respectively. Comparisons with KPIs from other taxa indicate that the sequences of these domains evolve rapidly. A few conserved positions, e.g. six invariant cysteines were present in all domain sequences whereas the position of P1 amino acid, a determinant for substrate specificity, varied highly. A study with a single crayfish animal suggested that even at the individual level considerable sequence variability among hemocyte KPIs produced exist. Expression analysis of four crayfish KPI transcripts in hematopoietic tissue cells and different hemocyte types suggest that some of these KPIs are likely to be involved in hematopoiesis or hemocyte release as they were produced in particular hemocyte types or maturation stages only. PMID:19715720

  7. 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.

  8. 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...

  9. 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

  10. 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

  11. Cysteine proteinase inhibitor in eccrine sweat is derived from sweat gland.

    Science.gov (United States)

    Yokozeki, H; Hibino, T; Takemura, T; Sato, K

    1991-02-01

    Although cysteine proteinases have been reported to be present in human eccrine sweat, their endogenous inhibitors, cysteine proteinase inhibitors (CPIs), have remained unstudied. We now present evidence that CPIs are indeed a true ingredient of human eccrine sweat. Sweat induced in sauna was collected over a Vaseline barrier placed on the skin to minimize epidermal contamination. The absence of major epidermal contamination of the sweat was further ensured by monitoring an epidermal marker, high-molecular-mass aminopeptidase. Sweat CPI was purified sequentially by chromatography with Sephacryl S-200, carboxymethylated papain-Sepharose, and anion-exchange Mono Q fast-protein liquid chromatography columns. Sweat CPI has a molecular mass of approximately 15 kDa, is stable for temperature (up to 80 degrees C) and pH (from 3 to 10), and inhibits papain, ficin, and sweat cathepsin B- and H-like enzymes. Sweat CPI may be of sweat gland origin because 1) the rate of CPI output in sweat (CPI concentration x sweat rate) is constant over 45 min; 2) antibody against epidermal CPI, which cross-reacts with sweat CPI, localized immunoreactivity in the sweat duct; 3) CPI activity was present in the glandular extracts of control and methacholine-stimulated (for 1 h in vitro) human sweat glands; and 4) the peaks of CPI activity in the glandular extract and sweat CPI were both eluted (by high-pressure liquid chromatography) at around 15 kDa. Sweat CPI may be very similar to epidermal CPI (which belongs to the stefin family of CPIs) because of many shared characteristics. The identity and function of sweat CPI remain to be studied. PMID:1899981

  12. 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

  13. 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.

  14. Differential induction of two procesing proteases controls the processing pattern of the trypsin proteinase inhibitor precursor in Nicotiana attenuata

    Czech Academy of Sciences Publication Activity Database

    Horn, Martin; Patankar, A. G.; Zavala, J. A.; Wu, J.; Marešová, Lucie; Vůjtěchová, Milana; Mareš, Michael; Baldwin, I. T.

    Ljubljana : -, 2005. s. 94. [International Symposium on Proteinase Inhibitors and Biological Control /9./. 25.06.2005-29.06.2005, Brdo Estate] R&D Projects: GA AV ČR(CZ) IAA4055303; GA ČR(CZ) GA522/04/1286 Institutional research plan: CEZ:AV0Z40550506 Keywords : posttranslational modifications * differential fragmentation * vacuolar processing enzyme Subject RIV: CE - Biochemistry

  15. 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

  16. Development and bioassay of transgenic Chinese cabbage expressing potato proteinase inhibitor II gene.

    Science.gov (United States)

    Zhang, Junjie; Liu, Fan; Yao, Lei; Luo, Chen; Yin, Yue; Wang, Guixiang; Huang, Yubi

    2012-06-01

    Lepidopteran larvae are the most injurious pests of Chinese cabbage production. We attempted the development of transgenic Chinese cabbage expressing the potato proteinase inhibitor II gene (pinII) and bioassayed the pest-repelling ability of these transgenic plants. Cotyledons with petioles from aseptic seedlings were used as explants for Agrobacterium-mediated in vitro transformation. Agrobacterium tumefaciens C58 contained the binary vector pBBBasta-pinII-bar comprising pinII and bar genes. Plants showing vigorous PPT resistance were obtained by a series concentration selection for PPT resistance and subsequent regeneration of leaf explants dissected from the putative chimera. Transgenic plants were confirmed by PCR and genomic Southern blotting, which showed that the bar and pinII genes were integrated into the plant genome. Double haploid homozygous transgenic plants were obtained by microspore culture. The pinII expression was detected using quantitative real time polymerase chain reaction (qRT-PCR) and detection of PINII protein content in the transgenic homozygous lines. Insect-feeding trials using the larvae of cabbage worm (Pieris rapae) and the larvae of the diamondback moth (Plutella xylostella) showed higher larval mortality, stunted larval development, and lower pupal weights, pupation rates, and eclosion rates in most of the transgenic lines in comparison with the corresponding values in the non-transformed wild-type line. PMID:23136521

  17. Human cysteine-proteinase inhibitors: nucleotide sequence analysis of three members of the cystatin gene family.

    Science.gov (United States)

    Saitoh, E; Kim, H S; Smithies, O; Maeda, N

    1987-01-01

    Three genes from the human cystatin gene family of cysteine-proteinase inhibitors have been isolated from a bacteriophage lambda library containing HindIII digests of human genomic DNA. Two of the genes code for salivary cystatin SN and SA, the third is a pseudogene. The cloned genes were identified with a probe made from a salivary cystatin cDNA. The complete nucleotide sequence of the gene that codes for the precursor form of the neutral salivary protein, cystatin SN, was determined. The gene, which we name CST1, contains three exons and two intervening sequences. The expected CAT and ATA boxes are present in the 5'-flanking region of the gene. Partial nucleotide sequence determination of a second gene revealed that it codes for the precursor form of the acidic salivary protein, cystatin SA. This gene, which we name CST2, has the same gene organization as CST1. The complete nucleotide sequence of a third gene was determined. It does not contain a typical ATA box, and in addition, a premature stop codon and a frameshift deletion mutation occur within the gene. These inactivation mutations show that this gene, which we name CSTP1, is a cystatin pseudogene. These data combined with our genomic Southern-blot analyses show that the cystatin genes form a multigene family with at least seven members. PMID:3446578

  18. 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

  19. Biochemical and immunological characterization of a recombinantly-produced antifungal cysteine proteinase inhibitor from green kiwifruit (Actinidia deliciosa).

    Science.gov (United States)

    Popovic, Milica; Andjelkovic, Uros; Burazer, Lidija; Lindner, Buko; Petersen, Arnd; Gavrovic-Jankulovic, Marija

    2013-10-01

    Plant proteinase inhibitors are considered important defense molecules against insect and pathogen attack. The cysteine proteinase inhibitor (CPI) from green kiwifruit (Actinidia deliciosa) belongs to the cystatin family and shows potent antifungal activity (in vitro and in vivo). However, the low abundance of this molecule in fruit (6μg/g of fresh fruit) seems to limit further investigations on the interaction between phytocystatin and photopathogenic fungi. In this paper the cDNA of the kiwi CPI was expressed in Escherichia coli. Fifteen N-terminal amino acids were identified by Edman degradation, and 77% of the rCPI primary structure was confirmed by mass fingerprint. The structural homology of recombinant CPI (rCPI) to its natural counterpart has been clearly demonstrated in immunological assays (immunoblot and ELISA inhibition). Biological activity of rCPI was demonstrated in inhibition assay with cysteine proteinase papain (EC50 2.78nM). In addition, rCPI reveals antifungal properties toward pathogenic fungi (Alternaria radicina and Botrytis cinerea), which designates it as an interesting model protein for the exploration of plant phytocystatins - pathogen interactions. Understanding the molecular mechanisms of natural plant resistance could lead to the development of ecologically safe fungicides for controlling post-harvest diseases and maintaining food quality. PMID:23830694

  20. Selective loss of cysteine residues and disulphide bonds in a potato proteinase inhibitor II family.

    Directory of Open Access Journals (Sweden)

    Xiu-Qing Li

    Full Text Available Disulphide bonds between cysteine residues in proteins play a key role in protein folding, stability, and function. Loss of a disulphide bond is often associated with functional differentiation of the protein. The evolution of disulphide bonds is still actively debated; analysis of naturally occurring variants can promote understanding of the protein evolutionary process. One of the disulphide bond-containing protein families is the potato proteinase inhibitor II (PI-II, or Pin2, for short superfamily, which is found in most solanaceous plants and participates in plant development, stress response, and defence. Each PI-II domain contains eight cysteine residues (8C, and two similar PI-II domains form a functional protein that has eight disulphide bonds and two non-identical reaction centres. It is still unclear which patterns and processes affect cysteine residue loss in PI-II. Through cDNA sequencing and data mining, we found six natural variants missing cysteine residues involved in one or two disulphide bonds at the first reaction centre. We named these variants Pi7C and Pi6C for the proteins missing one or two pairs of cysteine residues, respectively. This PI-II-7C/6C family was found exclusively in potato. The missing cysteine residues were in bonding pairs but distant from one another at the nucleotide/protein sequence level. The non-synonymous/synonymous substitution (Ka/Ks ratio analysis suggested a positive evolutionary gene selection for Pi6C and various Pi7C. The selective deletion of the first reaction centre cysteine residues that are structure-level-paired but sequence-level-distant in PI-II illustrates the flexibility of PI-II domains and suggests the functionality of their transient gene versions during evolution.

  1. Proteinase treatment of intact hepatic mitochondria has differential effects on inhibition of carnitine palmitoyltransferase by different inhibitors.

    Science.gov (United States)

    Kashfi, K; Cook, G A

    1992-01-01

    Proteolysis of intact mitochondria by Nagarse (subtilisin BPN') and papain resulted in limited loss of activity of the outer-membrane carnitine palmitoyltransferase, but much greater loss of sensitivity to inhibition by malonyl-CoA. In contrast with a previous report [Murthy & Pande (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 378-382], we found that trypsin had no effect on malonyl-CoA sensitivity. Even when 80% of activity was destroyed by trypsin, there was no difference in the malonyl-CoA sensitivity of the enzyme remaining. Trypsin caused release of the intermembrane-space enzyme adenylate kinase, indicating loss of integrity of the mitochondrial outer membrane, whereas Nagarse and papain caused no release of that enzyme. Citrate synthase was not released by any of the three proteinases, indicating no damage to the mitochondrial inner membrane. When we examined the effects of proteolysis on the inhibition of carnitine palmitoyltransferase by a wide variety of inhibitors having different mechanisms of inhibition, we found differential proteolytic effects that were specific for those inhibitors (malonyl-CoA and hydroxyphenylglyoxylate) that have their inhibitory potencies diminished by changes in physiological state. Both of those inhibitors protected carnitine palmitoyltransferase from the effects of proteolysis, but did not inhibit the proteinases directly. Inhibition by two other inhibitors (DL-2-bromopalmitoyl-CoA and N-benzyladriamycin 14-valerate) was not altered by proteinase treatment, even when most of the enzyme activity had been destroyed. Inhibition by glyburide, which is minimally affected by physiological state, was affected only to a slight extent at the highest concentration of trypsin tested. Proteolysis by Nagarse appeared to produce loss of co-operativity in malonyl-CoA inhibition. The effects of proteolysis are discussed and compared with changes in Ki occurring with changing physiological states. PMID:1554374

  2. Effects of proteinase inhibitor from Adenanthera pavonina seeds on short- and long term larval development of Aedes aegypti.

    Science.gov (United States)

    Sasaki, Daniele Yumi; Jacobowski, Ana Cristina; de Souza, Antônio Pancrácio; Cardoso, Marlon Henrique; Franco, Octávio Luiz; Macedo, Maria Lígia Rodrigues

    2015-05-01

    Currently, one of the major global public health concerns is related to the transmission of dengue/yellow fever virus by the vector Aedes aegypti. The most abundant digestive enzymes in Ae. aegypti midgut larvae are trypsin and chymotrypsin. Since protease inhibitors have the capacity to bind to and inhibit the action of insect digestive proteinases, we investigated the short- and long-term effects of Adenanthera pavonina seed proteinase inhibitor (ApTI) on Ae. aegypti larvae, as well as a possible mechanism of adaptation. ApTI had a significant effect on Ae. aegypti larvae exposed to a non-lethal concentration of ApTI during short- and long-duration assays, decreasing survival, weight and proteinase activities of midgut extracts of larvae. The zymographic profile of ApTI demonstrated seven bands; three bands apparently have trypsin-like activity. Moreover, the peritrophic membrane was not disrupted. The enzymes of ApTI-fed larvae were found to be sensitive to ApTI and to have a normal feedback mechanism; also, the larval digestive enzymes were not able to degrade the inhibitor. In addition, ApTI delayed larval development time. Histological studies demonstrated a degeneration of the microvilli of the posterior midgut region epithelium cells, hypertrophy of the gastric caeca cells and an augmented ectoperitrophic space in larvae. Moreover, Ae. aegypti larvae were incapable of overcoming the negative effects of ApTI, indicating that this inhibitor might be used as a promising agent against Ae. aegypti. In addition, molecular modeling and molecular docking studies were also performed in order to construct three-dimensional theoretical models for ApTI, trypsin and chymotrypsin from Ae. aegypti, as well as to predict the possible interactions and affinity values for the complexes ApTI/trypsin and ApTI/chymotrypsin. In this context, this study broadens the base of our understanding about the modes of action of proteinase inhibitors in insects, as well as the way insects

  3. Inactivation of α1-proteinase inhibitor by Candida albicans aspartic proteases favors the epithelial and endothelial cell colonization in the presence of neutrophil extracellular traps.

    Science.gov (United States)

    Gogol, Mariusz; Ostrowska, Dominika; Klaga, Kinga; Bochenska, Oliwia; Wolak, Natalia; Aoki, Wataru; Ueda, Mitsuyoshi; Kozik, Andrzej; Rapala-Kozik, Maria

    2016-01-01

    Candida albicans, a causative agent of opportunistic fungal infections in immunocompromised patients, uses ten secreted aspartic proteases (SAPs) to deregulate the homeostasis of the host organism on many levels. One of these deregulation mechanisms involves a SAP-dependent disturbance of the control over proteolytic enzymes of the host by a system of dedicated proteinase inhibitors, with one important example being the neutrophil elastase and alpha1-proteinase inhibitor (A1PI). In this study, we found that soluble SAPs 1-4 and the cell membrane-anchored SAP9 efficiently cleaved A1PI, with the major cleavage points located at the C-terminal part of A1PI in a close vicinity to the reactive-site loop that plays a critical role in the inhibition mechanism. Elastase is released by neutrophils to the environment during fungal infection through two major processes, a degranulation or formation of neutrophil extracellular traps (NET). Both, free and NET-embedded elastase forms, were found to be controlled by A1PI. A local acidosis, resulting from the neutrophil activity at the infection sites, favors A1PI degradation by SAPs. The deregulation of NET-connected elastase affected a NET-dependent damage of epithelial and endothelial cells, resulting in the increased susceptibility of these host cells to candidal colonization. Moreover, the SAP-catalyzed cleavage of A1PI was found to decrease its binding affinity to a proinflammatory cytokine, interleukin-8. The findings presented here suggest a novel strategy used by C. albicans for the colonization of host tissues and overcoming the host defense. PMID:26641639

  4. Bauhinia proteinase inhibitor-based synthetic fluorogenic substrates for enzymes isolated from insect midgut and caterpillar bristles.

    Science.gov (United States)

    Andrade, Sonia A; Santomauro-Vaz, Eugênio M; Lopes, Adriana R; Chudzinski-Tavassi, Ana M; Juliano, Maria A; Terra, Walter R; Sampaio, Misako U; Sampaio, Claudio A M; Oliva, Maria Luiza V

    2003-03-01

    Bauhinia ungulata factor Xa inhibitor (BuXI) inactivates factor Xa and LOPAP, a prothrombin activator proteinase isolated from the venom of Lonomia obliqua caterpillar bristles. The reactive site of the enzyme-inhibitor interaction was explored to design specific substrates for both enzymes. Methionine is crucial for LOPAP and factor Xa substrate interaction, since the change of both Met residues in the substrates abolished the hydrolysis. Synthetic substrates containing the sequence around the reactive site of BbKI, a plasma kallikrein inhibitor, were shown to be specific for trypsin hydrolysis. Therefore, these substrates may be an alternative in studies aiming at a characterization of trypsin-like enzyme activities, especially non-mammalian enzymes. PMID:12715900

  5. Cysteine proteinase inhibitor level in tumor and normal tissues in control and cured mice.

    Science.gov (United States)

    Poteryaeva, O N; Falameyeva, O V; Korolenko, T A; Kaledin, V I; Djanayeva, S J; Nowicky, J W; Sandula, J

    2000-01-01

    Cystatin C is the best known extracellular endogenous cysteine proteinase inhibitor and has been studied as a possible index of tumor growth and as a marker of the effectiveness of antitumor therapy. The aim of this study was to evaluate cystatin C concentrations in murine tumor tissues (compared with other organs not directly involved with tumor development, such as the liver and spleen) during treatment with several antitumor drugs (Ukrain and/or cyclophosphane). Cystatin C concentrations in murine tissues and biological fluids was determined by enzyme-linked immunosorbent (ELISA) assay. The cystatin C ELISA test is a sandwich immunoassay, which uses immobilized rabbit antihuman cystatin C Pab and mouse antihuman cystatin C Mab-HRP (monoclonal antibodies, conjugated with horseradish peroxidase). We observed decreased serum cystatin C concentrations compared with controls in all nontreated tumor models: HA-1 hepatoma (solid and ascitic forms), lung adenocarcinoma (solid and ascitic forms) and LS lymphosarcoma. In the ascitic fluid of mice with HA-1 hepatoma the cystatin C concentration was much lower than in the serum of the same mice (about 20-fold lower). In the HA-1 model of hepatoma cells cystatin C concentration decreased about 2-3-fold compared with the control (intact liver) and Ukrain significantly increased the cystatin C concentration. Cyclophosphane treatment of LS lymphosarcoma significantly increased the cystatin C concentration in serum. Cyclophosphane treatment (50 mg/kg, single injection) increased cystatin C by up to 8-fold more in tumor issue. Ukrain treatment of LS lymphosarcoma was also followed by increased levels of cystatin C in tumor tissue (4-fold); cyclophosphane plus Ukrain had a similar positive effect. In the group with LS lymphosarcoma Ukrain or cyclophosphane plus Ukrain treatment induced a significant increase in cystatin C concentration in liver. Liver cystatin C concentration decreased in the HA-1 hepatoma group and treatment with

  6. 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

  7. 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

  8. 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

  9. Concurrent occurrence of insect proteinases and their inhibitors in insect midgut

    Czech Academy of Sciences Publication Activity Database

    Taranushenko, J.; Sehnal, František

    Izmir : Entomological Society of Turkey , 2006. s. 134-134. [European Congress of Entomology /8./. 17.09.2006-22.09.2006, Izmir] R&D Projects: GA ČR(CZ) GA522/06/1591 Institutional research plan: CEZ:AV0Z50070508 Keywords : serin proteinases Subject RIV: ED - Physiology

  10. 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.

  11. Growth and development of Colorado potato beetle larvae, Leptinotarsa decemlineata, on potato plants expressing the oryzacystatin II proteinase inhibitor.

    Science.gov (United States)

    Cingel, Aleksandar; Savić, Jelena; Vinterhalter, Branka; Vinterhalter, Dragan; Kostić, Miroslav; Jovanović, Darka Šešlija; Smigocki, Ann; Ninković, Slavica

    2015-08-01

    Plant proteinase inhibitors (PIs) are attractive tools for crop improvement and their heterologous expression can enhance insect resistance in transgenic plants. PI oryzacystatin II (OCII), isolated from rice, showed potential in controlling pests that utilize cysteine proteinases for protein digestion. To evaluate the applicability of the OCII gene in enhancing plant defence, OCII-transformed potatoes were bioassayed for resistance to Colorado potato beetle (Leptinotarsa decemlineata Say). Feeding on transformed leaves of potato cultivars Desiree and Jelica significantly affected larval growth and development, but did not change mortality rates. During the L2 and L3 developmental stages larvae consumed the OCII-transformed foliage faster as compared to the nontransformed control. Also these larvae reached the prepupal stage (end of L4 stage) 2 days earlier than those fed on control leaves. However, the total amounts of consumed OCII-transformed leaves were up to 23% lower than of control, and the maximal weights of prepupal larvae were reduced by up to 18% as compared to larvae fed on nontransformed leaves. The reduction in insect fitness reported in this study in combination with other control measures, could lead to improved CPB resistance management in potato. PMID:25820664

  12. 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...

  13. 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

  14. 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....

  15. 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.

  16. 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.

  17. Selective cell adhesion inhibitors: Barbituric acid based alpha4beta7--MAdCAM inhibitors.

    Science.gov (United States)

    Harriman, Geraldine C; Brewer, Matthias; Bennett, Robert; Kuhn, Cyrille; Bazin, Marc; Larosa, Greg; Skerker, Paul; Cochran, Nancy; Gallant, Debra; Baxter, Deborah; Picarella, Dominic; Jaffee, Bruce; Luly, Jay R; Briskin, Michael J

    2008-04-01

    A novel series of barbituric acid derivatives were identified as selective inhibitors of alpha4beta7 MAdCAM (mucosal addressin cell adhesion molecule-1) interactions via a high throughput screening exercise. These inhibitors were optimized to submicromolar potencies in whole cell adhesion assays, retaining their selectivity over alpha4beta1 VCAM. PMID:18331794

  18. 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.

  19. 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

  20. 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.

  1. An overview on 5alpha-reductase inhibitors.

    Science.gov (United States)

    Aggarwal, Saurabh; Thareja, Suresh; Verma, Abhilasha; Bhardwaj, Tilak Raj; Kumar, Manoj

    2010-02-01

    Benign prostatic hyperplasia (BPH) is the noncancerous proliferation of the prostate gland associated with benign prostatic obstruction and lower urinary tract symptoms (LUTS) such as frequency, hesitancy, urgency, etc. Its prevalence increases with age affecting around 70% by the age of 70 years. High activity of 5alpha-reductase enzyme in humans results in excessive dihydrotestosterone levels in peripheral tissues and hence suppression of androgen action by 5alpha-reductase inhibitors is a logical treatment for BPH as they inhibit the conversion of testosterone to dihydrotestosterone. Finasteride (13) was the first steroidal 5alpha-reductase inhibitor approved by U.S. Food and Drug Administration (USFDA). In human it decreases the prostatic DHT level by 70-90% and reduces the prostatic size. Dutasteride (27) another related analogue has been approved in 2002. Unlike Finasteride, Dutasteride is a competitive inhibitor of both 5alpha-reductase type I and type II isozymes, reduced DHT levels >90% following 1 year of oral administration. A number of classes of non-steroidal inhibitors of 5alpha-reductase have also been synthesized generally by removing one or more rings from the azasteroidal structure or by an early non-steroidal lead (ONO-3805) (261). In this review all categories of inhibitors of 5alpha-reductase have been covered. PMID:19879888

  2. Alpha 1-blockers vs 5 alpha-reductase inhibitors in benign prostatic hyperplasia. A comparative review

    DEFF Research Database (Denmark)

    Andersen, J T

    1995-01-01

    During recent years, pharmacological treatment of symptomatic benign prostatic hyperplasia (BPH) has become the primary treatment choice for an increasing number of patients. The 2 principal drug classes employed are alpha 1-blockers and 5 alpha-reductase inhibitors. Current information from...... patients who will respond well to alpha 1-blockers have yet to be identified, and data concerning the long term effects of these drugs are not yet available. 5 alpha-Reductase inhibitors have a slow onset of effect, but treatment leads to improvement in symptoms, reduction of the size of the prostate gland...... and improvement in objective parameters for bladder outflow obstruction. Approximately 30 to 50% of patients will respond to treatment with 5 alpha-reductase inhibitors. The definitive role of pharmacological treatment in symptomatic BPH remains to be established, although it seems that patients unfit...

  3. 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.

  4. Glycosaminoglycan-bound and free inter-alpha-trypsin inhibitor components of follicular fluid.

    Science.gov (United States)

    Odum, L; Jessen, T E; Andersen, C Y

    2001-11-01

    The proteinase inhibitor inter-alpha trypsin inhibitor (ITI) is a blood-derived protein necessary for normal female fertility. Absence of ITI leads to ovulation of naked oocytes that cannot fertilise. ITI consists of two heavy chains (ITI-HC) and bikunin linked by a chrondroitin sulphate. By binding to hyaluronate, ITI-HC stabilises the extracellular matrix, but ITI-HC also binds to proteoglycans in follicular fluid. In vivo concentrations of ITI components in preovulatory follicular fluid, free as well as bound to hyaluronate or proteoglycan, are unknown. In order to quantify these components, 58 follicular fluids and 13 blood samples were collected in connection with in vitro fertilisation and embryo transfer treatment of 13 women. Quantitation of glycosaminoglycan-bound ITI-HC was performed after separation from free ITI in agarose gel. ITI components were determined by immunoelectrophoresis and hyaluronate by an ELISA method. The follicular fluid concentration of ITI was on average 70% of that in plasma and the concentration of hyaluronate remained low despite follicular production, suggesting that the production of hyaluronate is the rate-limiting step in the formation of the extracellular matrix of the oocyte-cumulus complex. In follicular fluid, the concentration of free ITI-HC was higher than that of glycosaminoglycan-bound ITI-HC. Addition of exogeneous hyaluronate doubled the amount of hyaluronate-bound ITI-HC, further supporting the notion that ITI in follicular fluid is not rate-limiting for cumulus expansion in vivo. PMID:11771893

  5. Zinc oxide nanoparticles as novel alpha-amylase inhibitors

    Science.gov (United States)

    Dhobale, Sandip; Thite, Trupti; Laware, S. L.; Rode, C. V.; Koppikar, Soumya J.; Ghanekar, Ruchika-Kaul; Kale, S. N.

    2008-11-01

    Amylase inhibitors, also known as starch blockers, contain substances that prevent dietary starches from being absorbed by the body via inhibiting breakdown of complex sugars to simpler ones. In this sense, these materials are projected as having potential applications in diabetes control. In this context, we report on zinc oxide nanoparticles as possible alpha-amylase inhibitors. Zinc oxide nanoparticles have been synthesized using soft-chemistry approach and 1-thioglycerol was used as a surfactant to yield polycrystalline nanoparticles of size ˜18 nm, stabilized in wurtzite structure. Conjugation study and structural characterization have been done using x-ray diffraction technique, Fourier transform infrared spectroscopy, UV-visible spectroscopy, and transmission electron microscopy. Cytotoxicity studies on human fibrosarcoma (HT-1080) and skin carcinoma (A-431) cell lines as well as mouse primary fibroblast cells demonstrate that up to a dose of 20 μg/ml, ZnO nanoparticles are nontoxic to the cells. We report for the first time the alpha-amylase inhibitory activity of ZnO nanoparticles wherein an optimum dose of 20 μg/ml was sufficient to exhibit 49% glucose inhibition at neutral pH and 35 °C temperature. This inhibitory activity was similar to that obtained with acarbose (a standard alpha-amylase inhibitor), thereby projecting ZnO nanoparticles as novel alpha-amylase inhibitors.

  6. 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

  7. Antisense-mediated depletion of a potato lipoxygenase reduces wound induction of proteinase inhibitors and increases weight gain of insect pests

    OpenAIRE

    Royo, Joaquín; León, José; Vancanneyt, Guy; Albar, Juan Pablo; Rosahl, Sabine; Ortego, Félix; Castañera, Pedro; Sánchez-Serrano, José J.

    1999-01-01

    De novo jasmonic acid (JA) synthesis is required for wound-induced expression of proteinase inhibitors and other defense genes in potato and tomato. The first step in JA biosynthesis involves lipoxygenase (LOX) introducing molecular oxygen at the C-13 position of linolenic acid. We previously have shown that, in potato, at least two gene families code for 13-LOX proteins. We have now produced transgenic potato plants devoid of one specific 13-LOX isoform (LOX-H3) through antisense-mediated de...

  8. 21 CFR 866.5890 - Inter-alpha trypsin inhibitor immunological test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Inter-alpha trypsin inhibitor immunological test... Systems § 866.5890 Inter-alpha trypsin inhibitor immunological test system. (a) Identification. An inter-alpha trypsin inhibitor immunological test system is a device that consists of the reagents used...

  9. Barley alpha-amylase/subtilisin inhibitor: structure, biophysics and protein engineering

    DEFF Research Database (Denmark)

    Nielsen, P.K.; Bønsager, Birgit Christine; Fukuda, Kenji; Svensson, Birte

    2004-01-01

    Bifunctional alpha-amylase/subtilisin inhibitors have been implicated in plant defence and regulation of endogenous alpha-amylase action. The barley alpha-amylase/subtilisin inhibitor (BASI) inhibits the barley alpha-amylase 2 (AMY2) and subtilisin-type serine proteases. BASI belongs to the Kunit...

  10. 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

  11. 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.

  12. Binding of [alpha, alpha]-Disubstituted Amino Acids to Arginase Suggests New Avenues for Inhibitor Design

    Energy Technology Data Exchange (ETDEWEB)

    Ilies, Monica; Di Costanzo, Luigi; Dowling, Daniel P.; Thorn, Katherine J.; Christianson, David W. (MIT); (Episcopal U); (Rutgers); (Drexel); (Penn)

    2011-10-21

    Arginase is a binuclear manganese metalloenzyme that hydrolyzes L-arginine to form L-ornithine and urea, and aberrant arginase activity is implicated in various diseases such as erectile dysfunction, asthma, atherosclerosis, and cerebral malaria. Accordingly, arginase inhibitors may be therapeutically useful. Continuing our efforts to expand the chemical space of arginase inhibitor design and inspired by the binding of 2-(difluoromethyl)-L-ornithine to human arginase I, we now report the first study of the binding of {alpha},{alpha}-disubstituted amino acids to arginase. Specifically, we report the design, synthesis, and assay of racemic 2-amino-6-borono-2-methylhexanoic acid and racemic 2-amino-6-borono-2-(difluoromethyl)hexanoic acid. X-ray crystal structures of human arginase I and Plasmodium falciparum arginase complexed with these inhibitors reveal the exclusive binding of the L-stereoisomer; the additional {alpha}-substituent of each inhibitor is readily accommodated and makes new intermolecular interactions in the outer active site of each enzyme. Therefore, this work highlights a new region of the protein surface that can be targeted for additional affinity interactions, as well as the first comparative structural insights on inhibitor discrimination between a human and a parasitic arginase.

  13. 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.

  14. 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.

  15. Off-label use of TNF-alpha inhibitors in a dermatological university department

    DEFF Research Database (Denmark)

    Sand, Freja Lærke; Thomsen, Simon Francis

    2015-01-01

    Tumor necrosis factor-alpha (TNF)-alpha inhibitors are licensed for patients with severe refractory psoriasis and psoriatic arthritis. However, TNF-alpha inhibitors have also been used off-label for various recalcitrant mucocutaneous diseases. This study aimed to evaluate the efficacy and safety...... of TNF-alpha inhibitors used for off-label dermatological indications. We retrospectively evaluated patient records of 118 patients treated off-label with TNF-alpha inhibitors in a dermatological university department. Patients presented with severe aphthous stomatitis/genital aphthous lesions (26...

  16. Screening alpha-glucosidase and alpha-amylase inhibitors from natural compounds by molecular docking in silico.

    Science.gov (United States)

    Jhong, Chien-Hung; Riyaphan, Jirawat; Lin, Shih-Hung; Chia, Yi-Chen; Weng, Ching-Feng

    2015-01-01

    The alpha-glucosidase inhibitor is a common oral anti-diabetic drug used for controlling carbohydrates normally converted into simple sugars and absorbed by the intestines. However, some adverse clinical effects have been observed. The present study seeks an alternative drug that can regulate the hyperglycemia by down-regulating alpha-glucosidase and alpha-amylase activity by molecular docking approach to screen the hyperglycemia antagonist against alpha-glucosidase and alpha-amylase activities from the 47 natural compounds. The docking data showed that Curcumin, 16-hydroxy-cleroda-3,13-dine-16,15-olide (16-H), Docosanol, Tetracosanol, Antroquinonol, Berberine, Catechin, Quercetin, Actinodaphnine, and Rutin from 47 natural compounds had binding ability towards alpha-amylase and alpha-glucosidase as well. Curcumin had a better biding ability of alpha-amylase than the other natural compounds. Analyzed alpha-glucosidase activity reveals natural compound inhibitors (below 0.5 mM) are Curcumin, Actinodaphnine, 16-H, Quercetin, Berberine, and Catechin when compared to the commercial drug Acarbose (3 mM). A natural compound with alpha-amylase inhibitors (below 0.5 mM) includes Curcumin, Berberine, Docosanol, 16-H, Actinodaphnine/Tetracosanol, Catechin, and Quercetin when compared to Acarbose (1 mM). When taken together, the implication is that molecular docking is a fast and effective way to screen alpha-glucosidase and alpha-amylase inhibitors as lead compounds of natural sources isolated from medicinal plants. PMID:26154585

  17. 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.

  18. The human cystatin gene family: cloning of three members and evolutionary relationship between cystatins and Bowman-Birk type proteinase inhibitors.

    Science.gov (United States)

    Saitoh, E; Isemura, S; Sanada, K; Ohnishi, K

    1991-01-01

    Three genes from the human cystatin gene family have been isolated from a bacteriophage lambda library containing Hind III digests of human genomic DNA. The cloned genes were identified with three DNA probes each containing exon 1, exon 2 and exon 3 of the CST1 gene for cystatin SN. The genes, which we name CST2B, CST4, and CST5, are 6.8 kb, 5.4 kb and 12.5 kb in size, respectively. Statistical analysis of DNA sequence homology elucidated that the second and third exons of cystatin (family II) genes and three cystatin (family II) gene like segments in the kininogen (family III) genes are significantly homologous to the gene segments coding for the inhibitory domains of Bowman-Birk type proteinase inhibitors. PMID:1801729

  19. Reactive oxygen species and anti-proteinases.

    Science.gov (United States)

    Siddiqui, Tooba; Zia, Mohammad Khalid; Ali, Syed Saqib; Rehman, Ahmed Abdur; Ahsan, Haseeb; Khan, Fahim Halim

    2016-01-01

    Reactive oxygen species (ROS) cause damage to macromolecules such as proteins, lipids and DNA and alters their structure and function. When generated outside the cell, ROS can induce damage to anti-proteinases. Anti-proteinases are proteins that are involved in the control and regulation of proteolytic enzymes. The damage caused to anti-proteinase barrier disturbs the proteinase-anti-proteinases balance and uncontrolled proteolysis at the site of injury promotes tissue damage. Studies have shown that ROS damages anti-proteinase shield of the body by inactivating key members such as alpha-2-macroglobulin, alpha-1-antitrypsin. Hypochlorous acid inactivates α-1-antitrypsin by oxidizing a critical reactive methionine residue. Superoxide and hypochlorous acid are physiological inactivators of alpha-2-macroglobulin. The damage to anti-proteinase barrier induced by ROS is a hallmark of diseases such as atherosclerosis, emphysema and rheumatoid arthritis. Thus, understanding the behaviour of ROS-induced damage to anti-proteinases may helps us in development of strategies that could control these inflammatory reactions and diseases. PMID:26699123

  20. Barley alpha-amylase/subtilisin inhibitor: structure, biophysics and protein engineering

    DEFF Research Database (Denmark)

    Nielsen, P.K.; Bønsager, Birgit Christine; Fukuda, Kenji;

    2004-01-01

    Bifunctional alpha-amylase/subtilisin inhibitors have been implicated in plant defence and regulation of endogenous alpha-amylase action. The barley alpha-amylase/subtilisin inhibitor (BASI) inhibits the barley alpha-amylase 2 (AMY2) and subtilisin-type serine proteases. BASI belongs to the Kunitz...... Ca2+-modulated kinetics of the AMY2/BASl interaction and found that the complex formation involves minimal structural changes. The modulation of the interaction by calcium ions makes it unique among the currently known binding mechanisms of proteinaceous alpha-amylase inhibitors....

  1. The impact of single nucleotide polymorphism in monomeric alpha-amylase inhibitor genes from wild emmer wheat, primarily from Israel and Golan

    Directory of Open Access Journals (Sweden)

    Yan Ze-Hong

    2010-06-01

    Full Text Available Abstract Background Various enzyme inhibitors act on key insect gut digestive hydrolases, including alpha-amylases and proteinases. Alpha-amylase inhibitors have been widely investigated for their possible use in strengthening a plant's defense against insects that are highly dependent on starch as an energy source. We attempted to unravel the diversity of monomeric alpha-amylase inhibitor genes of Israeli and Golan Heights' wild emmer wheat with different ecological factors (e.g., geography, water, and temperature. Population methods that analyze the nature and frequency of allele diversity within a species and the codon analysis method (comparing patterns of synonymous and non-synonymous changes in protein coding sequences were used to detect natural selection. Results Three hundred and forty-eight sequences encoding monomeric alpha-amylase inhibitors (WMAI were obtained from 14 populations of wild emmer wheat. The frequency of SNPs in WMAI genes was 1 out of 16.3 bases, where 28 SNPs were detected in the coding sequence. The results of purifying and the positive selection hypothesis (p Conclusions Great diversity at the WMAI locus, both between and within populations, was detected in the populations of wild emmer wheat. It was revealed that WMAI were naturally selected for across populations by a ratio of dN/dS as expected. Ecological factors, singly or in combination, explained a significant proportion of the variations in the SNPs. A sharp genetic divergence over very short geographic distances compared to a small genetic divergence between large geographic distances also suggested that the SNPs were subjected to natural selection, and ecological factors had an important evolutionary role in polymorphisms at this locus. According to population and codon analysis, these results suggested that monomeric alpha-amylase inhibitors are adaptively selected under different environmental conditions.

  2. 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.; Hejgaard, Jørn

    2003-01-01

    and vascular tissues of roots, and to the phloem of coleoptiles and leaves. The identification of BSZ4 in vegetative tissues by western blotting was confirmed for the roots by purification and amino acid sequencing, and for the leaves by in vitro reactive-centre loop cleavage studies. Plant serpins...... 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...... centres in vitro, were ubiquitous at low levels, but the protein could not be detected. EST analysis showed that expression of genes for serpins with BSZx-type reactive centres in vegetative tissues is widespread in the plant kingdom, suggesting a common regulatory function. For BSZ4 and BSZ7, expression...

  3. 5-Amino-pyrazoles as potent and selective p38[alpha] inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Das, Jagabandhu; Moquin, Robert V.; Dyckman, Alaric J.; Li, Tianle; Pitt, Sidney; Zhang, Rosemary; Shen, Ding Ren; McIntyre, Kim W.; Gillooly, Kathleen; Doweyko, Arthur M.; Newitt, John A.; Sack, John S.; Zhang, Hongjian; Kiefer, Susan E.; Kish, Kevin; McKinnon, Murray; Barrish, Joel C.; Dodd, John H.; Schieven, Gary L.; Leftheris, Katerina (BMS)

    2012-02-07

    The synthesis and structure-activity relationships (SAR) of p38{alpha} MAP kinase inhibitors based on a 5-amino-pyrazole scaffold are described. These studies led to the identification of compound 2j as a potent and selective inhibitor of p38{alpha} MAP kinase with excellent cellular potency toward the inhibition of TNF{alpha} production. Compound 2j was highly efficacious in vivo in inhibiting TNF{alpha} production in an acute murine model of TNF{alpha} production. X-ray co-crystallography of a 5-amino-pyrazole analog 2f bound to unphosphorylated p38{alpha} is also disclosed.

  4. [Neurological complications during treatment of the tumor necrosis alpha inhibitors].

    Science.gov (United States)

    Piusińska-Macoch, Renata

    2013-05-01

    Medications with TNF-alpha inhibitors family are successfully applicable in rheumatology, gastroenterology, dermatology and neurology. Still, the ongoing research on the safety assessment of their application, also due to neurological complications. The vast majority of these complications is associated with an increased risk of serious virus (Herpes simplex--JC) and bacterial (Listeria monocytogenes) neuroinfections. They can cause the occurrence of progressive multifocal leukoencephalopathy--PML with a severe clinical course and poor prognosis or herpes simplex encephalitis--HSE. Meta-analysis revealed a number of cases of PML and the HSE in the first 6 months of treatment with natalizumab, efalizumab, rituximab, abatacept and infliximab. Common complication occasionally turning on this biologics is chronic demyelinating polyneuropathy or Lewis-Sumner syndrome. Described are cases of central and peripheral demyelination typical of multiple sclerosis (MS). Are also reported cases of motor multifocal neuropathy with conduction block acute encephalithis with polyneuropathy or mononeuropathy in the form of anterior optic neuropathy Guillen-Barre' syndrome and its variant, Miller-Fisher syndrome have been confirmed as adverse events following treatment with infliximab. Also revealed several cases of myasthenia gravis after using etanercept. In the few cases of systemic lupus CNS involvement caused by treatment with TNF inhibitors, the mechanism of these disorders is still considered too vague. Due to the emerging reports on the number of neurological adverse events of TNF antagonists, significantly higher than those described in the literature, the safety of their use requires further monitoring and multicenter studies. PMID:23894783

  5. Isolation and characterization of selective and potent human Fab inhibitors directed to the active-site region of the two-component NS2B-NS3 proteinase of West Nile virus.

    Science.gov (United States)

    Shiryaev, Sergey A; Radichev, Ilian A; Ratnikov, Boris I; Aleshin, Alexander E; Gawlik, Katarzyna; Stec, Boguslaw; Frisch, Christian; Knappik, Achim; Strongin, Alex Y

    2010-05-01

    There is a need to develop inhibitors of mosquito-borne flaviviruses, including WNV (West Nile virus). In the present paper, we describe a novel and efficient recombinant-antibody technology that led us to the isolation of inhibitory high-affinity human antibodies to the active-site region of a viral proteinase. As a proof-of-principal, we have successfully used this technology and the synthetic naive human combinatorial antibody library HuCAL GOLD(R) to isolate selective and potent function-blocking active-site-targeting antibodies to the two-component WNV NS (non-structural protein) 2B-NS3 serine proteinase, the only proteinase encoded by the flaviviral genome. First, we used the wild-type enzyme in antibody screens. Next, the positive antibody clones were counter-screened using an NS2B-NS3 mutant with a single mutation of the catalytically essential active-site histidine residue. The specificity of the antibodies to the active site was confirmed by substrate-cleavage reactions and also by using proteinase mutants with additional single amino-acid substitutions in the active-site region. The selected WNV antibodies did not recognize the structurally similar viral proteinases from Dengue virus type 2 and hepatitis C virus, and human serine proteinases. Because of their high selectivity and affinity, the identified human antibodies are attractive reagents for both further mutagenesis and structure-based optimization and, in addition, for studies of NS2B-NS3 activity. Conceptually, it is likely that the generic technology reported in the present paper will be useful for the generation of active-site-specific antibody probes for multiple enzymes. PMID:20156198

  6. Molecular characterization and mapping of murine genes encoding three members of the stefin family of cysteine proteinase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Tsui, F.W.L.; Hingwo Tsui; Mok, S. (Univ. of Toronto, Ontario (Canada) Toronto Hospital, Ontario (Canada)); Mlinaric, I.; Siminovitch, K.A. (Univ. of Toronto, Ontario (Canada) Mount Sinai Hospital, Toronto, Ontario (Canada)); Copeland, N.G.; Gilbert, D.J.; Jenkins, N.A. (NCI-Frederick Cancer Research and Development Center, MD (United States))

    1993-03-01

    Stefins or Type 1 cystatins belong to a large, evolutionarily conserved protein superfamily, the members of which inhibit the papain-like cysteine proteinases. The authors report here on the molecular cloning and chromosomal localization of three newly identified members of the murine stefin gene family. These genes, designated herein as mouse stefins 1, 2, and 3, were isolated on the basis of their relatively increased expression in moth-eaten viable compared to normal congenic mouse bone marrow cells. The open reading frames of the stefin cDNAs encode proteins of approximately 11.5 kDa that show between 50 and 92% identity to sequences of stefins isolated from various other species. Data from Southern analysis suggest that the murine stefin gene family encompasses at least 6 and possible 10-20 membranes, all of which appear to be clustered in the genome. Analysis of interspecific backcross mice indicates that the genes encoding the three mouse stefins all map to mouse chromosome 16, a localization that is consistent with the recent assignment of the human stefin A gene to a region of conserved homology between human chromosome 3q and the proximal region of mouse chromosome 16. 51 refs., 7 figs.

  7. 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 ...

  8. 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

  9. 12-o-Tetradecanoyl-phorbol-13-acetate-differentiated U937 cells express a macrophage-like profile of neutral proteinases. High levels of secreted collagenase and collagenase inhibitor accompany low levels of intracellular elastase and cathepsin G.

    Science.gov (United States)

    Welgus, H G; Connolly, N L; Senior, R M

    1986-05-01

    Human monocytic tumor cells of the U937 cell line contain substantial quantities of two neutrophil neutral proteinases, elastase and cathepsin G, raising the question of whether their presence reflects an expression of transformation or whether normal monocytes undergo a developmental stage in which they produce certain neutrophil proteinases. To address this issue, we examined U937 cells for production of collagenase, since human alveolar macrophages release fibroblast-like collagenase, an enzyme that is distinct from neutrophil collagenase. Using an immunoassay that utilized antibody to skin fibroblast collagenase, we found that U937 cells secreted barely detectable quantities of enzyme, 10-12 ng/10(6) cells per 24 h, under basal conditions. Upon incubation with 10 nM 12-o-tetradecanoyl-phorbol-13-acetate (TPA), however, collagenase release increased 200-fold, comparable to the amount secreted by phorbol-stimulated human fibroblasts. Metabolic labeling and immunoprecipitation confirmed the enhanced synthesis of U937 cell collagenase upon TPA exposure. This enzyme activity further resembled fibroblast collagenase and differed from neutrophil collagenase by exhibiting preferential cleavage of monomeric type III collagen relative to type I. As previously observed with human alveolar macrophages, U937 cells also released a protein identical to the collagenase inhibitor produced by human skin fibroblasts, a molecule not associated with neutrophils. Release of this inhibitor increased 10-fold with TPA exposure. In contrast to collagenase and collagense inhibitor, TPA-treated U937 cells contained only 10-15% as much elastase and cathepsin G activities as control cells. Thus, TPA-induced differentiation modified the presence of these enzymes in the direction of their content in normal monocytes. Since the neutral proteinase profile of undifferentiated U937 cells resembles that of neutrophils and changes markedly after cellular differentiation to one that is

  10. The use of 5-alpha reductase inhibitors for the prevention of prostate cancer.

    Science.gov (United States)

    Yu, Eun-mi; El-Ayass, Walid; Aragon-Ching, Jeanny B

    2010-07-01

    The use of 5-alpha-reductase inhibitors has been studied not only in benign prostatic hyperplasia, but as a chemopreventive strategy in prostate cancer. Both finasteride and dutasteride, 5 alpha-reductase inhibitors (5ARI), have been shown to decrease the risk of prostate cancer. The results of the REDUCE trial using the dual alpha-reductase isoenzyme inhibitor dutasteride, has recently been published by Andriole et al. in the New England Journal of Medicine. Certain considerations regarding its use and applicability to men with high risk of developing prostate cancer are herein discussed. PMID:20574153

  11. 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.

  12. 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.

  13. 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

  14. Binding of carbohydrates and protein inhibitors to the surface of alpha-amylases

    DEFF Research Database (Denmark)

    Bozonnet, Sophie; Bønsager, Birgit Christine; Kramhoft, B.;

    2005-01-01

    This review on barley alpha-amylases 1 (AMY1) and 2 (AMY2) addresses rational mutations at distal subsites to the catalytic site, polysaccharide hydrolysis, and interactions with proteinaceous inhibitors. Subsite mapping of barley alpha-amylases revealed 6 glycone and 4 aglycone substrate subsite...

  15. Binding of carbohydrates and protein inhibitors to the surface of alpha-amylases

    DEFF Research Database (Denmark)

    Bozonnet, Sophie; Bønsager, Birgit Christine; Kramhoft, B.; Mori, H.; Abou Hachem, Maher; Willemoes, Martin; Jensen, M.T.; Fukuda, Kenji; Nielsen, P.K.; Juge, N.; Aghajari, N.; Tranier, S.; Robert, X.; Haser, R.; Svensson, Birte

    2005-01-01

    This review on barley alpha-amylases 1 (AMY1) and 2 (AMY2) addresses rational mutations at distal subsites to the catalytic site, polysaccharide hydrolysis, and interactions with proteinaceous inhibitors. Subsite mapping of barley alpha-amylases revealed 6 glycone and 4 aglycone substrate subsites...

  16. Activation of bean (Phaseolus vulgaris) [alpha]-amylase inhibitor requires proteolytic processing of the proprotein

    Energy Technology Data Exchange (ETDEWEB)

    Pueyo, J.J.; Hunt, D.C.; Chrispeels, M.J. (Univ. of California, San Diego, La Jolla (United States))

    1993-04-01

    Seeds of the common bean (Phaseolus vulgaris) contain a plant defense protein that inhibits the [alpha]-amylases of mammals and insects. This [alpha]-amylase inhibitor ([alpha]Al) is synthesized as a proprotein on the endoplasmic reticulum and is proteolytically processed after arrival in the protein storage vacuoles to polypeptides of relative molecular weight (M[sub r]) 15,000 to 18,000. The authors report two types of evidence that proteolytic processing is linked to activation of the inhibitory activity. First, by surveying seed extracts of wild accessions of P. vulgaris and other species in the genus Phaseolus, they found that antibodies to [alpha]Al recognize large (M[sub r] 30,000-35,000) polypeptides as well as typical [alpha]Al processing products (M[sub r] 15,000-18,000). [alpha]Al activity was found in all extracts that had the typical [alpha]Al processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, they made a mutant [alpha]Al in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-[alpha]Al when the gene is expressed in tobacco. When pro-[alpha]Al was separated from mature [alpha]Al by gel filtration, pro-[alpha]Al was found not to have [alpha]-amylase inhibitory activity. The authors interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. They suggest that the polypeptide cleavage removes a conformation constraint on the precursor to produce the biochemically active molecule. 43 refs., 5 figs., 1 tab.

  17. 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

  18. 3D QSAR Study on Alpha Keto Amide Derivatives as gp120-CD4 Inhibitors

    Directory of Open Access Journals (Sweden)

    Vinayak D. More

    2012-01-01

    Full Text Available The present communication deals with 3D QDAR analysis on series of Alpha keto amide derivatives some for the designing of new GP120-CD4 inhibitors with anti HIV activity. The four different QSAR models are generated using data set of 32 molecules as gp120-CD4 inhibitors from literature studies. The 3D QSAR result gives insights for understanding of the relationship between structural features of substituted alpha keto amide derivatives and their activities which should be useful to design newer potential anti-HIV agents.

  19. 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

  20. An alpha-glucosidase inhibitor from an endophytic Cladosporium sp. with potential as a biocontrol agent.

    Science.gov (United States)

    Singh, Bahaderjeet; Kaur, Tamanreet; Kaur, Sanehdeep; Manhas, Rajesh K; Kaur, Amarjeet

    2015-02-01

    This study highlights the importance of alpha-glucosidase inhibitors as mechanisms for endophyte-mediated resistance to insect pests. One of the major benefits which endophytes confer on plants is providing resistance against insect pests. This built-in defense mechanism of the plant can be used for exploring ecofriendly strategies for pest control. In the present study, 34 endophytic fungi were isolated from Tinospora cordifolia and screened for their ability to produce alpha-glucosidase inhibitors. Maximum inhibitory activity was observed in an isolate from T. cordifolia (TN-9S), identified to be Cladosporium sp. The inhibitor was purified using chromatographic techniques. The insecticidal activity of the purified inhibitor was evaluated against Spodoptera litura. The inhibitor induced a significant mortality in the larvae of S. litura and adversely affected its survival and development. It also inhibited the activity of α-glycosidases in vivo in the gut of the larvae. The purified inhibitor was determined to be a phenolic compound with amine groups, demonstrating a noncompetitive type of inhibition in vitro. The production of the inhibitor was optimized. Response surface methodology (RSM) analysis revealed a significant interaction between dextrose and malt extract, with first-order effect of pH. PMID:25432333

  1. Synthesis and in vitro activity of some epimeric 20 alpha-hydroxy, 20-oxime and aziridine pregnene derivatives as inhibitors of human 17 alpha-hydroxylase/C17,20-lyase and 5 alpha-reductase.

    Science.gov (United States)

    Ling, Y Z; Li, J S; Kato, K; Liu, Y; Wang, X; Klus, G T; Marat, K; Nnane, I P; Brodie, A M

    1998-10-01

    Some epimeric 20-hydroxy, 20-oxime, 16 alpha, 17 alpha-, 17,20- and 20,21-aziridine derivatives of progesterone were synthesized and evaluated as inhibitors of human 17 alpha-hydroxylase/C17,20-lyase (P450(17) alpha) and 5 alpha-reductase (5 alpha-R). The reduction of 16-dehydropregenolone acetate (3a) was reinvestigated. NaBH4 in the presence of CeCl3 gave better stereo-selectivity for 20 beta-ol [20 alpha/20 beta-OH (4 alpha/4 beta) = 1/2.7] than LTBAH or the Meerwein-Pondroff method reported; reduction with Zn in HOAc formed exclusively 20 alpha-ol (4 alpha b). The 20 alpha- and 20 beta-hydroxy-4,16-pregnadien-3-one (9 alpha) and (9 beta) were synthesized from the alcohols 4 alpha b and 4 beta b. Several 20-oxime pregnadienes and 16 alpha, 17 alpha-, 17,20- and 20,21-aziridinyl-5-pregnene derivatives were also synthesized. LiAlH4 reduction of the 16-en-20-oxime (12b) yielded 20 (R)-(13a) and 20(S)-17 alpha,20-aziridine (13b) and 20(R)-17 beta,20-aziridine (14a). Several compounds inhibited the human P450(17) alpha with greater potency than ketoconzole. The 5 alpha-R enzyme assay showed that while (9 alpha) did not have any activity, (9 beta) and (3b) were potent 5 alpha-reductase (IC50 = 21 and 31 nM) inhibitors with activities similar to finasteride. The 20-oximes (17a) and (17b) were potent dual inhibitors for both 5 alpha-R (IC50 = 63 and 115 nM, compared to 33 nM for finasteride) and P450(17) alpha (IC50 = 43 and 25 nM, compared to 78 nM for ketoconazole). PMID:9839000

  2. Intracellular modifications of rat alpha 1 inhibitor3. Formation of disulphides, internal thiolester and sulphation.

    Science.gov (United States)

    Sjöberg, M; Esnard, F; Fries, E

    1991-04-10

    alpha 1 Inhibitor3 (alpha 1I3) is a monomeric protease inhibitor of about 190 kDa which is secreted by rodent hepatocytes. We have studied intracellular modifications of this protein in [35]methionine-labelled rat hepatocytes by pulse/chase experiments followed by immunoprecipitation and gel electrophoresis under reducing and nonreducing conditions. Directly after the pulse, most of the unreduced alpha 1I3 migrated faster than the reduced form, indicating that disulphide bridges are formed during or shortly after synthesis yielding a compact structure. With increasing chase time however, an increasing portion of the unreduced alpha 1I3 migrated with a mobility lower than that of the reduced protein, half-maximal conversion occurring after about 10 min. This finding suggests that alpha 1I3 undergoes a conformational change in the endoplasmic reticulum, possibly becoming more elongated. During 10-30 min of chase, the protein acquired the capacity to undergo autolytic cleavage upon heating, a property due to the existence of an internal thiolester bond [Howard, J. B., Vermeulen, M. & Swenson, R. P. (1980) J. Biol. Chem. 255, 3820-3823; Esnard, F., Gutman, N., El Moujahed, A. & Gauthier, F. (1985) FEBS Lett. 182, 125-129]. Analysis by subcellular fractionation indicated that this bond is formed in the endoplasmic reticulum. Finally, we show that secreted alpha 1I3 is sulphated, presumably at Tyr618. PMID:2015826

  3. Characterization of the pattern of alphas1- and beta-casein breakdown and release of a bioactive peptide by a cell envelope proteinase from Lactobacillus delbrueckii subsp. lactis CRL 581.

    Science.gov (United States)

    Hebert, Elvira María; Mamone, Gianfranco; Picariello, Gianluca; Raya, Raúl R; Savoy, Graciela; Ferranti, Pasquale; Addeo, Francesco

    2008-06-01

    The cell envelope-associated proteinases (CEPs) of the lactobacilli have key roles in bacterial nutrition and contribute to the development of the organoleptic properties of fermented milk products as well, as they can release bioactive health-beneficial peptides from milk proteins. The influence of the peptide supply, carbohydrate source, and osmolites on the CEP activity of the cheese starter Lactobacillus delbrueckii subsp. lactis CRL 581 was investigated. The CEP activity levels were controlled by the peptide content of the growth medium. The maximum activity was observed in a basal minimal defined medium, whereas in the presence of Casitone, Casamino Acids, or yeast extract, the synthesis of CEP was inhibited 99-, 70-, and 68-fold, respectively. The addition of specific di- or tripeptides containing branched-chain amino acids, such as leucylleucine, prolylleucine, leucylglycylglycine, or leucylproline, to the growth medium negatively affected CEP activity, whereas dipeptides without branched-chain amino acids had no effect on the enzyme's production. The carbon source and osmolites did not affect CEP activity. The CEP of L. delbrueckii subsp. lactis CRL 581 exhibited a mixed-type CEP(I/III) variant caseinolytic specificity. Mass-spectrometric screening of the main peptide peaks isolated by reverse-phase high-pressure liquid chromatography allowed the identification of 33 and 32 peptides in the alpha(s1)- and beta-casein hydrolysates, respectively. By characterizing the peptide sequence in these hydrolysates, a pattern of alpha(s1)- and beta-casein breakdown was defined and is reported herein, this being the first report for a CEP of L. delbrueckii subsp. lactis. In this pattern, a series of potentially bioactive peptides (antihypertensive and phosphopeptides) which are encrypted within the precursor protein could be visualized. PMID:18424544

  4. High-level expression of the native barley alpha-amylase/subtilisin inhibitor in Pichia pastoris

    DEFF Research Database (Denmark)

    Micheelsen, Pernille Ollendorff; Ostergaard, Peter Rahbek; Lange, Lene;

    2008-01-01

    An expression system for high-level expression of the native Hordeum vulgare alpha-amylase/subtilisin inhibitor (BASI) has been developed in Pichia pastoris, using the methanol inducible alcohol oxidase 1 (AOX1) promoter. To optimize expression, two codon-optimized coding regions have been designed...... and expressed alongside the wild-type coding region. To ensure secretion of the native mature protein, a truncated version of the alpha mating factor secretion signal from Saccharomyces cerevisiae was used. In order to be able to compare expression levels from different clones, single insertion...

  5. 5-alpha reductase inhibitors, benign prostatic hyperplasia, and risk of male breast cancer

    OpenAIRE

    Robinson, David; Garmo, Hans; Holmberg, Lars; Stattin, Pär

    2015-01-01

    5-alpha reductase inhibitors (5-ARI) have been suggested to increase the risk of male breast cancer. The aim of this study was to study the risk of breast cancer in men on 5-ARI, in men with benign prostatic hyperplasia (BPH) not on 5-ARI, and in men without BPH. We performed a population-based cohort study in Sweden with data from The Prescribed Drug Register, The Patient Register, and The Cancer Register. Men on 5-ARI, men on alpha-blockers, or men who had undergone a transurethral resectio...

  6. Effect of 5-alpha Reductase Inhibitor on Storage Symptoms in Patients with Benign Prostatic Hyperplasia

    OpenAIRE

    Cho, Kang Jun; Kang, Se Hee; Kim, Hyo Sin; Koh, Jun Sung; Kim, Joon Chul

    2011-01-01

    Purpose Many patients with benign prostatic hyperplasia (BPH) have storage symptoms. The aim of this study was to evaluate the effects of treatment with a 5-alpha reductase inhibitor (5ARI) on storage symptoms in patients with BPH. Methods This study was conducted in 738 patients with lower urinary tract symptoms secondary to BPH. Patients with a prostate volume of higher than 30 mL on the transrectal ultrasound were classified into two groups: group A, in which an alpha blocker was solely ad...

  7. BstXI RFLP in the human inter-alpha-trypsin inhibitor light chain gene

    Energy Technology Data Exchange (ETDEWEB)

    Leveillard, T.; Bourguignon, J.; Sesbouee, R.; Hanauer, A.; Salier, J.P.; Diarra-Mehrpour, M.; Martin, J.P.

    1988-03-25

    The 1.2 kb EcoRI/SmaI fragment of lambdaHuLITI2 was used as probe. lambdaHuLITI2 is a full length cDNA clone coding for human inter-alpha-trypsin inhibitor light chain isolated from immunochemical screening of a lambdagt11 library. Its sequence coding for HI-30 and alpha-1-microglobulin is in agreement. BstXI identifies five invariant bands at 5.0 kb, 2.3 kb, 1.5 kb, 1.1 kb, and 0.7 kb and a diallelic polymorphism with DNA fragments at 2.0 kb or 1.7 kb.

  8. 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

  9. Prospeção de inibidores de serinoproteinases em folhas de leguminosas arbóreas da floresta Amazônica Prospecting serine proteinase inhibitors in leaves from leguminous trees of the Amazon forest

    Directory of Open Access Journals (Sweden)

    Larissa Ramos Chevreuil

    2011-03-01

    Full Text Available Os inibidores de proteinases são proteínas extensivamente investigadas nos tecidos de estocagem, mas pouco prospectadas em outros tecidos vegetais. O objetivo deste estudo foi detectar a presença de inibidores de serinoproteinases em extratos foliares de quinze espécies de leguminosas arbóreas da Amazônia. As espécies estudadas foram: Caesalpinia echinata, C. ferrea, Cedrelinga cateniformis, Copaifera multijuga, Dinizia excelsa, Enterolobium contortisiliquum, E. maximum, E. schomburgkii, Leucaena leucocephala, Ormosia paraensis, Parkia multijuga, P. pendula, P. platycephala, Swartzia corrugata e S. polyphylla. Folhas foram coletadas, secas a 30ºC durante 48 h, trituradas e submetidas à extração com NaCl (0,15 M, 10% p/v resultando no extrato total. Ensaios foram executados para determinar a concentração de proteínas e detectar a atividade inibitória contra a tripsina e quimotripsina bovina. Os teores de proteínas bruta e solúvel nos extratos foliares variaram de 7,9 a 31,2% e 1,3 a 14,8%, respectivamente. A atividade inibitória sobre a tripsina e quimotripsina foi observada em todos os extratos foliares. Contudo, nos extratos de E. maximum, L. leucocephala, P. pendula, S. corrugata e S. polyphylla a inibição foi maior sobre a tripsina, enquanto o extrato de P. multijuga foi mais efetivo contra a quimotripsina. Nós concluímos que nos extratos foliares de leguminosas arbóreas têm inibidores de serinoproteinases e exibem potencial aplicações biotecnológicas.The proteinase inhibitors are proteins extensively investigated in tissue storage, but few prospected in other plant tissues. The aim of this study was to detect the presence of serine proteinase inhibitors in leaf extracts from fifteen species of leguminous trees of the Amazon forest. The species studied were Caesalpinia echinata, C. ferrea, Cedrelinga cateniformis, Copaifera multijuga, Dinizia excelsa, Enterolobium contortisiliquum, E. maximum, E. schomburgkii

  10. 5alpha-Reductase inhibitor treatment of prostatic diseases: background and practical implications.

    Science.gov (United States)

    Dörsam, J; Altwein, J

    2009-01-01

    This literature review discusses the theoretical background of 5alpha-reductase inhibitor (5ARI) treatment and the resulting clinical implications. A Medline-based search for peer-reviewed articles addressing 5ARIs, benign prostatic hyperplasia and prostate cancer was performed. The 5ARIs Finasteride and Dutasteride, which specifically inhibit the production of dihydrotestosterone by acting as competitive inhibitors of 5alpha-reductase, are clinically well tolerated and represent an effective treatment option for benign prostatic obstruction. Finasteride is the first compound which has a proven efficacy in chemoprevention of prostate cancer. The aim of this review was to elucidate, if there are sufficient data available to point out clinically relevant differences between the drugs. Both compounds achieve a significant reduction of prostate volume, an improvement of symptoms and a lower risk of acute urinary retention. Whether the different pharmacokinetic and pharmacodynamic properties of Finasteride and Dutasteride are of clinical importance cannot be judged at this time. PMID:19030020

  11. The p53 inhibitor, pifithrin-{alpha}, suppresses self-renewal of embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Abdelalim, Essam Mohamed, E-mail: essam_abdelalim@yahoo.com [Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192 (Japan); Department of Cytology and Histology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522 (Egypt); Tooyama, Ikuo [Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu, Shiga 520-2192 (Japan)

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer We determine the role of p53 in ES cells under unstressful conditions. Black-Right-Pointing-Pointer PFT-{alpha} suppresses ES cell proliferation. Black-Right-Pointing-Pointer PFT-{alpha} induces ES cell cycle arrest. Black-Right-Pointing-Pointer PFT-{alpha} downregulates Nanog and cyclin D1. -- Abstract: Recent studies have reported the role of p53 in suppressing the pluripotency of embryonic stem (ES) cells after DNA damage and blocking the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. However, to date no evidence has been presented to support the function of p53 in unstressed ES cells. In this study, we investigated the effect of pifithrin (PFT)-{alpha}, an inhibitor of p53-dependent transcriptional activation, on self-renewal of ES cells. Our results revealed that treatment of ES cells with PFT-{alpha} resulted in the inhibition of ES cell propagation in a dose-dependent manner, as indicated by a marked reduction in the cell number and colony size. Also, PFT-{alpha} caused a cell cycle arrest and significant reduction in DNA synthesis. In addition, inhibition of p53 activity reduced the expression levels of cyclin D1 and Nanog. These findings indicate that p53 pathway in ES cells rather than acting as an inactive gene, is required for ES cell proliferation and self-renewal under unstressful conditions.

  12. Bean alpha-amylase inhibitor: A perspective transgene for plant biotechnology applications

    Czech Academy of Sciences Publication Activity Database

    Hýblová, Jana; Kluh, Ivan; Horn, Martin; Hubert, J.; Marešová, Lucie; Voburka, Zdeněk; Kudlíková, I.; Kocourek, F.; Mareš, Michael

    České Budějovice : Institute of Plant Molecular Biology ASCR, 2005. s. 16. ISBN 80-86778-16-9. [International Symposium Recent Advances in Plant Biotechnology: From Laboratory to Business /6./. 12.09.2005-16.09.2005, České Budějovice] Institutional research plan: CEZ:AV0Z40550506 Keywords : alpha-amylase inhibitors Subject RIV: CE - Biochemistry

  13. Battling Prostate Cancer with 5-Alpha-Reductase Inhibitors: a Pyrrhic Victory?

    OpenAIRE

    Hoffman, Richard M.; Richard G. Roberts; Barry, Michael J.

    2011-01-01

    Given the relatively small impact of prostate cancer screening on cancer mortality, experts are now suggesting that chemoprevention with 5-alpha-reductase inhibitors (5-ARI) may be a more effective strategy for cancer control. Two large placebo-controlled randomized trials found that men receiving 5-ARI were about 25% less likely than controls to be detected with cancer. However, most cancers were detected on routine biopsies required by study protocols. The benefit from receiving 5-ARI was m...

  14. Clinical Effects of Discontinuing 5-Alpha Reductase Inhibitor in Patients With Benign Prostatic Hyperplasia

    OpenAIRE

    Kim, Won; Jung, Jae Hung; Kang, Tae Wook; Song, Jae Mann; Chung, Hyun Chul

    2014-01-01

    Purpose To assess changes in lower urinary tract symptoms (LUTS), prostate volume, and serum prostate-specific antigen (PSA) after discontinuation of 5-alpha reductase inhibitor (5ARI) combination therapy in patients with benign prostatic hyperplasia (BPH). Materials and Methods From December 2003 to December 2012, data were collected retrospectively from 81 men more than 40 years of age with moderate to severe BPH symptoms (International Prostate Symptom Score [IPSS]≥8). The men were classif...

  15. Specific inhibitors of eukaryotic DNA synthesis and DNA polymerase alpha, 3-deoxyaphidicolin and aphidicolin-17-monoacetate.

    OpenAIRE

    Haraguchi, T; Oguro, M; Nagano, H; Ichihara, A; Sakamura, S

    1983-01-01

    Of several phytotoxins isolated from culture filtrates of Phoma betae Frank PS-13, an incitant of leaf spot disease of sugar beet, three have been identified as aphidicolin, 3-deoxyaphidicolin and aphidicolin-17-monoacetate. Aphidicolin is a selective inhibitor of eukaryotic DNA polymerase alpha (Ikegami et al. (1978) Nature 275, 458-460). Consequently, we studied the action mechanism of 3-deoxyaphidicolin and aphidicolin-17-monoacetate. These aphidicolin analogues markedly inhibited the in v...

  16. Role of 5 alpha-reductase inhibitors in the management of prostate cancer

    OpenAIRE

    Hudak, Steven J.; Hernandez, Javier; Thompson, Ian M

    2006-01-01

    Prostate cancer is one of the most complex and enigmatic oncologic problems in medicine. It is highly prevalent, particularly in elderly males. Unfortunately, its generally protracted and variable clinical course and high association with treatment-related morbidity raise serious questions about the ideal treatment strategy for the individual patient. 5 alpha-reductase (5AR) inhibitors have a dramatic effect on benign prostatic disease with low toxicity. Thus, there is much interest in the po...

  17. Effect of bilineobin, a thrombin-like proteinase from the venom of common cantil (Agkistrodon bilineatus).

    Science.gov (United States)

    Komori, Y; Nikai, T; Ohara, A; Yagihashi, S; Sugihara, H

    1993-03-01

    A thrombin-like proteinase, named bilineobin, was isolated from Agkistrodon bilineatus venom by Sephadex G-75, DEAE-Sephacel and Heparin-Sepharose CL-6B column chromatography. The purified enzyme has a mol. wt of 57,000 and catalysed the hydrolysis of arginine esters and thrombin substrates Boc-Val-Pro-Arg-MCA and Boc-Asp(OBz)-Pro-Arg-MCA. Although bilineobin converted fibrinogen into fibrin resulting in the production of fibrinopeptides, the activity was relatively low (0.65 NIH units/mg). Fibrinopeptides released upon hydrolysis by this proteinase were identified as fibrinopeptide A (FpA) and fibrinopeptide B (FpB) by measuring fast atom bombardment (FAB) mass spectra and amino acid sequence. This indicates that bilineobin hydrolyses the Arg(19)-Gly(20) bond in the A alpha chain and the Arg(21)-Gly(22) bond in the B beta chain of the bovine fibrinogen molecule. Kinetic study of FpA and FpB release reveals that bilineobin has a preference for cleaving the B beta chain. In addition, bilineobin is resistant to thrombin inhibitors such as hirudin. These suggest that the mechanism of action of bilineobin is similar but not identical to that of thrombin. It was demonstrated that the NH2-terminal region of bilineobin has significant similarities in sequence with thrombin-like proteinases from other snake venoms; however, only three residues were common with thrombin up to residue number 24. PMID:8470131

  18. Synthesis of novel 21-trifluoropregnane steroids: inhibitors of 17 alpha-hydroxylase/17,20-lyase (17 alpha-lyase).

    Science.gov (United States)

    Njar, V C; Klus, G T; Johnson, H H; Brodie, A M

    1997-06-01

    Novel 21-trifluoropregnenolone (6), 21-trifluoroprogesterone (7) and related compounds 4a and 8 have been synthesized in high yields from 3 beta-acetoxyandrost-5-ene-17 beta-carbaldehyde (3). The key reaction was the conversion of 3 into the 21-trifluoromethyl-20-alcohol as a diastereomeric mixture (4) by trifluoromethyltrimethylsilane (TMS-CF3) in the presence of tetrabutylammonium fluoride (TBAF). All compounds, including 6 and 7, were unambiguously characterized by IR, 1H and 19F NMR, high-resolution mass spectrometry (HRMS), and elemental analysis. On this basis, we concluded that the only report of an earlier synthesis of 6 and 7 is erroneous. Enzyme inhibition studies showed that 20 xi-hydroxy-21-trifluoropregn-4-en-3-one (8) is a potent inhibitor (IC50 value = 0.6 microM) of rat 17 alpha-hydroxylase/17,20-lyase. PMID:9185294

  19. Alternative Agents in Type 1 Diabetes in Addition to Insulin Therapy: Metformin, Alpha-Glucosidase Inhibitors, Pioglitazone, GLP-1 Agonists, DPP-IV Inhibitors, and SGLT-2 Inhibitors.

    Science.gov (United States)

    DeGeeter, Michelle; Williamson, Bobbie

    2016-04-01

    Insulin is the mainstay of current treatment for patients with type 1 diabetes mellitus (T1DM). Due to increasing insulin resistance, insulin doses are often continually increased, which may result in weight gain for patients. Medications currently approved for the treatment of type 2 diabetes offer varying mechanisms of action that can help to reduce insulin resistance and prevent or deter weight gain. A MEDLINE search was conducted to review literature evaluating the use of metformin, alpha-glucosidase inhibitors, pioglitazone, glucagon-like peptide 1 agonists, dipeptidyl peptidase, and sodium-dependent glucose transporter 2 inhibitors, in patients with T1DM. Varying results were found with some benefits including reductions in hemoglobin A1c, decreased insulin doses, and favorable effects on weight. Of significance, a common fear of utilizing multiple therapies for diabetes treatment is the risk of hypoglycemia, and this review displayed limited evidence of hypoglycemia with multiple agents. PMID:25312263

  20. Crystal structure of two new bifunctional nonsubstrate type thrombin inhibitors complexed with human alpha-thrombin.

    Science.gov (United States)

    Féthière, J.; Tsuda, Y.; Coulombe, R.; Konishi, Y.; Cygler, M.

    1996-01-01

    The crystal structures of two new thrombin inhibitors, P498 and P500, complexed with human alpha-thrombin have been determined at 2.0 A resolution and refined to crystallographic R-factors of 0.170 and 0.169, respectively. These compounds, with picomolar binding constants, belong to a family of potent bifunctional inhibitors that bind thrombin at two remote sites: the active site and the fibrinogen recognition exosite (FRE). The inhibitors incorporate a nonsubstrate type active site binding fragment: Dansyl-Arg-(D)Pipecolic acid (Dns-Arg-(D)Pip), reminiscent of the active-site directed inhibitors MD-805 and MQPA, rendering them resistant to thrombin-induced hydrolysis. The FRE binding fragment of these inhibitors corresponds to the hirudin55-65 sequence. They differ in the chemical nature of the nonpeptidyl linker bridging these two functional activities. In both cases, the active site binding fragment is well defined in the electron density. The DnsH1, ArgH2, and (D)PipH3 groups occupy the S3, S1, and S2 subsites of thrombin, respectively, in a way similar to that observed in the thrombin-MQPA complexes. Binding in the active site of thrombin is characterized by numerous van der Waals contacts and ring-ring system interactions. Unlike in the substrate-like inhibitors, ArgH2 enters the S1 specificity pocket from the P2 position and adopts a bent conformation to make an hydrogen bond to the carboxylate of Asp189. In this noncanonical position, its carbonyl points away from the oxyanion hole, which is now occupied by well-ordered solvent molecules. The linkers fit in the groove extending from the active site to the FRE. The C-terminal fragments of both inhibitors bind in the same way as analogous FRE binding elements in previously described complexes. PMID:8762149

  1. Mutational analysis of target enzyme recognition of the beta-trefoil fold barley alpha-amylase/subtilisin inhibitor

    DEFF Research Database (Denmark)

    Bønsager, Birgit Christine; Nielsen, Per K.; Abou Hachem, Maher;

    2005-01-01

    The barley alpha-amylase/subtilisin inhibitor ( BASI) inhibits alpha-amylase 2 (AMY2) with subnanomolar affinity. The contribution of selected side chains of BASI to this high affinity is discerned in this study, and binding to other targets is investigated. Seven BASI residues along the AMY2-BASI...... interface and four residues in the putative protease-binding loop on the opposite side of the inhibitor were mutated. A total of 15 variants were compared with the wild type by monitoring the alpha-amylase and protease inhibitory activities using Blue Starch and azoalbumin, respectively, and the kinetics of...

  2. 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

  3. Midgut proteinases of Sitotroga cerealella (Oliver) (Lepidoptera:Gelechiidae): Characterization and relationship to resistance in cereals

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Lan.

    1989-01-01

    Midgut proteinases are vital to the insects which digest ingested food in the midgut. Insect midgut proteinases, therefore, have been considered as possible targets for the control of insect pests. Proteinaceous proteinase inhibitors are very attractive for their potential use in developing insect resistant plant varieties via genetic engineering. Sitotroga cerealella is one of the major storage pests of cereals, and no antibiotic resistance in wheat against this insect has been identified to date. A series of diagnostic inhibitors, thiol-reducing agents and a metal-ion chelator were used in the identification of proteinases in crude extracts from S. cerealella larval midguts with both protein and ester substrates. The partial inhibition of proteolytic activity in crude midgut extract toward ({sup 3}H)-methemoglobin by pepstatin A suggested the presence of another proteinase which was sensitive to pepstatin A. The optimum pH range for the proteolytic activity, however, indicated that the major midgut proteinases were not carboxyl proteinases. Two proteinases were successfully purified by a combination of fractionation with ammonium sulfate, gel permeation and anion exchange chromatography. Characterization of the enzymes with the purified enzyme preparations confirmed that the two major proteinases were serine endoproteinases with trypsin-like and chymotrypsin-like specificities respectively. Bioassays were conducted using the artificial seeds to test naturally occurring proteinaceous proteinase inhibitors of potential value. Soybean trypsin inhibitor and the Bowman-Birk proteinase inhibitor had adverse effects on the development of the insect. A predictive model was constructed to evaluate effects of seed resistance in conjunction with other control methods on S. cerealella population dynamics.

  4. Midgut proteinases of Sitotroga cerealella (Oliver) (Lepidoptera:Gelechiidae): Characterization and relationship to resistance in cereals

    International Nuclear Information System (INIS)

    Midgut proteinases are vital to the insects which digest ingested food in the midgut. Insect midgut proteinases, therefore, have been considered as possible targets for the control of insect pests. Proteinaceous proteinase inhibitors are very attractive for their potential use in developing insect resistant plant varieties via genetic engineering. Sitotroga cerealella is one of the major storage pests of cereals, and no antibiotic resistance in wheat against this insect has been identified to date. A series of diagnostic inhibitors, thiol-reducing agents and a metal-ion chelator were used in the identification of proteinases in crude extracts from S. cerealella larval midguts with both protein and ester substrates. The partial inhibition of proteolytic activity in crude midgut extract toward [3H]-methemoglobin by pepstatin A suggested the presence of another proteinase which was sensitive to pepstatin A. The optimum pH range for the proteolytic activity, however, indicated that the major midgut proteinases were not carboxyl proteinases. Two proteinases were successfully purified by a combination of fractionation with ammonium sulfate, gel permeation and anion exchange chromatography. Characterization of the enzymes with the purified enzyme preparations confirmed that the two major proteinases were serine endoproteinases with trypsin-like and chymotrypsin-like specificities respectively. Bioassays were conducted using the artificial seeds to test naturally occurring proteinaceous proteinase inhibitors of potential value. Soybean trypsin inhibitor and the Bowman-Birk proteinase inhibitor had adverse effects on the development of the insect. A predictive model was constructed to evaluate effects of seed resistance in conjunction with other control methods on S. cerealella population dynamics

  5. Dra I polymorphism in the human inter-alpha-tryspin inhibitor heavy chain gene ITIH1

    Energy Technology Data Exchange (ETDEWEB)

    Leveillard, T.; Salier, J.P.; Sesbouee, R.; Bourguignon, J.; Diarra-Mehrpour, M.; Martin, J.P. (Institut National de la Sante et de la Recherche Medicale, St. Etienne Rouvray (France))

    1989-07-11

    The 1.0 kb insert of lambda HuHITI-19, used as a probe, codes for the heavy chain H1 of the human inter-alpha-tryspin inhibitor. Dra I (TTT/AAA) identifies one invariant band at 5.8 kb and a diallelic polymorphism with DNA fragments at 4.0 kb or 2.4 kb and 1.6 kb. The allele frequency was studied in 24 healthy caucasians. The ITIH1 gene has been mapped to 3p211-p212 by in situ hybridization. Co-dominant segregation was found in three informative families (11 individuals).

  6. An hypervariable polymorphism detected in the human inter-. alpha. -trypsin inhibitor heavy chain gene ITIH2

    Energy Technology Data Exchange (ETDEWEB)

    Leveillard, T.; Sesbouee, R.; Bourguignon, J.; Diarra-Mehrpour, M.; Salier, J.P.; Martin, J.P. (Inst. National de la Sante et de la Recherche Medicale, Rouvray (France)); Sirugo, G.; Hanauer, A. (Inst. National de la Sante et de la Recherche Medicale, Strasbourg (France))

    1990-03-11

    The 112 bp BamHi/Bst YI fragment of lambda HuHITI-9 used as a probe codes for the heavy chain H2 of human inter-{alpha}-trypsin inhibitor. BstXI (CCAN{sub 5}/NTGG) identifies a 5 allele VNTR polymorphism with bands between 2.6 kb and 3.0 kb. DraI, MspI, PstI and TaqI also detect the same polymorphism. The ITH2 gene has been mapped to 10p15 by in situ hybridization.

  7. [A case report of hereditary angioedema and studies on the serum components of complement, C1-inactivator and proteinase inhibitors during edema attack].

    Science.gov (United States)

    Mikami, A; Kohno, M

    1987-05-01

    Sixteen years old girl was admitted because of for the past ten years' frequent edema attack and abdominal pain. Laboratory examination revealed hypocomplementemia, marked depletion of the fourth component of complement and low level of C1-inactivator. Familial studies revealed that her mother was also hypocomplementemic and in low level of C1-inactivator. Serial studies performed on the alterlation of components of complement, C1-inactivator, alpha 1-antitrypsin, antithrombin III, and alpha 2-macroglobulin during edema attack. The fourth component of complement and C1-inactivator were markedly depleted in remission and attack. Remarkable depletion was found in antithrombin III and esterase inhibition activity of C1-inactivator during attack. In contrast, alpha 1-antitrypsin and alpha 2-macroglobulin did not change. The present study may explain that Hageman factor fragments, activated by C1s, promotes kinin generation via kalikrein activation. And the condition that complete functional deficiency of C1-inactivator was main role in this circuit. Fibrynolysis and late components of complement was less influence on edema attack. PMID:3610041

  8. 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.

  9. 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

  10. Preliminary neutron and ultrahigh-resolution X-ray diffraction studies of the aspartic proteinase endothiapepsin cocrystallized with a gem-diol inhibitor

    International Nuclear Information System (INIS)

    Three data sets have been collected on endothiapepsin complexed with the gem-diol inhibitor PD-135,040: a high-resolution synchrotron X-ray data set, a room-temperature X-ray data set and a neutron diffraction data set. Until recently, it has been impossible to grow large protein crystals of endothiapepsin with any gem-diol inhibitor that are suitable for neutron diffraction. Endothiapepsin has been cocrystallized with the gem-diol inhibitor PD-135,040 in a low solvent-content (39%) unit cell, which is unprecedented for this enzyme–inhibitor complex and enables ultrahigh-resolution (1.0 Å) X-ray diffraction data to be collected. This atomic resolution X-ray data set will be used to deduce the protonation states of the catalytic aspartate residues. A room-temperature neutron data set has also been collected for joint refinement with a room-temperature X-ray data set in order to locate the H/D atoms at the active site

  11. 5alpha-reductase inhibitors in benign prostatic hyperplasia and prostate cancer risk reduction.

    Science.gov (United States)

    Rittmaster, Roger S

    2008-04-01

    Androgens play an essential role in prostatic development and function, but are also involved in prostate disease pathogenesis. The primary prostatic androgen, dihydrotestosterone (DHT), is synthesized from testosterone by 5alpha-reductase types 1 and 2. Inhibition of the 5alpha-reductase isoenzymes therefore has potential therapeutic benefit in prostate disease. The two currently approved 5alpha-reductase inhibitors (5ARIs), finasteride and dutasteride, have demonstrated long-term efficacy and safety in the treatment of benign prostatic hyperplasia. Finasteride, a type-2 5ARI, has also been studied for its ability to reduce the incidence of biopsy-detectable prostate cancer in the Prostate Cancer Prevention Trial. Treatment with dutasteride, a dual 5ARI, has been shown to result in a greater degree and consistency of DHT suppression compared with finasteride. Two large-scale studies of dutasteride are currently investigating the role of near-maximal DHT suppression in the settings of prostate cancer risk reduction and expectant management of localized prostate cancer. PMID:18471794

  12. Long-term therapy of interferon-alpha induced pulmonary arterial hypertension with different PDE-5 inhibitors: a case report

    Directory of Open Access Journals (Sweden)

    Baumann Gert

    2005-09-01

    Full Text Available Abstract background Interferon alpha2 is widely used in hepatitis and high-risk melanoma. Interferon-induced pulmonary arterial hypertension as a side effect is rare. Case presentation We describe a melanoma patient who developed severe pulmonary arterial hypertension 30 months after initiation of adjuvant interferon alpha2b therapy. Discontinuation of interferon did not improve pulmonary arterial hypertension. This patient could be treated successfully with phosphodiesterase-5 inhibitor therapy. Conclusion This is only the 5th case of interferon-induced pulmonary arterial hypertension and the first documented case where pulmonary arterial hypertension was not reversible after termination of interferon alpha2 therapy. If interferon alpha2 treated patients develop respiratory symptoms, pulmonary arterial hypertension should be considered in the differential diagnosis. For these patients phosphodiesterase-5 inhibitors, e.g. sildenafil or vardenafil, could be an effective therapeutic approach.

  13. 5-Alpha reductase inhibitor use and prostate cancer survival in the Finnish Prostate Cancer Screening Trial.

    Science.gov (United States)

    Murtola, Teemu J; Karppa, Elina K; Taari, Kimmo; Talala, Kirsi; Tammela, Teuvo Lj; Auvinen, Anssi

    2016-06-15

    Randomized clinical trials have shown that use of 5α-reductase inhibitors (5-ARIs) lowers overall prostate cancer (PCa) risk compared to placebo, while the proportion of Gleason 8-10 tumors is elevated. It is unknown whether this affects PCa-specific survival. We studied disease-specific survival by 5-ARI usage in a cohort of 6,537 prostate cancer cases diagnosed in the Finnish Prostate Cancer Screening Trial and linked to the national prescription database for information on medication use. Cox proportional hazards regression was used to estimate hazard ratios and 95% confidence intervals for prostate cancer-specific deaths. For comparison, survival among alpha-blocker users was also evaluated. During the median follow-up of 7.5 years after diagnosis a total of 2,478 men died; 617 due to prostate cancer and 1,861 due to other causes. The risk of prostate cancer death did not differ between 5-ARI users and nonusers (multivariable adjusted HR 0.94, 95% CI 0.72-1.24 and HR 0.98, 95% CI 0.69-1.41 for usage before and after the diagnosis, respectively). Alpha-blocker usage both before and after diagnosis was associated with increased risk of prostate cancer death (HR 1.29, 95% CI 1.08-1.54 and HR 1.56, 95% CI 1.30-1.86, respectively). The risk increase vanished in long-term alpha-blocker usage. Use of 5-ARIs does not appear to affect prostate cancer mortality when used in management of benign prostatic hyperplasia. Increased risk associated with alpha-blocker usage should prompt further exploration on the prognostic role of lower urinary tract symptoms. PMID:26804670

  14. Molecular Docking Based Virtual Design of Polysubstituted Triazoles as Cytochrome P-450 14-Alpha-Sterol Demethylase (Cyp51) Inhibitor

    OpenAIRE

    Bibek Pati*,1; Subhasis Banerjee2

    2012-01-01

    Computational ligand docking methodology, AutoDock 4.0, based on Lamarckian genetic algorithm was employed for virtual screening of a compound library with 13 entries including reference compound as fluconazole with the enzyme Cytochrome P-450-14-Alpha-Sterol Demethylase (Cyp51), a potential antifungal drug target. Considering free energy of binding as a criteria of evaluation, a total of 12 compounds were predicted to be potential inhibitors of Cytochrome P-450-14-Alpha-Sterol Demethylase (C...

  15. Digestive alpha-amylases of the flour moth Ephestia kuehniella - adaptation to alkaline environment and plant inhibitors

    Czech Academy of Sciences Publication Activity Database

    Pytelková, Jana; Hubert, J.; Lepšík, Martin; Šobotník, Jan; Šindelka, Radek; Křížková, I.; Horn, Martin; Mareš, Michael

    2009-01-01

    Roč. 276, č. 13 (2009), s. 3531-3546. ISSN 1742-464X R&D Projects: GA AV ČR IAA400550617; GA MŠk LC512; GA ČR GA301/09/1752 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520701 Keywords : alkaline adaptation * alpha -amylase * alpha-amylase inhibitor * Ephestia kuehniella * plant-insect interaction Subject RIV: CE - Biochemistry Impact factor: 3.042, year: 2009

  16. Molecular Docking Based Virtual Design of Polysubstituted Triazoles as Cytochrome P-450 14-Alpha-Sterol Demethylase (Cyp51 Inhibitor

    Directory of Open Access Journals (Sweden)

    Bibek Pati*,1

    2012-01-01

    Full Text Available Computational ligand docking methodology, AutoDock 4.0, based on Lamarckian genetic algorithm was employed for virtual screening of a compound library with 13 entries including reference compound as fluconazole with the enzyme Cytochrome P-450-14-Alpha-Sterol Demethylase (Cyp51, a potential antifungal drug target. Considering free energy of binding as a criteria of evaluation, a total of 12 compounds were predicted to be potential inhibitors of Cytochrome P-450-14-Alpha-Sterol Demethylase (Cyp51 and 10 compounds displayed greater binding affinities than fluconazole as Cytochrome P-450-14-Alpha-Sterol Demethylase (Cyp51 Inhibitor. Compound 1a & 1b were the most potent in inhibiting the Cytochrome P-450-14-Alpha-Sterol Demethylase (Cyp51, in silico. Putative interactions between Cytochrome P-450-14-Alpha-Sterol Demethylase (Cyp51 and inhibitors were identified by inspection of docking-predicted poses. This understanding of protein–ligand interaction and value of binding energy imparts impetus to the rapid development of novel Cytochrome P-450-14-Alpha-Sterol Demethylase (Cyp51.

  17. The kinetics of interaction of porcine - alpha-, and porcine - beta -trypsin with intact and modified soybean trypsin inhibitor (kunitz)

    International Nuclear Information System (INIS)

    The association of porcine trypsin with soybean trypsin inhibitor (Kunitz) resulted in characteristic changes in absorption spectrum, indicating an alteration of the micro environments of the enzyme chromophores as a consequence of the interaction. The rates of formation of the stable trypsin - inhibitor complexes from porcine - alpha - trypsin and soybean trypsin inhibitor and from porcine - beta - trypsin and either intact or modified soybean trypsin inhibitor were measured by mixing the equimolar concentration of the reactants in a Stopped - Flow apparatus at pH (4.5 to 10.0). The reaction of trypsin with soybean trypsin inhibitor was of first order with respect to the concentration of the reactants used. The rates of dissociation of the stable complexes, alpha - trypsin - soybean trypsin inhibitor, beta -trypsin - soybean trypsin inhibitor and beta -trypsin modified soybean trypsin inhibitor were also measured at pH (1.92 to 3.58). The values of first order rate constant, k/sub D/ obtained for the dissociation of all the three complexes were identical with one another. The kinetics results obtained for the porcine trypsin were compared with those of bovine trypsin system and it was suggested that the reaction mechanisms in both these systems were identical. (author)

  18. Calcineurin inhibitor tacrolimus does not interfere with the suppression of hepatitis C virus infection by interferon-alpha.

    Science.gov (United States)

    Pan, Qiuwei; Metselaar, Herold J; de Ruiter, Petra; Kwekkeboom, Jaap; Tilanus, Hugo W; Janssen, Harry L A; van der Laan, Luc J W

    2010-04-01

    Immunosuppression considerably affects hepatitis C virus (HCV) recurrence and the outcome of antiviral treatment after liver transplantation. Recent findings have suggested that the calcineurin inhibitor tacrolimus (Tac), unlike cyclosporine A (CsA), interferes with the antiviral activity of interferon-alpha (IFN-alpha) in vitro. The aim of this study was to more extensively investigate the effects of calcineurin inhibitors on IFN-alpha signaling and antiviral activity in subgenomic and infectious HCV models. Treatment with Tac and CsA did not affect Huh7 cell proliferation at doses of 10 to 500 ng/mL; however, it completely inhibited T cell proliferation. In contrast to previous reports, Tac had no effect on IFN-alpha-stimulated reporter gene expression, even at the dose of 5 microg/mL. Furthermore, in Huh7 subgenomic HCV replicon cells, treatment with Tac had no significant effect on the suppression of viral replication by IFN-alpha. In the infectious HCV model, treatment with IFN-alpha effectively inhibited both viral RNA replication and de novo production of virus particles, and neither was attenuated at any concentration of Tac. CsA had no significant effect on IFN-alpha-stimulated reporter gene expression; however, as shown previously, a combination of CsA (at 500 ng/mL and higher) and IFN-alpha resulted in enhanced inhibition of viral replication in both the subgenomic and infectious HCV models. In conclusion, our study shows no evidence that Tac or CsA interferes with IFN-alpha-mediated inhibition of HCV replication and virion production in vitro. Therefore, no further mechanistic arguments have been found to break the clinical controversy about the choice of calcineurin inhibitors during posttransplantation antiviral therapy. PMID:20373462

  19. 12-o-Tetradecanoyl-phorbol-13-acetate-differentiated U937 cells express a macrophage-like profile of neutral proteinases. High levels of secreted collagenase and collagenase inhibitor accompany low levels of intracellular elastase and cathepsin G.

    OpenAIRE

    Welgus, H G; Connolly, N L; Senior, R M

    1986-01-01

    Human monocytic tumor cells of the U937 cell line contain substantial quantities of two neutrophil neutral proteinases, elastase and cathepsin G, raising the question of whether their presence reflects an expression of transformation or whether normal monocytes undergo a developmental stage in which they produce certain neutrophil proteinases. To address this issue, we examined U937 cells for production of collagenase, since human alveolar macrophages release fibroblast-like collagenase, an e...

  20. Role of golimumab, a TNF-alpha inhibitor, in the treatment of the psoriatic arthritis

    Directory of Open Access Journals (Sweden)

    Melissa A Michelon

    2010-05-01

    Full Text Available Melissa A Michelon1, Alice B Gottlieb1,21Tufts University School of Medicine, 2Department of Dermatology, Tufts Medical Center, Boston, MA, USAAbstract: Psoriatic arthritis (PsA is an inflammatory arthritis that affects many psoriasis patients and can often have a debilitating disease progression. Golimumab is a new tumor necrosis factor (TNF antagonist recently approved by the FDA for controlling signs and symptoms of psoriatic arthritis. In a Phase III clinical trial in patients with PsA, patients receiving golimumab showed significant improvement in the signs and symptoms of disease. It was usually well tolerated, but adverse events generally occurred more in patients receiving golimumab compared to placebo. Golimumab has also recently shown efficacy in slowing structural damage in PsA. This new biologic therapy provides physicians with another option in the treatment of this inflammatory arthritis while offering patients certain advantages over other TNF antagonists.Keywords: golimumab, psoriatic arthritis, TNF-alpha inhibitor

  1. 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.

  2. The REDUCE trial: chemoprevention in prostate cancer using a dual 5alpha-reductase inhibitor, dutasteride.

    Science.gov (United States)

    Musquera, Mireia; Fleshner, Neil E; Finelli, Antonio; Zlotta, Alexandre R

    2008-07-01

    Dutasteride, a dual 5alpha-reductase inhibitor, is used in the treatment of benign prostatic hyperplasia (BPH). It reduces serum prostate-specific antigen levels by approximately 50% at 6 months and total prostate volume by 25% after 2 years. Randomized placebo-controlled trials in BPH patients have shown the efficacy of dutasteride in symptomatic relief, improvements in quality of life and peak urinary flow rate. Side effects occurring with dutasteride are decreased libido, erectile dysfunction, ejaculation disorders and gynecomastia. Preliminary data from placebo-controlled BPH trials have shown a decrease in the detection of prostate cancer in patients treated with dutasteride, although these studies were not designed to look at this issue. Dutasteride differs from finasteride in that it inhibits both isoenzymes of 5alpha-reductase, type I and type II. The landmark Prostate Cancer Prevention Trial at the end of the 7-year study demonstrated a 24.8% reduction in the incidence of prostate cancer in the finasteride group compared with placebo. However, a 25.5% increase in the prevalence of high-grade Gleason tumors has been observed, the clinical significance of which has been debated. Preliminary data suggest a decrease in prostate cancer incidence in dutasteride-treated patients and demonstrate type I alphareductase enzyme expression in prostate cancer. As a result, dutasteride is being investigated for prostate cancer prevention in the ongoing Reduction by Dutasteride of Prostate Cancer Events (REDUCE) trial, which is discussed here. PMID:18588452

  3. Cicaprost and the type IV phosphodiesterase inhibitor, rolipram, synergize in suppression of tumor necrosis factor-alpha synthesis

    NARCIS (Netherlands)

    Greten, T F; Sinha, B; Haslberger, C; Eigler, A; Endres, S

    1996-01-01

    Suppression of tumor necrosis factor-alpha (TNF) synthesis is one major target in pharmacological immunomodulation. We now showed the synergistic suppressive effect of the specific type IV phosphodiesterase inhibitor, rolipram, and of the stable prostacyclin analogue, cicaprost, on TNF synthesis. Th

  4. The spectrum of low molecular weight alpha-amylase/protease inhibitor genes expressed in the US bread wheat Butte 86

    Science.gov (United States)

    The complement of genes encoding alpha-amylase/protease inhibitors expressed in Triticum aestivum cv. Butte 86 was characterized by transcript and proteomic analysis. Coding sequences for 18 distinct proteins were identified among a collection of expressed sequence tags (ESTs) from Butte 86 developi...

  5. Alpha adrenergic modulation on effects of norepinephrine transporter inhibitor reboxetine in five-choice serial reaction time task

    Directory of Open Access Journals (Sweden)

    Liu Yia-Ping

    2009-08-01

    Full Text Available Abstract The study examined the effects of a norepinephrine transporter (NET inhibitor reboxetine (RBX on an attentional performance test. Adult SD rats trained with five-choice serial reaction time task (5-CSRTT were administered with RBX (0, 3.0 and 10 mg/kg in the testing day. Alpha-1 adrenergic receptor antagonist PRA and alpha-2 adrenergic receptor antagonist RX821002 were used to clarify the RBX effect. Results revealed that rat received RBX at 10 mg/kg had an increase in the percentage of the correct response and decreases in the numbers of premature response. Alpha-1 adrenergic receptor antagonist Prazosin (PRA at 0.1 mg/kg reversed the RBX augmented correct responding rate. However, alpha-2 adrenergic receptor antagonist RX821002 at 0.05 and 0.1 mg/kg dose dependently reversed the RBX reduced impulsive responding. Our results suggested that RBX as a norepinephrine transporter inhibitor can be beneficial in both attentional accuracy and response control and alpha-1 and alpha-2 adrenergic receptors might be involved differently.

  6. Molecular characterization of two kazal-type serine proteinase inhibitor genes in the surf clam Mesodesma donacium exposed to Vibrio anguillarum.

    Science.gov (United States)

    Maldonado-Aguayo, Waleska; Núñez-Acuña, Gustavo; Valenzuela-Muñoz, Valentina; Chávez-Mardones, Jacqueline; Gallardo-Escárate, Cristian

    2013-06-01

    This study reports two kazal-type serine protease inhibitors (KPI) identified in a cDNA library from the surf clam Mesodesma donacium, and characterized through Rapid Amplification of cDNA Ends (RACE). The KPIs, denoted as MdSPI-1 and MdSPI-2, presented full sequences of 1139 bp and 781 bp respectively. MdSPI-1 had a 5'untranslated region (UTR) of 175 bp, a 3'UTR of 283 bp and an open reading frame (ORF) of 681 pb that encodes for 227 amino acids. MdSPI-2 showed a 5'UTR of 70 bp, a 3'UTR of 279 bp and an ORF of 432 bp that encodes for 144 amino acids. Both sequences presented two kazal-type tandem domains. Phylogenetic analysis of MdSPI-1 and MdSPI-2 shows a main clade composed by other bivalve species and closely related crustaceans. Real time PCR analysis showed that MdSPI-1 is mainly up-regulated in mantle, foot, gills and muscle tissues, while MdSPI-2 is expressed principally in foot tissue. Moreover, to evaluate the immune response of MdSPI-1 and MdSPI-2, infections with Vibrio anguillarum were performed. Herein, MdSPI-1 and MdSPI-2 transcription expression were significantly up-regulated at 2 and 8 h post-challenge. Our results suggest that MdSPI-1 and MdSPI-2 are important humoral factors of innate immunity in M. donacium. PMID:23528874

  7. The polyamine inhibitor alpha-difluoromethylornithine modulates hippocampus-dependent function after single and combined injuries.

    Directory of Open Access Journals (Sweden)

    Susanna Rosi

    Full Text Available Exposure to uncontrolled irradiation in a radiologic terrorism scenario, a natural disaster or a nuclear battlefield, will likely be concomitantly superimposed on other types of injury, such as trauma. In the central nervous system, radiation combined injury (RCI involving irradiation and traumatic brain injury may have a multifaceted character. This may entail cellular and molecular changes that are associated with cognitive performance, including changes in neurogenesis and the expression of the plasticity-related immediate early gene Arc. Because traumatic stimuli initiate a characteristic early increase in polyamine metabolism, we hypothesized that treatment with the polyamine inhibitor alpha-difluoromethylornithine (DFMO would reduce the adverse effects of single or combined injury on hippocampus structure and function. Hippocampal dependent cognitive impairments were quantified with the Morris water maze and showed that DFMO effectively reversed cognitive impairments after all injuries, particularly traumatic brain injury. Similar results were seen with respect to the expression of Arc protein, but not neurogenesis. Given that polyamines have been found to modulate inflammatory responses in the brain we also assessed the numbers of total and newly born activated microglia, and found reduced numbers of newly born cells. While the mechanisms responsible for the improvement in cognition after DFMO treatment are not yet clear, the present study provides new and compelling data regarding the potential use of DFMO as a potential countermeasure against the adverse effects of single or combined injury.

  8. Cost Effectiveness of 5-Alpha Reductase Inhibitors for the Prevention of Prostate Cancer in Multiple Patient Populations

    OpenAIRE

    Stephanie R. Earnshaw; McDade, Cheryl L; Libby K. Black; Christopher F. Bell; Kattan, Michael W.

    2010-01-01

    Background: Although 5-alpha reductase inhibitors (5ARIs) have demonstrated that they reduce the risk of prostate cancer (PCa), they have not demonstrated cost effectiveness in the patient populations in which they have been examined. Objective: A decision-analytic model was created to explore economic benefits from a third-party payer perspective of the use of 5ARIs in preventing PCa in men with different risk factors for developing the disease. Methods: A Markov model was developed to simul...

  9. Five-alpha Reductase Inhibitor Influences Expression of Androgen Receptor and HOXB13 in Human Hyperplastic Prostate Tissue

    OpenAIRE

    Chaeyong Jung; Youngwoong Park; Young-Rang Kim; Soo Bang Ryu; Taek Won Kang

    2013-01-01

    Objectives Five-alpha reductase inhibitors (5ARIs) are known as chemopreventive agents in prostate cancer with a risk of high-grade disease. This study evaluated the effects of 5ARI on androgen receptor (AR) and proteins involved in prostate cell growth such as HOXB13 expression in human prostate tissue and LNCaP prostate cancer cells. Materials and Methods We retrospectively selected 21 patients who underwent TURP between March 2007 and February 2010 for previously confirmed BPH by prostate...

  10. Potential of the bean alpha-amylase inhibitor alpha-AI-1 to inhibit alpha-amylase activity in true bugs(Hemiptera)

    Science.gov (United States)

    True bugs (Hemiptera) are an important pest complex not controlled by Bt crops. An alternative source of resistance includes inhibitors of digestive enzymes. aAI-1, an a-amylase inhibitor from the common bean, has been shown to inhibit a-amylases of bruchid pests of grain legumes. Here we quantify t...

  11. A structural model of picornavirus leader proteinases based on papain and bleomycin hydrolase

    OpenAIRE

    Skern, Tim; Fita, Ignacio; Guarné, Alba

    1998-01-01

    The leader (L) proteinases of aphthoviruses (foot-and-mouth disease viruses) and equine rhinovirus serotypes 1 and 2 cleave themselves from the growing polyprotein. This cleavage occurs intramolecularly between the C terminus of the L proteinases and the N terminus of the subsequent protein VP4. The foot-and-mouth disease virus enzyme has been shown, in addition, to cleave at least one cellular protein, the eukaryotic initiation factor 4G. Mechanistically, inhibitor studies and sequence analy...

  12. Inhibitors of lysosomal cysteine proteases

    OpenAIRE

    Lyanna O. L.; Chorna V. I.

    2011-01-01

    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 anal...

  13. 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.

  14. Seed-specific stable expression of the alpha-AI1 inhibitor in coffee grains and the in vivo implications for the development of the coffee berry borer

    OpenAIRE

    Albuquerque, E. V. S.; Bezerra, C. A.; Romero, J. V.; Valencia, J. W. A.; Valencia-Jimenez, A.; Pimenta, L. M.; Barbosa, Aead; Silva, M. C. M.; Meneguim, A. M.; Sa, M. E. L.; Engler, G.; de Almeida-Engler, J.; Fernandez, Diana; Grossi-De-Sa, M. F.

    2015-01-01

    Genetic transformation of coffee (Coffea spp.), the second most traded commodity worldwide, is an alternative approach to introducing features that cannot be introgressed by traditional crossings. The transgenic stability, heritability and quantitative and spatial expression patterns of the seed-specific promoter phytohemagglutinin (PHA-L) from Phaseolus vulgaris were characterized in genetically modified C. arabica expressing the alpha-amylase inhibitor-1 (alpha-AI1) gene. The alpha-AI1 inhi...

  15. Characterization of proteinases in trypanosomatids.

    Science.gov (United States)

    Branquinha, M H; Vermelho, A B; Goldenberg, S; Bonaldo, M C

    1994-02-01

    Proteinases are important factors in the pathogenicity of many parasitic diseases. In this study, the proteolytic activities of 10 trypanosomatids from five different genera (Crithidia, Phytomonas, Endotrypanum, Trypanosoma and Leishmania) were determined by SDS-PAGE containing copolymerized gelatin as substrate. In almost all species we could detect two proteolytic classes, cysteine- and metalloproteinases, based on the inhibition of their activities by E-64 and 1,10-phenanthroline, respectively. In all cases, the metalloproteinase activities did not change over a broad pH range (from 5.5 to 10). E. schaudinni, T. mega, T. dionisii, C. luciliae, C. fasciculata, C. oncopelti and C. guilhermei expressed one or two metalloproteinases of 45-66 kDa, whereas in P. serpens and P. hyssopifolia a double band of this endopeptidase was detected at 94 kDa. In contrast, no metalloproteinase activity was observed in L. tarentolae. The optimal pH for the cysteine-proteinase activities was acidic (about 5.5). In E. schaudinni, T. mega and in Crithidia sp., these proteinases had an apparent molecular weight of 66-94 kDa, while L. tarentolae expressed a broad band from 29 to 45 kDa. In Phytomonas sp., this class of endopeptidase showed a unique feature, in that major cysteine-proteinases were found at 29-66 kDa, but multiple, low-activity bands were detected from 116 to 200 kDa. The most striking characteristic, however, was the very intense cysteine-proteinase activity expressed by T. dionisii (29-66 kDa). We conclude that these differences in the proteolytic profiles could be useful markers to characterize and compare trypanosomatids. PMID:8081271

  16. Urine of patients with early prostate cancer contains lower levels of light chain fragments of inter-alpha-trypsin inhibitor and saposin B but increased expression of an inter-alpha-trypsin inhibitor heavy chain 4 fragment.

    Science.gov (United States)

    Jayapalan, Jaime J; Ng, Keng L; Shuib, Adawiyah S; Razack, Azad H A; Hashim, Onn H

    2013-06-01

    The present study was aimed at the identification of proteins that are differentially expressed in the urine of patients with prostate cancer (PCa), those with benign prostatic hyperplasia (BPH) and age-matched healthy male control subjects. Using a combination of 2DE and MS/MS, significantly lower expression of urinary saposin B and two different fragments of inter-alpha-trypsin inhibitor light chain (ITIL) was demonstrated in the PCa patients compared to the controls. However, only one of the ITIL fragments was significantly different between the PCa and BPH patients. When image analysis was performed on urinary proteins that were transferred onto NC membranes and detected using a lectin that binds to O-glycans, a truncated fragment of inter-alpha-trypsin inhibitor heavy chain 4 was the sole protein found to be significantly enhanced in the PCa patients compared to the controls. Together, these urinary peptide fragments might be useful complementary biomarkers to indicate PCa as well as to distinguish it from BPH, although further epidemiological evidence on the specificity and sensitivity of the protein candidates is required. PMID:23417432

  17. Lycopene is a more potent inhibitor of human cancer cell proliferation than either alpha-carotene or beta-carotene.

    Science.gov (United States)

    Levy, J; Bosin, E; Feldman, B; Giat, Y; Miinster, A; Danilenko, M; Sharoni, Y

    1995-01-01

    The antiproliferative properties of lycopene, the major tomato carotenoid, were compared with those of alpha- and beta-carotene. Lycopene, delivered in cell culture medium from stock solutions in tetrahydrofuran, strongly inhibited proliferation of endometrial (Ishikawa), mammary (MCF-7), and lung (NCI-H226) human cancer cells with half-maximal inhibitory concentration of 1-2 microM; alpha- and beta-carotene were far less effective inhibitors. For example, in Ishikawa cells, a 4-fold higher concentration of alpha-carotene or a 10-fold higher concentration of beta-carotene was needed for the same order of growth suppression. The inhibitory effect of lycopene was detected after 24 hours of incubation, and it was maintained for at least three days. In contrast to cancer cells, human fibroblasts were less sensitive to lycopene, and the cells gradually escaped growth inhibition over time. In addition to its inhibitory effect on basal endometrial cancer cell proliferation, lycopene also suppressed insulin-like growth factor-I-stimulated growth. Insulin-like growth factors are major autocrine/paracrine regulators of mammary and endometrial cancer cell growth. Therefore, lycopene interference in this major autocrine/paracrine system may open new avenues for research on the role of lycopene in the regulation of endometrial cancer and other tumors. PMID:8610045

  18. [Studies on human alpha-2 macroglobulin structure and its complexes with proteases, using polyacrylamide gel electrophoresis].

    Science.gov (United States)

    Fine, J M; Lambin, P; Steinbuch, M

    1975-09-01

    Pure alpha2M is prepared with fresh plasma as starting material, to prevent the interaction of alpha2M from proteolytic enzymes of plasma such as thrombin, plasmin and kallikrein. During the purification steps, polybrene and aprotin are used as inhibitors and plasminogen is absorbed onto bentonite. When alpha 2M is submitted to polyacrylamide gel electrophoresis (PAA) containing 0.1% SDS, a complete dissociation in two half-molecules of MW 380,000 occurs. When alpha2M is incubated in 1% SDS and 1% beta-mercaptoethanol as reducing agent, only one component of MW 190,000 is observed in PAA-SDS. This experiments show that the alpha2M molecule consist of two symetric halves of same MW (380,000) linked by non covalent bonds. Each two-half-molecules is made of two polypeptides chains MW 190,000 linked by disulfide bonds. Thus alpha2M molecule contains four polypeptides chains having a same MW. The same techniques were applied to the study of alaph2M proteinases complexes. Three different proteinases (plasmin, trypsin and papain) were used in these experiments. Trypsin and papain are commercialy available. Plasminogen was obtained by affinity chromatography and activated into plasmin by insoluble streptokinase fixed on PAB cellulose. PMID:59941

  19. A heterotetrameric alpha-amylase inhibitor from emmer (Triticum dicoccon Schrank) seeds.

    Science.gov (United States)

    Capocchi, A; Muccilli, V; Cunsolo, V; Saletti, R; Foti, S; Fontanini, D

    2013-04-01

    Plants have developed a constitutive defense system against pest attacks, which involves the expression of a set of inhibitors acting on heterologous amylases of different origins. Investigating the soluble protein complement of the hulled wheat emmer we have isolated and characterized a heterotetrameric α-amylase inhibitor (ETI). Based on mass spectrometry data, it is an assembly of proteins highly similar to the CM2/CM3/CM16 found in durum wheat. Our data indicate that these proteins can also inhibit exogenous α-amylases in binary assemblies. The calculated dissociation constants (K(i)) for the pancreatic porcine amylase- and human salivary amylase-ETI complexes are similar to those found in durum and soft wheat. Homology modeling of the CM subunits indicate structural similarities with other proteins belonging to the cereal family of trypsin/α-amylase inhibitors; a possible homology modeled structure for a tetrameric assembly of the subunits is proposed. PMID:23320956

  20. Reflection on design and testing of pancreatic alpha-amylase inhibitors: an in silico comparison between rat and rabbit enzyme models

    Directory of Open Access Journals (Sweden)

    Khalil-Moghaddam Shiva

    2012-11-01

    Full Text Available Abstract Background Inhibitors of pancreatic alpha-amylase are potential drugs to treat diabetes and obesity. In order to find compounds that would be effective amylase inhibitors, in vitro and in vivo models are usually used. The accuracy of models is limited, but these tools are nonetheless valuable. In vitro models could be used in large screenings involving thousands of chemicals that are tested to find potential lead compounds. In vivo models are still used as preliminary mean of testing compounds behavior in the whole organism. In the case of alpha-amylase inhibitors, both rats and rabbits could be chosen as in vivo models. The question was which animal could present more accuracy with regard to its pancreatic alpha-amylase. Results As there is no crystal structure of these enzymes, a molecular modeling study was done in order to compare the rabbit and rat enzymes with the human one. The overall result is that rabbit enzyme could probably be a better choice in this regard, but in the case of large ligands, which could make putative interactions with the −4 subsite of pancreatic alpha-amylase, interpretation of results should be made cautiously. Conclusion Molecular modeling tools could be used to choose the most suitable model enzyme that would help to identify new enzyme inhibitors. In the case of alpha-amylase, three-dimensional structures of animal enzymes show differences with the human one which should be taken into account when testing potential new drugs.

  1. 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

  2. The spectrum of low molecular weight alpha-amylase/protease inhibitor genes expressed in the US bread wheat cultivar Butte 86

    Directory of Open Access Journals (Sweden)

    Vensel William H

    2011-07-01

    Full Text Available Abstract Background Wheat grains accumulate a variety of low molecular weight proteins that are inhibitors of alpha-amylases and proteases and play an important protective role in the grain. These proteins have more balanced amino acid compositions than the major wheat gluten proteins and contribute important reserves for both seedling growth and human nutrition. The alpha-amylase/protease inhibitors also are of interest because they cause IgE-mediated occupational and food allergies and thereby impact human health. Results The complement of genes encoding alpha-amylase/protease inhibitors expressed in the US bread wheat Butte 86 was characterized by analysis of expressed sequence tags (ESTs. Coding sequences for 19 distinct proteins were identified. These included two monomeric (WMAI, four dimeric (WDAI, and six tetrameric (WTAI inhibitors of exogenous alpha-amylases, two inhibitors of endogenous alpha-amylases (WASI, four putative trypsin inhibitors (CMx and WTI, and one putative chymotrypsin inhibitor (WCI. A number of the encoded proteins were identical or very similar to proteins in the NCBI database. Sequences not reported previously included variants of WTAI-CM3, three CMx inhibitors and WTI. Within the WDAI group, two different genes encoded the same mature protein. Based on numbers of ESTs, transcripts for WTAI-CM3 Bu-1, WMAI Bu-1 and WTAI-CM16 Bu-1 were most abundant in Butte 86 developing grain. Coding sequences for 16 of the inhibitors were unequivocally associated with specific proteins identified by tandem mass spectrometry (MS/MS in a previous proteomic analysis of milled white flour from Butte 86. Proteins corresponding to WDAI Bu-1/Bu-2, WMAI Bu-1 and the WTAI subunits CM2 Bu-1, CM3 Bu-1 and CM16 Bu-1 were accumulated to the highest levels in flour. Conclusions Information on the spectrum of alpha-amylase/protease inhibitor genes and proteins expressed in a single wheat cultivar is central to understanding the importance of

  3. Carboxy-terminal truncation of oryzacystatin II by oryzacystatin-insensitive insect digestive proteinases.

    Science.gov (United States)

    Michaud, D; Cantin, L; Vrain, T C

    1995-10-01

    The biochemical interactions between digestive proteinases of the Coleoptera pest black vine weevil (Otiorynchus sulcatus) and two plant cysteine proteinase inhibitors, oryzacystatin I (OCI) and oryzacystatin II (OCII), were assessed using gelatin-polyacrylamide gel electrophoresis, OCI-affinity chromatography, and recombinant forms of the two plant inhibitors. The insect proteinases were resolved in gelatin-containing polyacrylamide gels as five major bands, only three of them being totally or partially inactivated by OCI and OCII. The maximal inhibitory effect of both OCs at pH 5.0 was estimated at 40% and the inhibition was stable with time despite the presence of OC-insensitive proteases, indicating the stability of the OCI and OCII effects. After removing OC-sensitive proteinases from the insect crude extract by OCI-affinity chromatography, the effects of the insect cystatin-insensitive proteases on the structural integrity of the free OCs were analyzed. While OCI remained stable, OCII was subjected to limited proteolysis leading to its gradual transformation into a approximately 10.5-kDa unstable intermediate, OCIIi. As shown by the degradation pattern of a glutathione S-transferase (GST)/OCII fusion protein, the appearance of OCIIi resulted from the C-terminal truncation of OCII. Either free or linked to GST, OCIIi was as active against papain and human cathepsin H as OCII, and the initial specificities of the inhibitor for these two cysteine proteinases were conserved after cleavage. Although these observations indicate the high conformational stability of OCII near its active (inhibitory) site, they also suggest a general conformational destabilization of this inhibitor following its initial cleavage, subsequently leading to its complete hydrolysis. This apparent susceptibility of OCII to proteolytic cleavage by the insect proteinases could have major implications when planning the use of this plant cystatin for insect pest control. PMID:7574723

  4. Two RFLPs in human inter-alpha-trypsin inhibitor heavy chain gene ITIH2 on chromosome 10

    Energy Technology Data Exchange (ETDEWEB)

    Leveillard, T.; Bourguignon, J.; Salier, J.P.; Diarra-Mehrpour, M.; Sesbouee, R.; Martin, J.P. (Institut National de la Sante et de la Recherche Medicale, St. Etienne Rouvray (France))

    1989-07-11

    The 0.8 kb Eco RI/Bam HI fragment of lambda HuHITI-9 used as a probe codes for human heavy chain H2 of the inter-alpha-trypsin inhibitor. Kpn I (GGTAC/C) identifies one invariant band at 8.5 kb and a diallelic polymorphism with DNA fragments at 26.0 kb or 20.0 kb. Msp I (C/CGG) identifies three invariant bands at 2.35 kb, 2.1 kb and 1.0 kb and a diallelic polymorphism with DNA fragments at 5.0 kb or 2.8 kb and 2.2 kb. The allele frequency for Kpn I and Msp I were determined. The ITIH2 gene has been mapped to 10p15 by in situ hybridization. Co-dominant segregation was found for each polymorphism in two informative families.

  5. Impact of tobacco smoking on response to tumour necrosis factor-alpha inhibitor treatment in patients with ankylosing spondylitis

    DEFF Research Database (Denmark)

    Glintborg, Bente; Højgaard, Pil; Lund Hetland, Merete;

    2016-01-01

    .29-4.95)), P < 0.0001). Similar results were found in multivariate analyses (current versus never smokers, HR 1.41 (95% CI 1.21-1.65), P < 0.001), most pronounced among men. Current smokers had poorer 6 months' BASDAI50%/20 mm-response rate than never smokers (42%/58%, P < 0.001). In multivariate analyses......OBJECTIVES: To investigate the association between tobacco smoking and disease activity, treatment adherence and treatment responses in patients with AS treated with their first tumour necrosis factor-alpha inhibitor (TNFi) therapy in routine care. METHODS: Observational cohort study based on the...... Danish nationwide DANBIO registry. Kaplan-Meier plots, Cox and logistic regression analyses by smoking status (current/never/previous) were calculated for treatment adherence and BASDAI 50%/20 mm-response. Additional stratified analyses were performed for gender and TNFi-type. RESULTS: Of 1576 AS...

  6. Tissue factor pathway inhibitor-alpha inhibits prothrombinase during the initiation of blood coagulation

    OpenAIRE

    Wood, Jeremy P.; Bunce, Matthew W.; Maroney, Susan A.; Tracy, Paula B.; Camire, Rodney M.; Mast, Alan E.

    2013-01-01

    The generation of thrombin by prothrombinase, a complex composed of activated (a) factors X (FXa) and V (FVa), is a final step in blood coagulation. We demonstrate that tissue factor pathway inhibitor (TFPI) blocks thrombin generation by prothrombinase at physiologically relevant rates and concentrations, but only during the initiation of clot formation. TFPI mediates this inhibitory activity through two high-affinity interactions, one with FXa and one with FVa. This is the first description ...

  7. De novo design of alpha-amylase inhibitor: A small linear mimetic of macromolecular proteinaceous ligands

    Czech Academy of Sciences Publication Activity Database

    Marešová, Lucie; Pavlík, Manfred; Horn, Martin; Mareš, Michael

    2005-01-01

    Roč. 12, č. 12 (2005), 1349-1357. ISSN 1074-5521 R&D Projects: GA ČR(CZ) GP203/02/P081; GA MŠk(CZ) OC D16.001 Institutional research plan: CEZ:AV0Z40550506 Keywords : amylase * peptide inhibitor * combinatorial chemistry Subject RIV: CE - Biochemistry Impact factor: 6.138, year: 2005

  8. N-Acetyl-Beta-D-Glucopyranosylamine - a Potent T-State Inhibitor of Glycogen-Phosphorylase - a Comparison with Alpha-D-Glucose

    OpenAIRE

    Oikonomakos, Nikos G.; Kontou, M.; Zographos, Spyros E.; Watson, K. A.; Johnson, L N; Bichard, C. J. F.; Fleet, G. W. J.; Acharya, K. R.

    2008-01-01

    Structure-based drug design has led to the discovery of a number of glucose analogue inhibitors of glycogen phosphorylase that have an increased affinity compared to alpha-D-glucose (Ki = 1.7 mM). The best inhibitor in the class of N-acyl derivatives of beta-D-glucopyranosylamine, N-acetyl-beta-D-glucopyranosylamine (1-GlcNAc), has been characterized by kinetic, ultracentrifugation, and crystallographic studies. 1-GlcNAc acts as a competitive inhibitor for both the b (Ki = 32 microM) and the ...

  9. 5-Alpha reductase inhibitors in men with an enlarged prostate: an evaluation of outcomes and therapeutic alternatives.

    Science.gov (United States)

    Naslund, Michael; Regan, Timothy S; Ong, Christine; Hogue, Susan L

    2008-05-01

    This article presents background information and highlights key findings from a managed care perspective related to enlarged prostate (EP) in Medicare-eligible patients. This article does not provide a comprehensive review of EP but instead attempts to increase the current understanding of EP through discussion of its prevalence in men aged > or =65 years, its associated economic burden, and some available treatment options. This supplement includes 3 additional articles, all of which present data from a naturalistic, managed care setting. The article by Fenter et al assesses differences in outcomes between elderly EP patients treated with finasteride and those treated with dutasteride in relation to the risks of acute urinary retention and prostate-related surgery. Issa et al conduct a comparative analysis of the combined use of alpha-blockers and 5-alpha reductase inhibitors to treat EP. The final article compares medical costs incurred within the first year of initiating treatment for EP patients receiving finasteride versus dutasteride. This supplement is intended to assist managed care formulary decision makers in evaluating key clinical and economic data that differentiate dutasteride and finasteride within the Medicare-aged population. Although the information presented is not designed to illustrate the superiority of one product over the other, it answers important questions in relation to treating EP in elderly men and raises substantial issues beyond medication costs. PMID:18611088

  10. Integrin alpha3beta1, a novel receptor for alpha3(IV) noncollagenous domain and a trans-dominant Inhibitor for integrin alphavbeta3.

    Science.gov (United States)

    Borza, Corina M; Pozzi, Ambra; Borza, Dorin-Bogdan; Pedchenko, Vadim; Hellmark, Thomas; Hudson, Billy G; Zent, Roy

    2006-07-28

    Exogenous soluble human alpha3 noncollagenous (NC1) domain of collagen IV inhibits angiogenesis and tumor growth. These biological functions are attributed to the binding of alpha3NC1 to integrin alphavbeta3. However, in some tumor cells that express integrin alphavbeta3, the alpha3NC1 domain does not inhibit proliferation, suggesting that integrin alphavbeta3 expression is not sufficient to mediate the anti-tumorigenic activity of this domain. Therefore, in the present study, we searched for novel binding receptors for the soluble alpha3NC1 domain in cells lacking alphavbeta3 integrin. In these cells, soluble alpha3NC1 bound integrin alpha3beta1; however, unlike alphavbeta3, alpha3beta1 integrin did not mediate cell adhesion to immobilized alpha3NC1 domain. Interestingly, in cells lacking integrin alpha3beta1, adhesion to the alpha3NC1 domain was enhanced due to activation of integrin alphavbeta3. These findings indicate that integrin alpha3beta1 is a receptor for the alpha3NC1 domain and transdominantly inhibits integrin alphavbeta3 activation. Thus integrin alpha3beta1, in conjunction with integrin alphavbeta3, modulates cellular responses to the alpha3NC1 domain, which may be pivotal in the mechanism underpinning its anti-angiogenic and anti-tumorigenic activities. PMID:16731529

  11. Heterologous expression and biochemical and functional characterization of a recombinant alpha-type myotoxin inhibitor from Bothrops alternatus snake.

    Science.gov (United States)

    Santos-Filho, Norival A; Boldrini-França, Johara; Santos-Silva, Ludier K; Menaldo, Danilo L; Henrique-Silva, Flávio; Sousa, Tiago S; Cintra, Adélia C O; Mamede, Carla C N; Oliveira, Fábio; Arantes, Eliane C; Antunes, Lusânia M Greggi; Cilli, Eduardo M; Sampaio, Suely V

    2014-10-01

    Venomous and non-venomous snakes possess phospholipase A2 (PLA2) inhibitory proteins (PLIs) in their blood serum. This study shows the expression and biochemical and functional characterization of a recombinant alpha inhibitor from Bothrops alternatus snake, named rBaltMIP. Its expression was performed in Pichia pastoris heterologous system, resulting in an active recombinant protein. The expressed inhibitor was tested regarding its ability to inhibit the phospholipase activity of different PLA2s, showing slight inhibitions especially at the molar ratios of 1:1 and 1:3 (PLA2:PLI). rBaltMIP was also effective in decreasing the myotoxic activity of the tested toxins at molar ratios greater than 1:0.4 (myotoxin:PLI). The inhibition of the myotoxic activity of different Asp49 (BthTX-II and PrTX-III) and Lys49 (BthTX-I and PrTX-I) myotoxins was also performed without the prior incubation of myotoxins/inhibitor in order to analyze the real possibility of using snake plasma inhibitors or recombinant inhibitors as therapeutic agents for treating envenomations. As a result, rBaltMIP was able to significantly inhibit the myotoxicity of Lys49 myotoxins. Histopathological analysis of the gastrocnemius muscles of mice showed that the myotoxins are able to induce severe damage to the muscle fibers of experimental animals by recruiting a large number of leukocyte infiltrates, besides forming an intense accumulation of intercellular fluid, leading to local edema. When those myotoxins were incubated with rBaltMIP, a reduction of the damage site could be observed. Furthermore, the cytotoxic activity of Asp49 PLA2s and Lys49 PLA2-like enzymes on C2C12 cell lines was decreased, as shown by the higher cell viabilities after preincubation with rBaltMIP. Heterologous expression would enable large-scale obtainment of rBaltMIP, thus allowing further investigations for the elucidation of possible mechanisms of inhibition of snake PLA2s, which have not yet been fully clarified. PMID:25047442

  12. Do rheumatoid arthritis patients have equal access to treatment with new medicines? Tumour necrosis factor-alpha inhibitors use in four European countries

    NARCIS (Netherlands)

    Hoebert, Joelle M.; Mantel-Teeuwisse, Aukje K.; van Dijk, Liset; Bijlsma, Johannes W. J.; Leufkens, Hubert G. M.

    2012-01-01

    Purpose: To explore the use of the biological tumour necrosis factor alpha (TNFalpha) inhibitors used in the treatment of rheumatoid arthritis as a measure of access to treatment with new medicines. In addition, characteristics both related to national health systems and spending will be assessed to

  13. Do rheumatoid arthritis patients have equal access to treatment with new medicines? Tumour necrosis factor-alpha inhibitors use in four European countries.

    NARCIS (Netherlands)

    Hoebert, J.M.; Mantel-Teeuwisse, A.K.; Dijk, L. van; Bijlsma, J.W.J.; Leufkens, H.G.M.

    2012-01-01

    Purpose: To explore the use of the biological tumour necrosis factor alpha (TNFalpha) inhibitors used in the treatment of rheumatoid arthritis as a measure of access to treatment with new medicines. In addition, characteristics both related to national health systems and spending will be assessed to

  14. 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. PMID:2327970

  15. Competitive inhibitor of cellular alpha-glucosidases protects mice from lethal dengue virus infection

    OpenAIRE

    Chang, Jinhong; Schul, Wouter; Yip, Andy; Xu, Xiaodong; Guo, Ju-Tao; Block, Timothy M.

    2011-01-01

    Dengue virus infection causes diseases in people, ranging from the acute febrile illness Dengue fever, to life-threatening Dengue Hemorrhagic Fever/Dengue Shock Syndrome. We previously reported that a host cellular α-glucosidases I and II inhibitor, imino sugar CM-10-18, potently inhibited dengue virus replication in cultured cells, and significantly reduced viremia in dengue virus infected AG129 mice. In this report we show that CM-10-18 also significantly protects mice from death and/or dis...

  16. [Effect of alpha-difluoromethylornithine and polyhexamethyleneguanidine (PMG), polyamine biosynthesis inhibitors on leucosis L1210 growth kinetics and life expectancy of animals with cancer].

    Science.gov (United States)

    Zaletok, S P; Berdinskikh, N K; Lialiushko, N M; Klenov, O A; Samoĭlenko, E A; Lidak, M Iu

    2005-12-01

    The article deals with the study of polyamines content and y-glutamiltranspeptidase (gamma-GTP) activity in leucosis L1210 cells under the influence of inhibitors of polyamines synthesis such as alpha-difluoromethylornithine (alpha-DFMO) and polyhexamethylenguanidine (PMG). Injections of alpha-DFMO and PMG to animals essentially reduce putrescine and spermidine concentrations, and the levels of spermine and gamma-GTP activity increase under this influence. These modulation were associated with L1210 leucosis growth retardation. Antiblastic effect was dependent on inhibitors' doses and mode of injections' course. Under the optimum conditions the retardation index was 90-98%. The animals with retarded tumor growth had essentially longer survival time frame than blank tumor-bearing animals (index was 37.2 for a-DFMO and 67.5 for PMG). PMID:16498791

  17. 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.

  18. Identification of stable plant cystatin/nematode proteinase complexes using mildly denaturing gelatin/polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Michaud, D; Cantin, L; Bonadé-Bottino, M; Jouanin, L; Vrain, T C

    1996-08-01

    The biochemical interactions between two cystatins from rice seeds, oryzacystatin I (OCI) and oryzacystatin II (OCII), and the cysteine proteinases from three plant parasitic nematodes, Meloidogyne hapla, M. incognita and M. javanica, were assessed using standard protease assays and mildly denaturing gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE). Activity detected in extracts of preparasitic second-stage larvae (J2) from M. hapla was optimal at pH 5.5 and was inhibited in vitro by the cysteine proteinase inhibitors trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane, hen egg cystatin, OCI, and OCII. As demonstrated by class-specific activity staining, all the activity measured between pH 3.5 and pH 7.5 was accounted for by a major proteinase form, Mhp1, and two minor forms, Mhp2 and Mhp3. Mhps were also detected in extracts and excretions of parasitic J2 and adult females, indicating their continuous expression throughout development of M. hapla, and their possible involvement in the extracellular degradation of proteins. Interestingly, the two plant cysteine proteinase inhibitors OCI and OCII showed different degrees of affinity for the major proteinase form, Mhp1. Both inhibitors almost completely inactivated this proteinase in native conditions but, unlike OCII, OCI conserved a high affinity for Mhp1 during mildly denaturing gelatin/PAGE, showing the differential stabilities of the OCI/Mhp1 and OCII/Mhp1 complexes. In contrast to Mhp1, the major cysteine proteinases detected in the two closely related species M. incognita and M. javanica were strongly inhibited by OCII, while the inhibition of OCI was partly prevented during electrophoresis. This species-related efficiency of plant cystatins against nematode cysteine proteinases could have practical implications when planning their use to control nematodes of the genus Meloidogyne. PMID:8874065

  19. 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 ...

  20. 17-Imidazolyl, pyrazolyl, and isoxazolyl androstene derivatives. Novel steroidal inhibitors of human cytochrome C17,20-lyase (P450(17 alpha).

    Science.gov (United States)

    Ling, Y Z; Li, J S; Liu, Y; Kato, K; Klus, G T; Brodie, A

    1997-09-26

    We recently described a number of inhibitors of P450(17 alpha), the key enzyme of androgen biosynthesis. Here, we report the synthesis and activity of novel 17-imidazolyl, pyrazolyl, and isoxazolyl androstene derivatives as potential agents for the treatment of prostatic cancer. A number of 17-(4'-Imidazolyl) derivatives were prepared by condensing the corresponding 17-ketol acetate side chain with aldehyde and ammonium hydroxide. The 17 beta-(4'imidazolyl) derivatives (2a, 2e, 4a, 4c) were found to be potent inhibitors of human testicular P450(17 alpha), with greater activity than ketoconazole. The juxtaposition between the imidazole ring and the steroid D ring appears to be important in contributing inhibitory properties, Compounds having a 17 beta-(2'-imidazolyl) ring (9a, 10) or a 20 beta-(2'-imidazolyl) ring (12), instead of the 17 beta-(4'-imidazolyl) ring (2a, 4a), are weak inhibitors. Among the 17-(4'-imidazolyl) derivatives, introduction of the 17 alpha-hydroxy group (4b) and 16 alpha,17 alpha-epoxide group (2d) diminished potency (2a-->2d; lC50 66-->430 nM; 4a-->4b; lC50 58-->1200 nM), while the 16,17 double bond increased the inhibitory activity by almost three times in the 5-en-3 beta-ol inhibitors (2a-->2e; lC50 60-->24 nM). There was virtually no difference in the inhibitory activity in the 4-en-3-one inhibitors (4a-->4c; IC50 58-->50 nM). The introduction of a methyl (2b) or phenyl group (2c) on the 2'-position of 4'-imidazolyl ring caused a dramatic decrease in the potency. As to modification of the A,B rings, the 3-acetate (2f, 2g) decreased the potency almost 3-fold compared with the 3-alcohol (2e-->2f, IC50 24-->75 nM; 2a-->2g, 66-->199 nM) and the conversion from the 5-en-3 beta-ol into the 4-en-3-one hardly affected the potency. As expected, 4c was more potent than 2e for the rat p450(17 alpha). 17-(3'Pyrazolyl)-(14b) and 17-(5'-isoxazolyl)-androsta-5,16-dien-3 beta-ol (15b) were also potent inhibitors of P450(17 alpha), whereas the 17

  1. Assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin-polyacrylamide gel electrophoresis.

    Science.gov (United States)

    Michaud, D; Cantin, L; Raworth, D A; Vrain, T C

    1996-01-01

    A method for assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin-polyacrylamide gel electrophoresis (gelatin-PAGE) is described. As suggested by the use of well-known cystatins (human stefins A and B, and oryzacystatins I and II) and the plant cysteine proteinase papain, the ability of cystatin/cysteine proteinase complexes to remain stable during electrophoresis is associated with the degree of affinity between the enzyme and the inhibitor (and inversely associated with the Ki values), at least with the disulfide bond-lacking cystatins. Complexes with Ki values > or = 10(-8) M (weak interactions) are partly or completely dissociated under the conditions used, while those with lower Ki values (strong interactions) remain stable. As shown by the differential effects of two plant cystatins, oryzacystatins I and II, against a cysteine proteinase present in crude (complex) extracts from a plant pest -- the two-spotted spider mite (Tetranychus urticae Koch), the gelatin-PAGE procedure is suitable for studying the ability of cystatins to form highly stable complexes with cysteine proteinases, without the need for prior purification steps. Considering the well-recognized potential of proteinase inhibitors for pest and pathogen control, this analytical approach will be useful for rapidly assessing the respective potential of various cystatins for protection of plants, animals, and humans. PMID:8907521

  2. Effects of a high-pressure treatment on the wheat alpha-amylase inhibitor and its relationship to elimination of allergenicity

    Energy Technology Data Exchange (ETDEWEB)

    Yamamoto, S [Food Science Center, Niigata University, Ikarashi, Niigata, 950-2181 (Japan); Takanohashi, K; Nishiumi, T [Graduate School of Science and Technology, Niigata University, Ikarashi, Niigata, 950-2181 (Japan); Hara, T [Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Ikarashi, Niigata, 950-2181 (Japan); Odani, S [Department of Living Science and Technology, Faculty of Education and Human Science, Ikarashi, Niigata, 950-2181 (Japan); Suzuki, A, E-mail: shuyama@agr.niigata-u.ac.j [Department of Health and Nutrition, Faculty of Medical Science for Health, Teikyo Heisei University, Ikebukuro, Tokyo, 170-0013 (Japan)

    2010-03-01

    In this study, the effects of high-pressure treatment on structure and allergeincity of alpha amylase inhibitor (a-AI) were investigated. The pressure-induced structural changes of {alpha}-AI were estimated by fluorescence spectra and by fourth derivative UV-spectroscopy for probed tyrosine residues and by circular dichroism (CD) spectroscopy. The changes in the tertiary structure detected by fluorescence spectra and by fourth derivative UV-spectroscopy under high pressure were indicated at over 300 MPa. Measurements of CD spectroscopy suggested that the effects of a high-pressure treatment on changes in the secondary structure of {alpha}-AI were little. From our results, pressure-induced changes of the {alpha}-AI structure were not apparent. On the other hands, the IgE-specific binding activities of pressurized {alpha}-AI to sera from allergic patients against wheat, which is estimated by observations of dot-blotting, were decreased by high-pressure treatment. It is known that the pressure-induced elimination of allergenicity is related to the tertiary structural changes of allergen molecules. This study are suspected that the epitopes of {alpha}-AI do not contain tyrosine residues, and thus the decrease of IgE-specific binding activities is probably caused by the tertiary structural changes of these parts of {alpha}-AI.

  3. Symptomatic benign prostatic hyperplasia: the role of 5-alpha-reductase inhibitors in the prevention of acute urinary retention and surgical therapy

    Directory of Open Access Journals (Sweden)

    Norma Marigliano

    2012-01-01

    Full Text Available Benign prostatic hyperplasia (BPH is a disease that affects over 50% of males aged 50 years or older. In men aged >80 years, the incidence is 90%. BPH occurs in 9-25% of males aged 40 to 79 years. Fifty percent of patients with BPH are symptomatic. The symptoms include reduced urinary flow, nocturia, defective bladder emptying, urinary hesitancy, and dysuria. Disease progression can be associated with acute urinary retention (AUR. Prostatic obstruction includes mechanical and dynamic components, the latter mediated by alpha-muscarinic receptors. Treatment with alpha-1-blockers (alfuzosin, doxazosin, tamsulosin, and terazosin leads to rapid amelioration of symptoms and urinary flow, usually within one or two weeks. The 5-alpha reductase inhibitors (5-ARIs are “disease-modifying drugs.” They control the growth of the prostate by blocking the conversion of testosterone into dihydrotestosterone (DHT. Finasteride is a 5–ARI that is selective for type 2 receptors. Dutasteride is a powerful inhibitor of both 5- alpha reductase isoforms (type 1 and 2 and produces more complete suppression of DHT synthesis than finasteride. Dutasteride also has a much longer half-life than finasteride (five weeks versus five to six hours. The authors review the results of clinical trials involving finasteride and dutasteride, with and without alpha-1-blockers, highlighting the important role of dutasteride in improving acute urinary retention and eliminating the need for surgical therapy.

  4. Diisopropyl fluorophosphate labeling of sperm-associated proteinases

    International Nuclear Information System (INIS)

    Proteinase inhibitors have been shown to be capable of preventing various aspects of fertilization. Diisopropyl fluorophosphate (DFP) is an irreversible inhibitor of trypsin-like enzymes that is commercially available in a radiolabeled form. The experiments described herein were designed to determine if DFP would prevent sperm function in live, motile sperm and to identify the sperm proteins bound with DFP. DFP at 5 mM concentrations had no observable effect on sperm motility, but inhibited the penetration of zona-free hamster ova by human sperm (5.5%) compared to controls (33.5%). Acid extracts of motile sperm that had been incubated with radiolabeled DFP and collected by the swim-up procedure demonstrated the presence of radiolabeled DFP, and the autoradiography of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of these extracts localized the uptake of radiolabeled DFP to proteins in the molecular weight region of the proacrosin-acrosin system. Acid-extracted proteinases from semen samples incubated with DFP demonstrated a concentration-dependent inhibition of both esterolytic hydrolysis of benzoyl-arginine ethyl ester on spectrophotometric analysis and proteolytic activity on gelatin SDS-PAGE zymography. DFP-labeled proteins were precipitated by highly specific antibodies to proacrosin. These results demonstrated that DFP is capable of inhibiting sperm function, and that it associates with the proacrosin-acrosin system in live motile sperm

  5. 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/.

  6. α-Blocker Monotherapy and α-Blocker Plus 5-Alpha-Reductase Inhibitor Combination Treatment in Benign Prostatic Hyperplasia; 10 Years' Long-Term Results

    OpenAIRE

    Shin, Teak Jun; Kim, Chun Il; Park, Choal Hee; Kim, Byung Hoon; Kwon, Young Kee

    2012-01-01

    Purpose We compared the effects of alpha-adrenergic receptor blocker (α-blocker) monotherapy with those of combination therapy with α-blocker and 5-alpha-reductase inhibitor (5-ARI) on benign prostatic hyperplasia (BPH) progression for over 10 years. Materials and Methods A total of 620 patients with BPH who received α-blocker monotherapy (α-blocker group, n=368) or combination therapy (combination group, n=252) as their initial treatment were enrolled from January 1989 to June 2000. The inci...

  7. Pneumatosis cystoides intestinalis following alpha-glucosidase inhibitor treatment: A case report and review of the literature

    Institute of Scientific and Technical Information of China (English)

    Tatsuhiro Tsujimoto; Hiroshi Fukui; Erika Shioyama; Kei Moriya; Hideto Kawaratani; Yasuyo Shirai; Masahisa Toyohara; Akira Mitoro; Jun-ichi Yarnao; Hisao Fujii

    2008-01-01

    A 69-year-old man was diagnosed as having myasthenia gravis (MG) in September 2004, and treated with thymectomy and prednisolone. He was then diagnosed as having steroid-induced diabetes mellitus, and received sulfonylurea (SU) therapy in May 2005. An alpha-glucosidase inhibitor (αGI) was added in March 2006, resulting in good glycemic control. He experienced symptoms of abdominal distention, increased flatus, and constipation in October 2007, and was admitted into our hospital in late November with hematochezia. Plain abdominal radiography revealed small linear radiolucent clusters in the wall of the colon. Computed tomography (CT) showed intramural air in the sigmoid colon. Colonoscopy revealed multiple smooth surfaced hemispherical protrusions in the sigmoid colon. The diagnosis of pneumatosis cystoides intestinalis (PCI) was made on the basis of these findings. As the αGI voglibose was suspected as the cause of this patient's PCI, treatment was conservative, ceasing voglibose, with fasting and fluid supplementation. The patient progressed well, and was discharged 2 wk later. Recently, several reports of PCI associated with αGI therapy have been published, predominantly in Japan where αGIs are commonly used. If the use of αGIs becomes more widespread, we can expect more reports of this condition on a global scale. The possibility of PCI should be considered in diabetic patients complaining of gastrointestinal symptoms, and the gastrointestinal tract should be thoroughly investigated in these patients.

  8. Enzyme-linked immunosorbent assays for doping control of 5alpha-reductase inhibitors finasteride and dutasteride.

    Science.gov (United States)

    Brun, Eva M; Torres, Ana; Ventura, Rosa; Puchades, Rosa; Maquieira, Angel

    2010-06-25

    Finasteride and dutasteride are 5alpha-reductase inhibitors included in the World Anti-Doping Agency's list of banned substances. Two highly sensitive and selective ELISA assays were developed for these compounds. Polyclonal rabbit antibodies were raised using synthesized haptens and other commercial products. The best immunoassay obtained, based on an antibody-coated format, showed a limit of detection of 0.01 microg L(-1) and an IC(50) of 0.75 microg L(-1) for finasteride (cross-reactivity with dutasteridefinasteride and dutasteride determination, with limits of detection of 0.013 and 0.021 microg L(-1), and IC(50) values 0.18 and 1.18 microg L(-1), respectively. Both assays were highly selective to a set of anabolic steroids, but they showed 37% and 30% cross-reactivity with the major urinary metabolite of finasteride, allowing its determination. The developed ELISA had better sensitivity than HPLC/MS/MS method and was applied as a screening technique to quantify dutasteride, finasteride, and its main metabolite in human urine without sample pre-treatment. Moreover, the analysis of dutasteride's excretion urines by ELISA was used to obtain its human excretion rate, essential to improve the analytical strategies about this type of drugs (permitted as medicines and prohibited in sport) and to establish an effective anti-doping policy. PMID:20541645

  9. 5-alpha reductase inhibitors and prostate cancer prevention%5α还原酶抑制剂与前列腺癌预防

    Institute of Scientific and Technical Information of China (English)

    万奔

    2012-01-01

    5-alpha reductase inhibitors decrease the level of dihydrotestosterone (DHT) by inhibiting 5-alpha reductase. Trials on 5-alpha reductase inhibitors in prostate cancer prevention showed that they could significantly decrease the incidence of prostate cancer, but meanwhile increase high-grade cases as well. Recent studies demonstrated that 5-alpha reductase inhibitors could reduce not only the prostate volume but also the volume of Gleason grade 3 prostate cancer, which made easier the detection of higher-grade prostate cancer in the second biopsy. 5-alpha reductase inhibitors could also increase the sensitivity of prostate biopsy in detecting prostate cancer, particularly that of a higher grade. The evidence we have obtained leads to the conclusion that 5-alpha reductase inhibitors do not increase the incidence of high-grade prostate cancer, but on the contrary help its earlier detection.%5α还原酶抑制剂通过抑制5α还原酶降低双氢睾酮水平.应用5α还原酶抑制剂进行的前列腺癌预防研究显示,5α还原酶抑制剂可以降低前列腺癌发病率,但同时也出现高级别前列腺癌病例增加的现象,引起广泛关注.有研究和分析显示,5α还原酶抑制剂使前列腺体积缩小,同时也缩小Gleason评分为3分患者的前列腺癌体积,使得再次穿刺更容易发现Gleason评分为4分以上的前列腺癌.另外5α还原酶抑制剂显著提高前列腺穿刺诊断前列腺癌和高级别前列腺癌敏感度.笔者认为5α还原酶抑制剂不但不增加高级别前列腺癌的发生率,反而会帮助我们更早发现高级别前列腺癌.

  10. Superior Orbital Fissure Syndrome and Ophthalmoplegia Caused by Varicella Zoster Virus with No Skin Eruption in a Patient Treated with Tumor Necrosis Alpha Inhibitor

    OpenAIRE

    Jensen, Helene; Thomsen, Sidsel Thorup; Hansen, Stine Scott; Munksgaard, Signe Bruun; Lindelof, Mette

    2015-01-01

    Varicella zoster virus lies dormant in the dorsal root ganglia after symptomatic chicken pox infection, usually in childhood. If the virus reactivates in the trigeminal ganglia, it can cause varicella zoster ophthalmicus, which can have severe ocular complications. We report a case of a 73-year-old woman in severe immunosuppression due to treatment with mycophenolate mofetil, glucocorticosteroids and a tumor necrosis factor alpha inhibitor. The reactivation caused superior orbital fissure syn...

  11. Use of 5 alpha-reductase inhibitors for lower urinary tract symptoms and risk of prostate cancer in Swedish men : nationwide, population based case-control study

    OpenAIRE

    Robinson, David; Garmo, Hans; Bill-Axelson, Anna; Mucci, Lorelei; Holmberg, Lars; Stattin, Pär

    2013-01-01

    Objective To assess the association between 5 alpha-reductase inhibitor (5-ARI) use in men with lower urinary tract symptoms and prostate cancer risk. Design Nationwide, population based case-control study for men diagnosed with prostate cancer in 2007-09 within the Prostate Cancer data Base Sweden 2.0. Setting The National Prostate Cancer Register, National Patient Register, census, and Prescribed Drug Register in Sweden, from which we obtained data on 5-ARI use before date of prostate cance...

  12. Do rheumatoid arthritis patients have equal access to treatment with new medicines? Tumour necrosis factor-alpha inhibitors use in four European countries.

    OpenAIRE

    Hoebert, J.M.; Mantel-Teeuwisse, A K; Van Dijk, L; Bijlsma, J.W.J.; Leufkens, H.G.M.

    2012-01-01

    Purpose: To explore the use of the biological tumour necrosis factor alpha (TNFalpha) inhibitors used in the treatment of rheumatoid arthritis as a measure of access to treatment with new medicines. In addition, characteristics both related to national health systems and spending will be assessed to explore possible differences in international utilisation. Methods: Data from four European countries were included: Ireland, The Netherlands, Norway and Portugal. Annual utilisation rates of TNFa...

  13. Proteinases and associated genes of parasitic helminths.

    Science.gov (United States)

    Tort, J; Brindley, P J; Knox, D; Wolfe, K H; Dalton, J P

    1999-01-01

    Many parasites have deployed proteinases to accomplish some of the tasks imposed by a parasitic life style, including tissue penetration, digestion of host tissue for nutrition and evasion of host immune responses. Information on proteinases from trematodes, cestodes and nematode parasites is reviewed, concentrating on those worms of major medical and economical importance. Their biochemical characterization is discussed, along with their putative biological roles and, where available, their associated genes. For example, proteinases expressed by the various stages of the schistosome life-cycle, in particular the well-characterized cercarial elastase which is involved in the penetration of the host skin and the variety of proteinases, such as cathepsin B (Sm31), cathepsin L1, cathepsin L2, cathepsin D, cathepsin C and legumain (Sm32), which are believed to be involved in the catabolism of host haemoglobin. The various endo- and exoproteinases of Fasciola hepatica, the causative agent of liver fluke disease, are reviewed, and recent reports of how these enzymes have been successfully employed in cocktail vaccines are discussed. The various proteinases of cestodes and of the diverse superfamilies of parasitic nematodes are detailed, with special attention being given to those parasites for which most is known, including species of Taenia, Echinococcus, Spirometra, Necator, Acylostoma and Haemonchus. By far the largest number of papers in the literature and entries to the sequence data bases dealing with proteinases of parasitic helminths report on enzymes belonging to the papain superfamily of cysteine proteinases. Accordingly, the final section of the review is devoted to a phylogenetic analysis of this superfamily using over 150 published sequences. This analysis shows that the papain superfamily can be divided into two major branches. Branch A contains the cathepin Bs, the cathepsin Cs and a novel family termed cathepsin Xs, while Branch B contains the cruzipains

  14. Prediction of novel and selective TNF-alpha converting enzyme (TACE) inhibitors and characterization of correlative molecular descriptors by machine learning approaches.

    Science.gov (United States)

    Cong, Yong; Yang, Xue-Gang; Lv, Wei; Xue, Ying

    2009-10-01

    The inhibition of TNF-alpha converting enzyme (TACE) has been explored as a feasible therapy for the treatment of rheumatoid arthritis (RA) and Crohn's disease (CD). Recently, large numbers of novel and selective TACE inhibitors have been reported. It is desirable to develop machine learning (ML) models for identifying the inhibitors of TACE in the early drug design phase and test the prediction capabilities of these ML models. This work evaluated four ML methods, support vector machine (SVM), k-nearest neighbor (k-NN), back-propagation neural network (BPNN) and C4.5 decision tree (C4.5 DT), which were trained and tested by using a diverse set of 443 TACE inhibitors and 759 non-inhibitors. A well-established feature selection method, the recursive feature elimination (RFE) method, was used to select the most appropriate descriptors for classification from a large pool of descriptors, and two evaluation methods, 5-fold cross-validation and independent evaluation, were used to assess the performances of these developed models. In this study, all these ML models have already achieved promising prediction accuracies. By using the RFE method, the prediction accuracies are further improved. In k-NN, the model gives the best prediction for TACE inhibitors (98.32%), and the SVM bears the best prediction for non-inhibitors (99.51%). Both the k-NN and SVM model give the best overall prediction accuracy (98.45%). To the best of our knowledge, the SVM model developed in this work is the first one for the classification prediction of TACE inhibitors with a broad applicability domain. Our study suggests that ML methods, particularly SVM, are potentially useful for facilitating the discovery of TACE inhibitors and for exhibiting the molecular descriptors associated with TACE inhibitors. PMID:19729328

  15. Interferon-alpha-2a is a potent inhibitor of hormone secretion by cultured human pituitary adenomas

    NARCIS (Netherlands)

    L.J. Hofland (Leo); W.W. de Herder (Wouter); M. Waaijers (Marlijn); J. Zuijderwijk; P. Uitterlinden (Piet); P.M. van Koetsveld (Peter); S.W.J. Lamberts (Steven)

    1999-01-01

    textabstractInterferon-alpha (IFN alpha) may exert direct inhibitory effects on cell proliferation and on the production of different peptide hormones. We investigated the effect of IFN alpha on hormone production by 15 GH-secreting pituitary adenomas, 4 clinically nonf

  16. Purification and characterization of a collagenolytic serine proteinase from the skeletal muscle of red sea bream (Pagrus major).

    Science.gov (United States)

    Wu, Guo-Ping; Chen, Su-Hua; Liu, Guang-Ming; Yoshida, Asami; Zhang, Ling-Jing; Su, Wen-Jin; Cao, Min-Jie

    2010-03-01

    A collagenolytic serine proteinase (CSP) was purified from red sea bream (Pagrus major) skeletal muscle to homogeneity by ammonium sulfate fractionation and chromatographies including DEAE-Sephacel, Phenyl Sepharose and Hydroxyapatite. The molecular mass of CSP was approximately 85 kDa as estimated by SDS-PAGE and gel filtration. Optimum temperature and pH of CSP were 40 degrees C and 8.0, respectively. CSP was specifically inhibited by serine proteinase inhibitors, while inhibitors to other type proteinases did not show much inhibitory effects. The K(m) and k(cat) values of CSP for Boc-Leu-Lys-Arg-MCA were 3.58 microM and 0.13 s(-1) at 37 degrees C, respectively. Furthermore, CSP hydrolyzed gelatin and native type I collagen effectively though its degradation on myosin heavy chain (MHC) was not significant, suggesting its involvement in the texture tenderization of fish muscle during the post-mortem stage. PMID:19945542

  17. Five-alpha Reductase Inhibitor Influences Expression of Androgen Receptor and HOXB13 in Human Hyperplastic Prostate Tissue

    Directory of Open Access Journals (Sweden)

    Chaeyong Jung

    2013-12-01

    Full Text Available Objectives Five-alpha reductase inhibitors (5ARIs are known as chemopreventive agents in prostate cancer with a risk of high-grade disease. This study evaluated the effects of 5ARI on androgen receptor (AR and proteins involved in prostate cell growth such as HOXB13 expression in human prostate tissue and LNCaP prostate cancer cells. Materials and Methods We retrospectively selected 21 patients who underwent TURP between March 2007 and February 2010 for previously confirmed BPH by prostate biopsy. They were grouped into control (group 1, n = 9 and 5ARI treatment (group 2, n = 12 before TURP. AR and HOXB13 expression in prostate tissue was evaluated by immunohistochemical staining. We tested the effect of 5ARI on the expression of AR, prostate specific antigen (PSA and HOXB13 in LNCaP cells. Cells were assessed by Western blot analysis, MTT in vitro proliferation assay, and ELISA. Results: Group 2 showed stronger reactivity for AR and HOXB13 than those of the group 1. MTT assay showed death of LNCaP cells at 25uM of 5ARI. At the same time, ELISA assay for PSA showed that 5ARI inhibited secretion of PSA in LNCaP cells. Western blot analysis showed that 5ARI did not greatly alter AR expression but it stimulated the expression of HOXB13. Conclusions These results demonstrated that 5ARI influences AR and HOXB13 expression in both LNCaP cells and human prostate tissue. In order to use 5ARI in chemoprevention of prostate cancer, we still need to clarify the influence of 5ARI in ARs and oncogenic proteins and its regulation pathway.

  18. Use of 5-alpha-reductase inhibitors did not increase the risk of cardiovascular diseases in patients with benign prostate hyperplasia: a five-year follow-up study.

    Directory of Open Access Journals (Sweden)

    Teng-Fu Hsieh

    Full Text Available This nationwide population-based study investigated the risk of cardiovascular diseases after 5-alpha-reductase inhibitor therapy for benign prostate hyperplasia (BPH using the National Health Insurance Research Database (NHIRD in Taiwan.In total, 1,486 adult patients newly diagnosed with BPH and who used 5-alpha-reductase inhibitors were recruited as the study cohort, along with 9,995 subjects who did not use 5-alpha-reductase inhibitors as a comparison cohort from 2003 to 2008. Each patient was monitored for 5 years, and those who subsequently had cardiovascular diseases were identified. A Cox proportional hazards model was used to compare the risk of cardiovascular diseases between the study and comparison cohorts after adjusting for possible confounding risk factors.The patients who received 5-alpha-reductase inhibitor therapy had a lower cumulative rate of cardiovascular diseases than those who did not receive 5-alpha-reductase inhibitor therapy during the 5-year follow-up period (8.4% vs. 11.2%, P=0.003. In subgroup analysis, the 5-year cardiovascular event hazard ratio (HR was lower among the patients older than 65 years with 91 to 365 cumulative defined daily dose (cDDD 5-alpha-reductase inhibitor use (HR=0.63, 95% confidence interval (CI 0.42 to 0.92; P=0.018, however there was no difference among the patients with 28 to 90 and more than 365 cDDD 5-alpha-reductase inhibitor use (HR=1.14, 95% CI 0.77 to 1.68; P=0.518 and HR=0.83, 95% CI 0.57 to 1.20; P=0.310, respectively.5-alpha-reductase inhibitor therapy did not increase the risk of cardiovascular events in the BPH patients in 5 years of follow-up. Further mechanistic research is needed.

  19. N-acetyl-beta-D-glucopyranosylamine: a potent T-state inhibitor of glycogen phosphorylase. A comparison with alpha-D-glucose.

    Science.gov (United States)

    Oikonomakos, N G; Kontou, M; Zographos, S E; Watson, K A; Johnson, L N; Bichard, C J; Fleet, G W; Acharya, K R

    1995-12-01

    Structure-based drug design has led to the discovery of a number of glucose analogue inhibitors of glycogen phosphorylase that have an increased affinity compared to alpha-D-glucose (Ki = 1.7 mM). The best inhibitor in the class of N-acyl derivatives of beta-D-glucopyranosylamine, N-acetyl-beta-D-glucopyranosylamine (1-GlcNAc), has been characterized by kinetic, ultracentrifugation, and crystallographic studies. 1-GlcNAc acts as a competitive inhibitor for both the b (Ki = 32 microM) and the a (Ki = 35 microM) forms of the enzyme with respect to glucose 1-phosphate and in synergism with caffeine, mimicking the binding of glucose. Sedimentation velocity experiments demonstrated that 1-GlcNAc was able to induce dissociation of tetrameric phosphorylase a and stabilization of the dimeric T-state conformation. Co-crystals of the phosphorylase b-1-GlcNAc-IMP complex were grown in space group P4(3)2(1)2, with native-like unit cell dimensions, and the complex structure has been refined to give a crystallographic R factor of 18.1%, for data between 8 and 2.3 A resolution. 1-GlcNAc binds tightly at the catalytic site of T-state phosphorylase b at approximately the same position as that of alpha-D-glucose. The ligand can be accommodated in the catalytic site with very little change in the protein structure and stabilizes the T-state conformation of the 280s loop by making several favorable contacts to Asn 284 of this loop. Structural comparisons show that the T-state phosphorylase b-1-GlcNAc-IMP complex structure is overall similar to the T-state phosphorylase b-alpha-D-glucose complex structure. The structure of the 1-GlcNAc complex provides a rational for the biochemical properties of the inhibitor. PMID:8580837

  20. 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.

  1. 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.

  2. Effect of tissue-specific acetylcholinesterase inhibitor C-547 on alpha 3 beta 4 and alpha beta epsilon delta acetylcholine receptors in COS cells

    Czech Academy of Sciences Publication Activity Database

    Lindovský, Jiří; Petrov, K.; Krůšek, Jan; Reznik, V.S.; Nikolsky, E. E.; Vyskočil, František

    2012-01-01

    Roč. 688, 1-3 (2012), s. 22-26. ISSN 0014-2999 R&D Projects: GA MŠk(CZ) LC554; GA ČR(CZ) GA202/09/0806; GA AV ČR(CZ) IAA500110905; GA AV ČR(CZ) IAA100110501; GA AV ČR(CZ) IAA5011411 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : nicotinic ACh receptor * alpha 3 beta 4 * alpha beta epsilon delta * C-547 * anti-cholinesterase Subject RIV: ED - Physiology Impact factor: 2.592, year: 2012

  3. 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

  4. Improved glucose regulation in type 2 diabetic patients with DPP-4 inhibitors: focus on alpha and beta cell function and lipid metabolism.

    Science.gov (United States)

    Ahrén, Bo; Foley, James E

    2016-05-01

    Inhibition of dipeptidyl peptidase-4 (DPP-4) is an established glucose-lowering strategy for the management of type 2 diabetes mellitus. DPP-4 inhibitors reduce both fasting and postprandial plasma glucose levels, resulting in reduced HbA1c with low risk for hypoglycaemia and weight gain. They act primarily by preventing inactivation of the incretin hormones glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1, thereby prolonging the enhanced endogenous levels of these hormones after meal ingestion. This in turn causes islet and extrapancreatic effects, including increased glucose sensing in islet alpha and beta cells. These effects result in increased insulin secretion and decreased glucagon secretion being more effective in hyperglycaemic states and reduced insulin secretion and increased glucagon secretion being more effective during hypoglycaemia. Other secondary pharmacological actions of DPP-4 inhibitors include mobilisation and burning of fat during meals, decrease in fat extraction from the gut, reduction of fasting lipolysis and liver fat and increase in LDL particle size. These actions contribute to the clinical effects of DPP-4 inhibition, and the reduced demand for insulin could also lead to a durability benefit. This review summarises the current knowledge of the secondary pharmacological actions of DPP-4 inhibitors that lead to improved glucose regulation in patients with type 2 diabetes, focusing on alpha and beta cell function and lipid metabolism. PMID:26894277

  5. Circulating levels of interleukin-6, vascular endothelial growth factor, YKL-40, matrix metalloproteinase-3, and total aggrecan in spondyloarthritis patients during 3 years of treatment with TNF alpha inhibitors

    DEFF Research Database (Denmark)

    Pedersen, S.J.; Hetland, M.L.; Sørensen, Inge Juul; Østergaard, Mikkel; Nielsen, H.J.; Johansen, J.S.

    2010-01-01

    The objectives of the study were to investigate short and long-term changes and relations to treatment response of plasma interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), YKL-40, matrix metalloproteinase-3 (MMP-3), and total aggrecan in patients with spondyloarthritis (SpA) treated...... with tumor necrosis factor-alpha (TNF alpha) inhibitors and to compare with levels in healthy subjects. Biomarkers were measured in an observational cohort of 49 SpA patients (ankylosing spondylitis, n = 32, and psoriatic arthritis, n = 17) initiating TNF alpha inhibitor therapy (infliximab, n = 38...

  6. Intrauterine coadministration of ERK1/2 inhibitor U0126 inhibits interferon TAU action in the endometrium and restores luteolytic PGF2alpha pulses in sheep.

    Science.gov (United States)

    Lee, JeHoon; Stanley, Jone A; McCracken, John A; Banu, Sakhila K; Arosh, Joe A

    2014-08-01

    In ruminants, prostaglandin F2 alpha (PGF2alpha) is synthesized and released in a pulsatile pattern from the endometrial luminal epithelial (LE) cells during the process of luteolysis. Interferon tau (IFNT) is a Type 1 IFN secreted by the trophoblast cells of the developing conceptus. IFNT acts locally on endometrial LE cells to inhibit pulsatile releases of PGF2alpha and thus establish an endocrine environment for recognition of pregnancy. Cell signaling pathways through which IFNT stimulates expression of multiple genes or proteins in endometrial LE are largely unknown. Results of the present investigation indicate that intrauterine administration of IFNT inhibits pulsatile release of PGF2alpha, while coadministration IFNT and ERK 1/2 inhibitor U0126 restores luteolytic PGF2alpha pulses in sheep. IFNT increases phosphorylation of ERK1/2 proteins and increases its interaction with PGT proteins in endometrial LE. Blockade of ERK1/2 pathways inhibits IFNT action, decreases pERK1/2 and PGT protein interactions, and re-establishes the spatial expression of the oxytocin receptor protein completely and the estrogen receptor protein partially without modulating the expression of interferon regulatory factor-2 (IRF-2) protein in endometrial LE. IFNT does not decrease expression of COX-2, PGDH, or PGT protein in endometrial LE. Our results provide important new insights into IFNT signaling and the molecular endocrine control of PGF2alpha release at the time of establishment of pregnancy in ruminants. This novel IFNT-ERK1/2 signaling module needs to be explored in future studies to understand molecular and cellular mechanisms of IFNT action in endometrial LE in ruminants. PMID:24876409

  7. Discovery of 4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-5-methyl-N-propylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (BMS-582949), a Clinical p38[alpha] MAP Kinase Inhibitor for the Treatment of Inflammatory Diseases

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Chunjian; Lin, James; Wrobleski, Stephen T.; Lin, Shuqun; Hynes, Jr., John; Wu, Hong; Dyckman, Alaric J.; Li, Tianle; Wityak, John; Gillooly, Kathleen M.; Pitt, Sidney; Shen, Ding Ren; Zhang, Rosemary F.; McIntyre, Kim W.; Salter-Cid, Luisa; Shuster, David J.; Zhang, Hongjian; Marathe, Punit H.; Doweyko, Arthur M.; Sack, John S.; Kiefer, Susan E.; Kish, Kevin F.; Newitt, John A.; McKinnon, Murray; Dodd, John H.; Barrish, Joel C.; Schieven, Gary L.; Leftheris, Katerina (BMS)

    2013-11-20

    The discovery and characterization of 7k (BMS-582949), a highly selective p38{alpha} MAP kinase inhibitor that is currently in phase II clinical trials for the treatment of rheumatoid arthritis, is described. A key to the discovery was the rational substitution of N-cyclopropyl for N-methoxy in 1a, a previously reported clinical candidate p38{alpha} inhibitor. Unlike alkyl and other cycloalkyls, the sp{sup 2} character of the cyclopropyl group can confer improved H-bonding characteristics to the directly substituted amide NH. Inhibitor 7k is slightly less active than 1a in the p38{alpha} enzymatic assay but displays a superior pharmacokinetic profile and, as such, was more effective in both the acute murine model of inflammation and pseudoestablished rat AA model. The binding mode of 7k with p38{alpha} was confirmed by X-ray crystallographic analysis.

  8. Synthesis Activity and Structural Analysis of Novel alpha-Hydroxytropolone Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase-Associated Ribonuclease H

    Energy Technology Data Exchange (ETDEWEB)

    S Chung; D Himmel; J Jiang; K Wojtak; J Bauman; J Rausch; J Wilson; J Beutler; C Thomas; et al.

    2011-12-31

    The {alpha}-hydroxytroplone, manicol (5,7-dihydroxy-2-isopropenyl-9-methyl-1,2,3,4-tetrahydro-benzocyclohepten-6-one), potently and specifically inhibits ribonuclease H (RNase H) activity of human immunodeficiency virus reverse transcriptase (HIV RT) in vitro. However, manicol was ineffective in reducing virus replication in culture. Ongoing efforts to improve the potency and specificity over the lead compound led us to synthesize 14 manicol derivatives that retain the divalent metal-chelating {alpha}-hydroxytropolone pharmacophore. These efforts were augmented by a high resolution structure of p66/p51 HIV-1 RT containing the nonnucleoside reverse transcriptase inhibitor (NNRTI), TMC278 and manicol in the DNA polymerase and RNase H active sites, respectively. We demonstrate here that several modified {alpha}-hydroxytropolones exhibit antiviral activity at noncytotoxic concentrations. Inclusion of RNase H active site mutants indicated that manicol analogues can occupy an additional site in or around the DNA polymerase catalytic center. Collectively, our studies will promote future structure-based design of improved {alpha}-hydroxytropolones to complement the NRTI and NNRTI currently in clinical use.

  9. Interaction between wheat alpha-amylase/trypsin bi-functional inhibitor and mammalian digestive enzymes: Kinetic, equilibrium and structural characterization of binding.

    Science.gov (United States)

    Cuccioloni, Massimiliano; Mozzicafreddo, Matteo; Ali, Ishtiaq; Bonfili, Laura; Cecarini, Valentina; Eleuteri, Anna Maria; Angeletti, Mauro

    2016-12-15

    Alpha-amylase/trypsin bi-functional inhibitors (ATIs) are non-gluten protein components of wheat and other cereals that can hypersensitise the human gastrointestinal tract, eventually causing enteropathies in predisposed individuals. These inhibitory proteins can act both directly by targeting specific pro-inflammatory receptors, and indirectly by impairing the activity of digestive enzymes, the latter event causing the accumulation of undigested peptides with potential immunogenic properties. Herein, according to a concerted approach based on in vitro and in silico methods we characterized kinetics, equilibrium parameters and modes of binding of the complexes formed between wheat ATI and two representative mammalian digestive enzymes, namely trypsin and alpha-amylase. Interestingly, we demonstrated ATI to target both enzymes with independent binding sites and with moderately high affinity. PMID:27451220

  10. Selective induction of apoptosis in the hamster flank sebaceous gland organ by a topical liposome 5-alpha-reductase inhibitor: a treatment strategy for acne.

    Science.gov (United States)

    Li, Lingna; Tang, Li; Baranov, Eugene; Yang, Meng; Amoh, Yasuyuki; Katsuoka, Kensei; Hoffman, Robert M

    2010-02-01

    Acne is a very widespread cosmesis problem. Isotretinoin, a synthetic oral retinoid is used to treat acne, which is androgen dependent. Numerous side-effects occur from this treatment. 5-alpha-Reductase plays a critical role in normal and pathological androgen-dependent processes. We have taken the approach to develop a selective, effective, topically-applied 5-alpha-reductase inhibitor to modify unwanted or pathological processes in the pilosebaceous unit such as acne. Toward this goal, we have previously developed a selective liposome hair follicle targeting system. We demonstrate in this report that the 5-alpha-reductase inhibitor N,N-diethyl-4-methyl-3-oxo-4-aza-5alpha-androstane-17beta-carboxamide (4-MA) incorporated into liposomes induces apoptosis and inhibits growth of the dihydrotestosterone (DHT)-dependent hamster flank organ sebaceous gland. We have compared topical application of liposome 4-MA and solvent-formulated 4-MA and observed selective efficacy of topical application of liposome 4-MA by the reduction of size and induction of apoptosis only in the treated hamster flank organ. Apoptosis induced by liposome 4-MA in the treated flank organ sebaceous gland cells was observed both by assays for DNA fragments (transferase deoxytidyl uridine end labeling) and by observation of condensed and fragmented nuclei. When 4-MA was topically applied formulated in ethanol and glycerol without liposomes, the selective efficacy was lost. Liposome 4-MA did not significantly affect prostate weight, testosterone/DHT ratios or bodyweight gain compared to controls indicating safety as well as efficacy of topical application of liposome 4-MA for pathological processes such as acne. PMID:20175850

  11. Superior Orbital Fissure Syndrome and Ophthalmoplegia Caused by Varicella Zoster Virus with No Skin Eruption in a Patient Treated with Tumor Necrosis Alpha Inhibitor.

    Science.gov (United States)

    Jensen, Helene; Thomsen, Sidsel Thorup; Hansen, Stine Scott; Munksgaard, Signe Bruun; Lindelof, Mette

    2015-01-01

    Varicella zoster virus lies dormant in the dorsal root ganglia after symptomatic chicken pox infection, usually in childhood. If the virus reactivates in the trigeminal ganglia, it can cause varicella zoster ophthalmicus, which can have severe ocular complications. We report a case of a 73-year-old woman in severe immunosuppression due to treatment with mycophenolate mofetil, glucocorticosteroids and a tumor necrosis factor alpha inhibitor. The reactivation caused superior orbital fissure syndrome, which has only rarely been described in relation to varicella zoster virus reactivation. In our case, the syndrome was seen along with severe encephalitis. PMID:26600786

  12. BgII reveals two polymorphic sites in the human inter-alpha-trypsin inhibitor heavy chain gene ITI H2

    Energy Technology Data Exchange (ETDEWEB)

    Leveillard, T.; Diarra-Mehrpour, M.; Salier, J.P.; Sesbouee, R.; Bourguignon, J.; Martin, J.P. (Institut National de la Sante et de la Recherche Medicale, St. Etienne Rouvray (France))

    1990-01-25

    The 0.8 kb EcoRI/BamHI fragment of lambda HuHITI-9 (1) used as probe codes for human heavy chain H2 of the inter-alpha-trypsin inhibitor. BgII (GCCN4/NGGC) identifies a three allele polymorphism with DNA fragments at 20.0 kb (A) or 11.0 kb (B) or 16.5 kb and 3.5 kb (C). The ITIH2 gene has been mapped to 10p15 by in situ hybridization. Co-dominant segregation was found for each polymorphism in one informative family.

  13. The role of 5-alpha reductase inhibitors in prostate pathophysiology: Is there an additional advantage to inhibition of type 1 isoenzyme?

    Science.gov (United States)

    Goldenberg, Larry; So, Alan; Fleshner, Neil; Rendon, Ricardo; Drachenberg, Darrel; Elhilali, Mostafa

    2009-06-01

    Normal growth and function of the prostate are contingent on the reduction of testosterone to dihydrotestosterone (DHT) by 5-alpha reductase (5-AR) enzymes types 1 and 2. It has been theorized that an overabundance of DHT may be implicated in the pathogenesis of both benign prostatic hyperplasia (BPH) and prostate cancer. Inhibitors of 5-AR such as dutasteride and finasteride may therefore have an important role in the prevention and treatment of BPH and prostate cancer. Dutasteride provides greater suppression of DHT than finasteride, thereby underlying the hypothesis that inhibition of both type 1 and type 2 would provide correspondingly greater protection than inhibition of type 2 alone. We review the potential significance of the 5-AR inhibitors in reducing the risk of prostate cancer according to the basic biology of prostate disease. PMID:19543428

  14. 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.

  15. 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.

  16. 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

  17. Isolation of linoleic and alpha-linolenic acids as COX-1 and -2 inhibitors in rose hip

    DEFF Research Database (Denmark)

    Jäger, Anna; Petersen, K N; Thomasen, G.;

    2008-01-01

    /2 activity-guided. The bioassay-guided fractionation led to the isolation of linoleic acid (the IC50 for COX-1 was 85 microm and 0.6 microM for COX-2) and alpha-linolenic acid (the IC50 for COX-1 was 52 microM and 12 microM for COX-2). The COX-2/COX-1 ratio was 0.007 for linoleic acid and 0.2 for alpha......-linolenic acid. Linoleic acid and alpha-linolenic acid contribute to the COX-1 and -2 inhibitory activity of rose hip....

  18. Proteinase, amylase, and proteinase-inhibitor activities in the gut of six cockroach species

    Czech Academy of Sciences Publication Activity Database

    Vinokurov, Konstantin; Taranushenko, Yuliya; Krishnan, Natraj; Sehnal, František

    2007-01-01

    Roč. 53, - (2007), s. 794-802. ISSN 0022-1910 R&D Projects: GA ČR(CZ) GA522/06/1591; GA MŠk 1M06030 Institutional research plan: CEZ:AV0Z50070508 Keywords : amylases * Blattodea * gut pH Subject RIV: CE - Biochemistry Impact factor: 2.294, year: 2007

  19. Individualized monitoring of drug bioavailability and immunogenicity in rheumatoid arthritis patients treated with the tumor necrosis factor alpha inhibitor infliximab

    DEFF Research Database (Denmark)

    Bendtzen, Klaus; Geborek, Pierre; Svenson, Morten;

    2006-01-01

    Infliximab, an anti-tumor necrosis factor alpha (anti-TNFalpha) antibody, is effective in the treatment of several immunoinflammatory diseases. However, many patients experience primary or secondary response failure, suggesting that individualization of treatment regimens may be beneficial. This ...

  20. 3- and 4-pyridylalkyl adamantanecarboxylates: inhibitors of human cytochrome P450(17 alpha) (17 alpha-hydroxylase/C17,20-lyase). Potential nonsteroidal agents for the treatment of prostatic cancer.

    Science.gov (United States)

    Chan, F C; Potter, G A; Barrie, S E; Haynes, B P; Rowlands, M G; Houghton, J; Jarman, M

    1996-08-16

    Various 3- and 4-pyridylalkyl 1-adamantanecarboxylates have been synthesized and tested for inhibitory activity toward the 17 alpha-hydroxylase and C17,20-lyase activities of human testicular cytochrome P450(17 alpha). The 4-pyridylalkyl esters were much more inhibitory than their 3-pyridylalkyl counterparts. The most potent was (S)-1-(4-pyridyl)ethyl 1-adamantanecarboxylate (3b; IC50 for lyase, 1.8 nM), whereas the (R)-enantiomer 3a was much less inhibitory (IC50 74 nM). Nearly as potent as 3b was the dimethylated counterpart, the 2-(4-pyridylpropan-2-yl) ester 5 (IC50 2.7 nM), which was also more resistant to degradation by esterases. In contrast to their 4-pyridyl analogs, the enantiomers of the 1-(3-pyridyl)ethyl ester were similarly inhibitory (IC50 for lyase; (R)-isomer 8a 150 nM, (S)-isomer 8b 230 nM). Amides corresponding to the 4-pyridylmethyl ester 1 and the (S)-1-(4-pyridyl)ethyl ester 3b, respectively 11 and 15b, were much less inhibitory than their ester counterparts. On the basis of a combination of inhibitory potency and resistance to esterases, the ester 5 was the best candidate for further development as a potential nonsteroidal inhibitor of cytochrome P450(17 alpha) for the treatment of prostate cancer. PMID:8765515

  1. 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

  2. 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.

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

    Science.gov (United States)

    Sun, Di; Chen, Shun; Cheng, Anchun; Wang, Mingshu

    2016-01-01

    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 (3C(pro)s) of picornaviruses share similar spatial structures and it is becoming apparent that 3C(pro) plays a significant role in the viral life cycle and virus host interaction. Importantly, the proteinase and RNA-binding activity of 3C(pro) are involved in viral polyprotein processing and the initiation of viral RNA synthesis. In addition, 3C(pro) 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 3C(pro) and these essential factors, 3C(pro) 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 3C(pro) are ongoing and a better understanding of the roles played by 3C(pro) 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 3C(pro) is summarized. PMID:26999188

  4. A proprietary alpha-amylase inhibitor from white bean (Phaseolus vulgaris: A review of clinical studies on weight loss and glycemic control

    Directory of Open Access Journals (Sweden)

    Barrett Marilyn L

    2011-03-01

    Full Text Available Abstract Obesity, and resultant health hazards which include diabetes, cardiovascular disease and metabolic syndrome, are worldwide medical problems. Control of diet and exercise are cornerstones of the management of excess weight. Foods with a low glycemic index may reduce the risk of diabetes and heart disease as well as their complications. As an alternative to a low glycemic index diet, there is a growing body of research into products that slow the absorption of carbohydrates through the inhibition of enzymes responsible for their digestion. These products include alpha-amylase and glucosidase inhibitors. The common white bean (Phaseolus vulgaris produces an alpha-amylase inhibitor, which has been characterized and tested in numerous clinical studies. A specific and proprietary product named Phase 2® Carb Controller (Pharmachem Laboratories, Kearny, NJ has demonstrated the ability to cause weight loss with doses of 500 to 3000 mg per day, in either a single dose or in divided doses. Clinical studies also show that Phase 2 has the ability to reduce the post-prandial spike in blood glucose levels. Experiments conducted incorporating Phase 2 into food and beverage products have found that it can be integrated into various products without losing activity or altering the appearance, texture or taste of the food. There have been no serious side effects reported following consumption of Phase 2. Gastro-intestinal side effects are rare and diminish upon extended use of the product. In summary, Phase 2 has the potential to induce weight loss and reduce spikes in blood sugar caused by carbohydrates through its alpha-amylase inhibiting activity.

  5. Crystal structures of the psychrophilic alpha-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor.

    OpenAIRE

    Aghajari, N.; Feller, G; Gerday, C.; Haser, R.

    1998-01-01

    Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the alpha-amylase (AHA) secreted by this bacterium, in its native state to 2.0 angstroms resolution as well as in complex with Tris to 1.85 angstroms resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known alpha-amy...

  6. Insights into affinity and specificity in the complexes of alpha-lytic protease and its inhibitor proteins: binding free energy from molecular dynamics simulation.

    Science.gov (United States)

    Deng, Nan-Jie; Cieplak, Piotr

    2009-07-01

    We report the binding free energy calculation and its decomposition for the complexes of alpha-lytic protease and its protein inhibitors using molecular dynamics simulation. Standard mechanism serine protease inhibitors eglin C and OMTKY3 are known to have strong binding affinity for many serine proteases. Their binding loops have significant similarities, including a common P1 Leu as the main anchor in the binding interface. However, recent experiments demonstrate that the two inhibitors have vastly different affinity towards alpha-lytic protease (ALP), a bacterial serine protease. OMTKY3 inhibits the enzyme much more weakly (by approximately 10(6) times) than eglin C. Moreover, a variant of OMTKY3 with five mutations, OMTKY3M, has been shown to inhibit 10(4) times more strongly than the wild-type inhibitor. The underlying mechanisms for the unusually large difference in binding affinities and the effect of mutation are not well understood. Here we use molecular dynamics simulation with molecular mechanics-Poisson Boltzmann/surface area method (MM-PB/SA) to investigate quantitatively the binding specificity. The calculated absolute binding free energies correctly differentiate the thermodynamic stabilities of these protein complexes, but the magnitudes of the binding affinities are systematically overestimated. Analysis of the binding free energy components provides insights into the molecular mechanism of binding specificity. The large DeltaDeltaG(bind) between eglin C and wild type OMTKY3 towards ALP is mainly attributable to the stronger nonpolar interactions in the ALP-eglin C complex, arising from a higher degree of structural complementarity. Here the electrostatic interaction contributes to a lesser extent. The enhanced inhibition in the penta-mutant OMTKY3M over its wild type is entirely due to an overall improvement in the solvent-mediated electrostatic interactions in the ALP-OMTKY3M complex. The results suggest that for these protein-complexes and

  7. Studies on the action of proteinase inhibitors in rats. 3

    International Nuclear Information System (INIS)

    Male Wistar rats (initial body weight 90 g) were fed ad libitum a whole-egg diet containing 10.5% crude protein. The animals of the experimental group received in each case 1 mg leupeptin per 100 g of body weight in 12 hrs intervals by i.p. injection (3 days of treatment). Control animals got a leupeptin-free solution. In addition, lysine dihydrochloride-α-15N was applied during the first three days of experiment to all animals and the nitrogen balance was determined. Urine from the N balance collection was analysed for 3-methyl-histidine excretion in order to calculate the degradation rate of myofibrillar proteins. On the fourth day the fractional rate of protein synthesis in several organs was estimated using the continuous infusion technique with 14C-leucine and 14C-lysine. The apparent biological half-lives of tissue protein were determined by a triple labelling technique, with (14C)-guanidino-L-arginine, L-5-3H-arginine and 15N-lysine. The short-term treatment (3 days) with leupeptin did not affect the weight gain, the apparent digestibility of nitrogen and the N balance. The fractional rate of protein synthesis was highest in the small intestine followed by the large intestine, liver and skeletal muscle and no influence of leupeptin treatment was observed. Furthermore no differences in the degradation rates of myofibrillar proteins between treated and untreated animals were found. The 3-methyl-histidine excretion via urine was 1.44 mgkg-1day-1 in both groups corresponding to a fractional rate of degradation of myofibrillar proteins of 2.5% per day. Half-lives of tissue proteins in intestine and liver were shortest when estimated from the decay curves for the 14C label and longest from the curves for the 15N label. Leupeptin treatment resulted in prolonged half-lives of the proteins in the large intestine and of the liver proteins with slow turnover. However, this effect seems to be caused rather by an increased reutilization of labelled amino acids than by a decreased protein degradation. (author)

  8. Dental Enamel Development: Proteinases and Their Enamel Matrix Substrates

    OpenAIRE

    Bartlett, John D.

    2013-01-01

    This review focuses on recent discoveries and delves in detail about what is known about each of the proteins (amelogenin, ameloblastin, and enamelin) and proteinases (matrix metalloproteinase-20 and kallikrein-related peptidase-4) that are secreted into the enamel matrix. After an overview of enamel development, this review focuses on these enamel proteins by describing their nomenclature, tissue expression, functions, proteinase activation, and proteinase substrate specificity. These protei...

  9. 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

    -expression with PEP4 leads to normal processing, i.e. the mutant zymogen is functional as a substrate for the maturation reaction in trans. We conclude that wild-type pro-proteinase A has the ability to mediate its own activation. Elimination of the co-expressed PEP4 gene did not effectively stop the processing...... 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....

  10. Digestive alpha-amylases of the flour moth Ephestia kuehniella - adaptation to alkaline environment and plant inhibitors

    Czech Academy of Sciences Publication Activity Database

    Pytelková, Jana; Hubert, J.; Lepšík, Martin; Šobotník, Jan; Šindelka, Radek; Křížková, I.; Horn, Martin; Mareš, Michael

    2009-01-01

    Roč. 276, Suppl. 1 (2009), s. 162-162. ISSN 1742-464X. [FEBS Congress /34/. 04.07.2009-09.07.2009, Praha] R&D Projects: GA ČR GP525/09/P600; GA AV ČR IAA400550617 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520701 Keywords : Ephestia kuehniella * alpha amylase * alkaline adaptation Subject RIV: CE - Biochemistry

  11. The gene signature in CCAAT-enhancer-binding protein alpha dysfunctional acute myeloid leukemia predicts responsiveness to histone deacetylase inhibitors

    Czech Academy of Sciences Publication Activity Database

    Liss, A.; Ooi, C.; Zjablovskaja, Polina; Benoukraf, T.; Radomska, H.S.; Ju, C.; Wu, M.C.; Balaštík, Martin; Delwel, R.; Brdička, Tomáš; Tan, P.; Tenen, D.G.; Alberich-Jorda, Meritxell

    2014-01-01

    Roč. 99, č. 4 (2014), s. 697-705. ISSN 0390-6078 R&D Projects: GA MŠk LK21307; GA MŠk(CZ) LK11213 Grant ostatní: NIH(US) CA66996; NIH(US) CA118316 Institutional support: RVO:68378050 Keywords : C/EBPa * histone deacetylase inhibitor * acute myeloid leukemia Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.814, year: 2014

  12. Is Transforming Growth Factor-β Signaling Activated in Human Hypertrophied Prostate Treated by 5-Alpha Reductase Inhibitor?

    OpenAIRE

    Jong Kwan Park; Do Sung Kim; Chen Zhao; Bo Ram Choi; Han Jung Chae; Hye Kyung Kim

    2013-01-01

    Background and Aim. It is well known that androgen deprivation relates to penile fibrosis, so we hypothesize that long-term treatment with 5-alphareductase inhibitors (5ARIs) may increase the risk of fibrosis of prostate. Patients and Methods. Thirty-two BPH patients who underwent transurethral resection of the prostate were enrolled. The patients were divided into two groups: group one, 16 patients underwent TURP who had been treated with tamsulosin for 2 years; group two, 16 patients underw...

  13. In silico study of curcumol, curcumenol, isocurcumenol, and β-sitosterol as potential inhibitors of estrogen receptor alpha of breast cancer

    Directory of Open Access Journals (Sweden)

    Resmi Mustarichiei

    2014-03-01

    Full Text Available Background: Based on data from the Hospital Information System (HIS in 2007, breast cancer is the top ranked diagnosed cancer in Indonesia. Estrogen receptor alpha (ERα is associated with breast cancer because it is found in high levels in cancer tissues. Curcumol, curcumenol, isocurcumenol of white tumeric rhizomes (Curcuma zedoaria (Christm. Roscoe, and β-sitosterol from seeds of pumpkin (Cucurbita pepo L. have been reported to have inhibitory activity against cancer cells. This study presents the in silico study of these compounds as inhibitors of ERα.Methods: Docking simulations are carried out in this paper to visualize molecular-level interactions between the four compounds with ERα. Docking simulations between estradiol and tamoxifen on ERα are carried out as well.Results: Docking results indicated that curcumol, curcumenol, isocurcumenol, and β-sitosterol showed inhibitory activity againts estrogen receptor alpha (ERα.  The order of potency is shown consecutively by isocurcumenol, curcumol, curcumenol, and β-sitosterol with values 0.584 M, 1.36 M, 1.61 M, and 7.35 M respectively. Curcumenol and estradiol interacts with ERα through hydrogen bonds and hydrophobic interactions, whereas curcumol, isocurcumenol, β-sitosterol and tamoxifen through hydrophobic interactions in succession. Conclusion: Natural products containing all four compounds have the potential to be used as drugs or adjuvant drugs in breast cancer therapy.Keywords: β-sitosterol, breast cancer, curcumol, curcumenol, estradiol, ERα, isocurcumenol

  14. Scale Alpha and Beta of Quantitative Convergence and Chemical Reactivity Analysis in Dual Cholinesterase/Monoamine Oxidase Inhibitors for the Alzheimer Disease Treatment Using Density Functional Theory (DFT

    Directory of Open Access Journals (Sweden)

    Alejandro Morales-Bayuelo

    2013-01-01

    Full Text Available Molecular quantum similarity descriptors and Density Functional Theory (DFT based reactivity descriptors were studied for a series of cholinesterase/monoamine oxidase inhibitors used for the Alzheimer's disease treatment (AD. This theoretical study is expected to shed some light onto some molecular aspects that could contribute to the knowledge of the molecular mechanics behind interactions of these molecules with acetylcholinesterase (AChE and butyrylcholinesterase (BuChE, as well as with monoamine oxidase (MAO A and B. The Topogeometrical Superposition Algorithm to handle flexible molecules (TGSA-Flex alignment method was used to solve the problem of the relative orientation in the quantum similarity (QS field. Using the molecular quantum similarity (MQS field and reactivity descriptors supported in the DFT was possible the quantification of the steric and electrostatic effects through of the Coulomb and Overlap quantitative convergence scales (alpha and beta. In addition, an analysis of reactivity indexes is development, using global and local descriptors, identifying the binding sites and selectivity in the (cholinesterase/monoamine oxidase inhibitors, understanding the retrodonor process, and showing new insight for drugs design in a disease of difficult control as Alzheimer.

  15. 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.

  16. Punica granatum flower extract, a potent alpha-glucosidase inhibitor, improves postprandial hyperglycemia in Zucker diabetic fatty rats.

    Science.gov (United States)

    Li, Yuhao; Wen, Suping; Kota, Bhavani Prasad; Peng, Gang; Li, George Qian; Yamahara, Johji; Roufogalis, Basil D

    2005-06-01

    Postprandial hyperglycemia plays an important role in the development of type 2 diabetes and has been proposed as an independent risk factor for cardiovascular diseases. The flowering part of Punica granatum Linn. (Punicaceae) (PGF) has been recommended in Unani literature as a remedy for diabetes. We investigated the effect and action mechanism of a methanolic extract from PGF on hyperglycemia in vivo and in vitro. Oral administration of PGF extract markedly lowered plasma glucose levels in non-fasted Zucker diabetic fatty rats (a genetic model of obesity and type 2 diabetes), whereas it had little effect in the fasted animals, suggesting it affected postprandial hyperglycemia in type 2 diabetes. In support of this conclusion the extract was found to markedly inhibit the increase of plasma glucose levels after sucrose loading, but not after glucose loading in mice, and it had no effect on glucose levels in normal mice. In vitro, PGF extract demonstrated a potent inhibitory effect on alpha-glucosidase activity (IC50: 1.8 microg/ml). The inhibition is dependent on the concentration of enzyme and substrate, as well as on the length of pretreatment with the enzyme. These findings strongly suggest that PGF extract improves postprandial hyperglycemia in type 2 diabetes and obesity, at least in part, by inhibiting intestinal alpha-glucosidase activity. PMID:15894133

  17. Structural and evolutionary insights into endogenous alpha-phospholipase A2 inhibitors of Latin American pit vipers.

    Science.gov (United States)

    Estevão-Costa, Maria Inácia; Fernandes, Carlos Alexandre H; Mudadu, Maurício de Alvarenga; Franco, Glória Regina; Fontes, Marcos Roberto M; Fortes-Dias, Consuelo Latorre

    2016-03-15

    Phospholipases A2 are major components of snake venoms (svPLA2s) and are able to induce multiple local and systemic deleterious effects upon envenomation. Several snake species are provided with svPLA2 inhibitors (sbPLIs) in their circulating blood, which confer a natural resistance against the toxic components of homologous and heterologous venoms. The sbPLIs belong to any of three structural classes named α, β and γ. In the present study, we identified, characterized and performed structural and evolutionary analyses of sbαPLIs transcripts and the encoded proteins, in the most common Latin American pit vipers belonging to Crotalus, Bothrops and Lachesis genera. Mutation data indicated that sbαPLIs from Latin American snakes might have evolved in an accelerated manner, similarly to that reported for sbαPLIs from Asian snakes, and possibly co-evoluted with svPLA2s in response to the diversity of target enzymes. The importance of sbαPLI trimerization for the effective binding and inhibition of acidic svPLA2s is discussed and conserved cationic residues located at the central pore of the inhibitor trimer are suggested to be a significant part of the binding site of sbαPLIs to acidic svPLA2s. Our data contribute to the current body of knowledge on the structural and evolutionary characteristics of sbPLIs, in general, and may assist in the future development of selective inhibitors for secretory PLA2 from several sources. PMID:26806211

  18. 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...

  19. [Hereditary deficiency of alpha 1- antitrypsin in rats due to evolving chronic lung pathology].

    Science.gov (United States)

    Soloveva, N A; Grishaeva, O N; Parik, Iu Ia; Kosova, E Iu; Korolenko, T A

    1994-01-01

    W/SSM rats which are characterized by hereditary abnormal changes in the lungs, hepato- and splenomegalia and some other disturbances have also alpha 1-antitrypsin (AAT) deficiency. A study of AAT in these rats by means of isoelectrofocusing and immunoblotting with anti-AAT antibodies labelled with peroxidase has demonstrated that deficiency of the protease inhibitor is not associated with any disturbances of its synthesis or any changes of its electrophoretic properties. A higher activity of lysosomal glycosidases and proteinases was found in the liver and leukocytes of W/SSM rats. It is suggested that AAT deficiency is due to its modification under the influence of lysosomal enzymes. The described biochemical distances seem to be associated with an increased hexose transport into the cells, which is controlled by a mutant gene. PMID:7513577

  20. Pharmacophore modeling, in silico screening, molecular docking and molecular dynamics approaches for potential alpha-delta bungarotoxin-4 inhibitors discovery

    Directory of Open Access Journals (Sweden)

    R Barani Kumar

    2015-01-01

    Full Text Available Background: The alpha-delta bungartoxin-4 (α-δ-Bgt-4 is a potent neurotoxin produced by highly venomous snake species, Bungarus caeruleus, mainly targeting neuronal acetylcholine receptors (nAchRs and producing adverse biological malfunctions leading to respiratory paralysis and mortality. Objective: In this study, we predicted the three-dimensional structure of α-δ-Bgt-4 using homology modeling and investigated the conformational changes and the key residues responsible for nAchRs inhibiting activity. Materials and Methods: From the selected plants, which are traditionally used for snake bites, the active compounds are taken and performed molecular interaction studies and also used for modern techniques like pharmacophore modeling and mapping and absorption, distribution, metabolism, elimination and toxicity analysis which may increase the possibility of success. Results: Moreover, 100′s of drug-like compounds were retrieved and analyzed through computational virtual screening and allowed for pharmacokinetic profiling, molecular docking and dynamics simulation. Conclusion: Finally the top five drug-like compounds having competing level of inhibition toward α-δ-Bgt-4 toxin were suggested based on their interaction with α-δ-Bgt-4 toxin.

  1. Alpha-amylase inhibitor, CS-1036 binds to serum amylase in a concentration-dependent and saturable manner.

    Science.gov (United States)

    Honda, Tomohiro; Kaneno-Urasaki, Yoko; Ito, Takashi; Kimura, Takako; Matsushima, Nobuko; Okabe, Hiromi; Yamasaki, Atsushi; Izumi, Takashi

    2014-03-01

    (2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxy-β-D-glucopyranosyl)-α-D-glucopyranoside (CS-1036), which is an α-amylase inhibitor, exhibited biphasic and sustained elimination with a long t1/2 (18.4-30.0 hours) in rats and monkeys, but exhibited a short t1/2 (3.7-7.9 hours) in humans. To clarify the species differences in the t1/2, the plasma protein binding of CS-1036 was evaluated by ultrafiltration. A concentration-dependent and saturable plasma protein binding of CS-1036 was observed in rats and monkeys with the dissociation rate constant (KD) of 8.95 and 27.2 nM, and maximal binding capacity (Bmax) of 52.8 and 22.1 nM, respectively. By the assessments of the recombinant amylase and immunoprecipitation, the major binding protein of CS-1036 in rats was identified as salivary amylase (KD 5.64 nM). CS-1036 also showed concentration-dependent and saturable binding to human salivary and pancreatic amylase, with similar binding affinity in rats. However, the protein binding of CS-1036 was constant in human plasma (≤10.2%) due to the lower serum amylase level compared with rats and monkeys. From the calculation of the unbound fraction (fu) in plasma based on in vitro KD and Bmax, the dose-dependent increase in fu after oral administration is speculated to lead to a dose-dependent increase in total body clearance and a high area under the curve/dose at lower doses, such as 0.3 mg/kg in rats. PMID:24319124

  2. Is Transforming Growth Factor-β Signaling Activated in Human Hypertrophied Prostate Treated by 5-Alpha Reductase Inhibitor?

    Directory of Open Access Journals (Sweden)

    Hye Kyung Kim

    2013-01-01

    Full Text Available Background and Aim. It is well known that androgen deprivation relates to penile fibrosis, so we hypothesize that long-term treatment with 5-alphareductase inhibitors (5ARIs may increase the risk of fibrosis of prostate. Patients and Methods. Thirty-two BPH patients who underwent transurethral resection of the prostate were enrolled. The patients were divided into two groups: group one, 16 patients underwent TURP who had been treated with tamsulosin for 2 years; group two, 16 patients underwent TURP who had been treated with combination of tamsulosin and dutasteride for at least 1 year. We evaluated the expressions of nNOS, iNOS, eNOS, TGF-β1, TGF-β2, phosphorylated-Smad2/3 (p-Smad2/3, E-cadherin, N-cadherin, and α-smooth muscle actin in the resected prostate tissues by western blotting, and the TGF-β concentration was determined by ELISA kit. Results. The expressions of 3 isoforms of NOS were significantly increased in group 2 except of eNOS in lateral prostate, and the expressions of TGF-β1, TGF-β2, and p-Smad2/3 increased about 2-fold compared with group 1. In group 2, the E-cadherin expression decreased while N-cadherin expression increased significantly. Conclusions. The overexpression of nNOS may contribute to prostate smooth muscle relaxation; however, long-time treatment with 5 ARI increases the risk of fibrosis of prostate.

  3. Effects of a high-pressure treatment on the wheat alpha-amylase inhibitor and its relationship to elimination of allergenicity

    International Nuclear Information System (INIS)

    In this study, the effects of high-pressure treatment on structure and allergeincity of alpha amylase inhibitor (a-AI) were investigated. The pressure-induced structural changes of α-AI were estimated by fluorescence spectra and by fourth derivative UV-spectroscopy for probed tyrosine residues and by circular dichroism (CD) spectroscopy. The changes in the tertiary structure detected by fluorescence spectra and by fourth derivative UV-spectroscopy under high pressure were indicated at over 300 MPa. Measurements of CD spectroscopy suggested that the effects of a high-pressure treatment on changes in the secondary structure of α-AI were little. From our results, pressure-induced changes of the α-AI structure were not apparent. On the other hands, the IgE-specific binding activities of pressurized α-AI to sera from allergic patients against wheat, which is estimated by observations of dot-blotting, were decreased by high-pressure treatment. It is known that the pressure-induced elimination of allergenicity is related to the tertiary structural changes of allergen molecules. This study are suspected that the epitopes of α-AI do not contain tyrosine residues, and thus the decrease of IgE-specific binding activities is probably caused by the tertiary structural changes of these parts of α-AI.

  4. Effects of a high-pressure treatment on the wheat alpha-amylase inhibitor and its relationship to elimination of allergenicity

    Science.gov (United States)

    Yamamoto, S.; Takanohashi, K.; Hara, T.; Odani, S.; Suzuki, A.; Nishiumi, T.

    2010-03-01

    In this study, the effects of high-pressure treatment on structure and allergeincity of alpha amylase inhibitor (a-AI) were investigated. The pressure-induced structural changes of α-AI were estimated by fluorescence spectra and by fourth derivative UV-spectroscopy for probed tyrosine residues and by circular dichroism (CD) spectroscopy. The changes in the tertiary structure detected by fluorescence spectra and by fourth derivative UV-spectroscopy under high pressure were indicated at over 300 MPa. Measurements of CD spectroscopy suggested that the effects of a high-pressure treatment on changes in the secondary structure of α-AI were little. From our results, pressure-induced changes of the α-AI structure were not apparent. On the other hands, the IgE-specific binding activities of pressurized α-AI to sera from allergic patients against wheat, which is estimated by observations of dot-blotting, were decreased by high-pressure treatment. It is known that the pressure-induced elimination of allergenicity is related to the tertiary structural changes of allergen molecules. This study are suspected that the epitopes of α-AI do not contain tyrosine residues, and thus the decrease of IgE-specific binding activities is probably caused by the tertiary structural changes of these parts of α-AI.

  5. Alpha-glucosidase inhibitor, acarbose, improves glycamic control and reduces body weight in type 2 diabetes: Findings on indian patients from the pooled data analysis

    Directory of Open Access Journals (Sweden)

    Sanjay Kalra

    2013-01-01

    Full Text Available Alpha-glucosidase inhibitors are widely used especially in Asian countries as a treatment option for type 2 diabetes patients with high postprandial glycemia (PPG. The higher carbohydrate in the Indian diets lead to greater prandial glycemic excursion, increased glucosidase, and incretin activity in the gut and may need special therapeutic strategies to tackle these glucose peaks. This is the subgroup analysis of Indian subjects who participated in the GlucoVIP study that investigated the effectiveness and tolerability of acarbose as add-on or monotherapy in a range of patients with type 2 diabetes mellitus. A total of 1996 Indian patients were included in the effectiveness analysis. After 12.5 weeks (mean, the mean change in 2-hour PPG from baseline was −74.4 mg/dl, mean HbA1c decreased by -1.0%, and mean fasting blood glucose decreased by -37.9 mg/dl. The efficacy of acarbose was rated "very good" or "good" in 91.1% of patients, and tolerability as "very good" or "good" in 88.0% of patients. The results of this observational study suggest that acarbose was effective and well tolerated in the Indian patients with T2DM.

  6. Imbalance between HAT and HDAC activities in the PBMCs of patients with ankylosing spondylitis or rheumatoid arthritis and influence of HDAC inhibitors on TNF alpha production.

    Directory of Open Access Journals (Sweden)

    Eric Toussirot

    Full Text Available OBJECTIVE: Acetylation or deacetylation of histone proteins may modulate cytokine gene transcription such as TNF alpha (TNF. We evaluated the balance between histone deacetytlase (HDAC and histone acetyltransferase (HAT in patients with rheumatoid arthritis (RA or ankylosing spondylitis (AS compared to healthy controls (HC and determined the influence of HDAC inhibitors (trichostatin A -TSA- or Sirtinol -Sirt- on these enzymatic activities and on the PBMC production of TNF. METHODS: 52 patients with RA, 21 with AS and 38 HC were evaluated. HAT and HDAC activities were measured on nuclear extracts from PBMC using colorimetric assays. Enzymatic activities were determined prior to and after ex vivo treatment of PBMC by TSA or Sirt. TNF levels were evaluated in PBMC culture supernatants in the absence or presence of TSA or Sirt. RESULTS: HAT and HDAC activities were significantly reduced in AS, while these activities reached similar levels in RA and HC. Ex vivo treatment of PBMC by HDACi tended to decrease HDAC expression in HC, but Sirt significantly reduced HAT in RA. TNF production by PBMC was significantly down-regulated by Sirt in HC and AS patients. CONCLUSION: HAT and HDAC were disturbed in AS while no major changes were found in RA. HDACi may modulate HDAC and HAT PBMC expression, especially Sirt in RA. Sirtinol was able to down regulate TNF production by PBMC in HC and AS. An imbalance between HAT and HDAC activities might provide the rationale for the development of HDACi in the therapeutic approach to inflammatory rheumatic diseases.

  7. Patients with ovarian carcinoma excrete different altered levels of urine CD59, kininogen-1 and fragments of inter-alpha-trypsin inhibitor heavy chain H4 and albumin

    Directory of Open Access Journals (Sweden)

    Hashim Onn H

    2010-11-01

    Full Text Available Abstract Background Diagnosis of ovarian carcinoma is in urgent need for new complementary biomarkers for early stage detection. Proteins that are aberrantly excreted in the urine of cancer patients are excellent biomarker candidates for development of new noninvasive protocol for early diagnosis and screening purposes. In the present study, urine samples from patients with ovarian carcinoma were analysed by two-dimensional gel electrophoresis and the profiles generated were compared to those similarly obtained from age-matched cancer negative women. Results Significant reduced levels of CD59, kininogen-1 and a 39 kDa fragment of inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4, and enhanced excretion of a 19 kDa fragment of albumin, were detected in the urine of patients with ovarian carcinoma compared to the control subjects. The different altered levels of the proteins were confirmed by Western blotting using antisera and a lectin that bind to the respective proteins. Conclusion CD59, kininogen-1 and fragments of ITIH4 and albumin may be used as complementary biomarkers in the development of new noninvasive protocols for diagnosis and screening of ovarian carcinoma.

  8. Overexpression of the calpain-specific inhibitor calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in [A30P]αSyn transgenic mice.

    Science.gov (United States)

    Diepenbroek, Meike; Casadei, Nicolas; Esmer, Hakan; Saido, Takaomi C; Takano, Jiro; Kahle, Philipp J; Nixon, Ralph A; Rao, Mala V; Melki, Ronald; Pieri, Laura; Helling, Stefan; Marcus, Katrin; Krueger, Rejko; Masliah, Eliezer; Riess, Olaf; Nuber, Silke

    2014-08-01

    Lewy bodies, a pathological hallmark of Parkinson's disease (PD), contain aggregated alpha-synuclein (αSyn), which is found in several modified forms and can be discovered phosphorylated, ubiquitinated and truncated. Aggregation-prone truncated species of αSyn caused by aberrant cleavage of this fibrillogenic protein are hypothesized to participate in its sequestration into inclusions subsequently leading to synaptic dysfunction and neuronal death. Here, we investigated the role of calpain cleavage of αSyn in vivo by generating two opposing mouse models. We crossed into human [A30P]αSyn transgenic (i) mice deficient for calpastatin, a calpain-specific inhibitor, thus enhancing calpain activity (SynCAST(-)) and (ii) mice overexpressing human calpastatin leading to reduced calpain activity (SynCAST(+)). As anticipated, a reduced calpain activity led to a decreased number of αSyn-positive aggregates, whereas loss of calpastatin led to increased truncation of αSyn in SynCAST(-). Furthermore, overexpression of calpastatin decreased astrogliosis and the calpain-dependent degradation of synaptic proteins, potentially ameliorating the observed neuropathology in [A30P]αSyn and SynCAST(+) mice. Overall, our data further support a crucial role of calpains, particularly of calpain 1, in the pathogenesis of PD and in disease-associated aggregation of αSyn, indicating a therapeutic potential of calpain inhibition in PD. PMID:24619358

  9. Proteomic demonstration of the recurrent presence of inter-alpha-inhibitor H4 heavy-chain during aspergillosis induced in an animal model.

    Science.gov (United States)

    Desoubeaux, Guillaume; Jourdan, Marie-Lise; Valera, Lionel; Jardin, Bénédicte; Hem, Sonia; Caille, Agnès; Cormier, Bénédicte; Marchand-Adam, Sylvain; Bailly, Éric; Diot, Patrice; Chandenier, Jacques

    2014-05-01

    Invasive pulmonary aspergillosis remains a matter of great concern in oncology/haematology, intensive care units and organ transplantation departments. Despite the availability of various diagnostic tools with attractive features, new markers of infection are required for better medical care. We therefore looked for potential pulmonary biomarkers of aspergillosis, by carrying out two-dimensional (2D) gel electrophoresis comparing the proteomes of bronchial-alveolar lavage fluids (BALF) from infected rats and from control rats presenting non-specific inflammation, both immunocompromised. A bioinformatic analysis of the 2D-maps revealed significant differences in the abundance of 20 protein spots (ANOVA P-value0.8). One of these proteins, identified by mass spectrometry, was considered of potential interest: inter-alpha-inhibitor H4 heavy-chain (ITIH4), characterised for the first time in this infectious context. Western blotting confirmed its overabundance in all infected BALF, particularly at early stages of murine aspergillosis. Further investigations were carried on rat serum, and confirmed that ITIH4 levels increased during experimental aspergillosis. Preliminary results in human samples strengthened this trend. To our knowledge, this is the first description of the involvement of ITIH4 in aspergillosis. PMID:24360996

  10. Link between allergic asthma and airway mucosal infection suggested by proteinase-secreting household fungi

    OpenAIRE

    Porter, P; Susarla, SC; Polikepahad, S; Qian, Y; HAMPTON, J.; Kiss, A; Vaidya, S; Sur, S.; Ongeri, V; Yang, T; Delclos, GL; Abramson, S.; Kheradmand, F.; Corry, DB

    2009-01-01

    Active fungal proteinases are powerful allergens that induce experimental allergic lung disease strongly resembling atopic asthma, but the precise relationship between proteinases and asthma remains unknown. Here, we analyzed dust collected from the homes of asthmatic children for the presence and sources of active proteinases to further explore the relationship between active proteinases, atopy, and asthma. Active proteinases were present in all houses and many were derived from fungi, espec...

  11. Costs and persistence of alpha-2 adrenergic agonists versus carbonic anhydrase inhibitors, both associated with prostaglandin analogues, for glaucoma as recorded by The United Kingdom General Practitioner Research Database

    Directory of Open Access Journals (Sweden)

    Philippe Denis

    2008-06-01

    Full Text Available Philippe Denis1, Antoine Lafuma2, Gilles Berdeaux31Hôpital Edouard Herriot, Lyon, France; 2Cemka, Bourg-la-Reine, France; 3Alcon France, Rueil-Malmaison, FranceAbstract: The persistence and costs of carbonic anhydrase inhibitors + prostaglandin analogues (CAIs + PGAs vs alpha-2 adrenergic agonists + prostaglandin analogues (alpha-2 agonists + PGAs were compared, based on The United Kingdom General Practitioner Research Database. Patients with a diagnosis of ocular hypertension, glaucoma, or treated for this, were selected. Selected patients were prescribed CAIs + PGAs or alpha-2 agonists + PGAs. Treatment failure was defined as a prescription change (adding, removing, or replacing glaucoma treatment, or initiating laser or surgery. Times to treatment failure were compared with a Cox model adjusted by a propensity score. Mean patient age was 69.0 years and 47.6% were males. Treatment failure at 1 year was experienced by 58.8% receiving CAIs + PGAs and 66.0% of patients receiving alpha-2 agonists + PGAs (p < 0.001. The hazard ratio for failure was 0.82 (p < 0.001 in favor of CAIs + PGAs after adjusting on age, gender, comorbidities, and duration of follow-up. Adjusted annual costs of glaucoma management did not differ significantly between treatments, £440.63 with alpha-2 agonists + PGAs and £413.37 with CAIs + PGAs. CAIs + PGAs therapies appear more persistent than alpha-2 agonist + PGA in everyday clinical practice, at a similar cost.Keywords: glaucoma, alpha-2 adrenergic agonists, carbonic anhydrase inhibitor, prostaglandin, effectiveness economics, costs

  12. 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

  13. 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...

  14. 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...

  15. Candida albicans Secreted Aspartyl Proteinases in Virulence and Pathogenesis

    OpenAIRE

    Naglik, Julian R.; Challacombe, Stephen J; Hube, Bernhard

    2003-01-01

    Candida albicans is the most common fungal pathogen of humans and has developed an extensive repertoire of putative virulence mechanisms that allows successful colonization and infection of the host under suitable predisposing conditions. Extracellular proteolytic activity plays a central role in Candida pathogenicity and is produced by a family of 10 secreted aspartyl proteinases (Sap proteins). Although the consequences of proteinase secretion during human infections is not precisely known,...

  16. Three distinct secreted aspartyl proteinases in Candida albicans.

    OpenAIRE

    White, T C; Miyasaki, S H; Agabian, N

    1993-01-01

    The secreted aspartyl proteinases of Candida albicans (products of the SAP genes) are thought to contribute to virulence through their effects on Candida adherence, invasion, and pathogenicity. From a single strain of C. albicans (WO-1) which expresses a phenotypic switching system, three secreted aspartyl proteinases have been identified as determined by molecular weight and N-terminal sequence. Each of the three identified proteins represents the mature form of one of three distinct protein...

  17. The induction of proteinases in corn and soybean by anoxia

    International Nuclear Information System (INIS)

    This study characterized the anaerobic changes in proteinase activities in corn and soybean roots and to investigate the possibility that these changes might contribute to the differential anaerobiosis tolerance of the two species. After 24 h of anoxia, crude protein extracts from H60 corn and Keller soybean root tips (10cm) were assayed for proteinase activities at pH range from 4.5 to 9.5. Turnover of aberrant proteins was studied in seedlings labelled with 3H-leucine for 12 h under: (a) puromycin (0.64 mM) in air, (b) ethanol (1%) in air, (c) nitrogen and (d) air. After the treatment, the labelled proteins remaining in roots were determined every 2 h for 6 h. In both corn and soybean, activities of alkali proteinases increased, and activities of acid proteinases declined under anoxia. Neutral proteinases increase in anoxic corn roots, but decline in anoxic soybean roots. The protein turnover rate in corn treated with puromycin, ethanol and nitrogen was much higher than in control roots. The protein turnover rate in soybean roots treated with puromycin, ethanol was similar to the rate of the control. The results indicated that: (a) anoxic corn can degrade aberrant proteins, but anoxic soybean cannot, (b) the degradation of aberrant proteins in anoxic corn is accomplished by neutral proteinases, and (c) the accumulation of aberrant proteins in soybean might contribute to the susceptibility of this species to anoxia

  18. Interaction of new kinase inhibitors cabozantinib and tofacitinib with human serum alpha-1 acid glycoprotein. A comprehensive spectroscopic and molecular Docking approach

    Science.gov (United States)

    Ajmal, Mohammad Rehan; Abdelhameed, Ali Saber; Alam, Parvez; Khan, Rizwan Hasan

    2016-04-01

    In the current study we have investigated the interaction of newly approved kinase inhibitors namely Cabozantinib (CBZ) and Tofacitinib (TFB) with human Alpha-1 acid glycoprotein (AAG) under simulated physiological conditions using fluorescence quenching measurements, circular dichroism, dynamic light scattering and molecular docking methods. CBZ and TFB binds to AAG with significant affinity and the calculated binding constant for the drugs lie in the order of 104. With the increase in temperature the binding constant values decreased for both CBZ and TFB. The fluorescence resonance energy transfer (FRET) from AAG to CBZ and TFB suggested the fluorescence intensity of AAG was quenched by the two studied drugs via the formation of a non-fluorescent complex in the static manner. The molecular distance r value calculated from FRET is around 2 nm for both drugs, fluorescence spectroscopy data was employed for the study of thermodynamic parameters, standard Gibbs free energy change at 300K was calculated as - 5.234 kcal mol- 1 for CBZ-AAG interaction and - 6.237 kcal mol- 1 for TFB-AAG interaction, standard enthalpy change and standard entropy change for CBZ-AAG interaction are - 9.553 kcal mol- 1 and - 14.618 cal mol- 1K- 1 respectively while for AAG-TFB interaction, standard enthalpy and standard entropy change was calculated as 4.019 kcal mol- 1 and 7.206 cal mol- 1K- 1 respectively. Protein binding of the two drugs caused the tertiary structure alterations. Dynamic light scattering measurements demonstrated the reduction in the hydrodynamic radii of the protein. Furthermore molecular docking results suggested the Hydrophobic interaction and hydrogen bonding were the interactive forces in the binding process of CBZ to AAG while in case of TFB only hydrophobic interactions were found to be involved, overlap of the binding site for two studied drugs on the AAG molecule was revealed by docking results.

  19. Effects of age, experience and inter-alpha inhibitor proteins on working memory and neuronal plasticity after neonatal hypoxia-ischemia.

    Science.gov (United States)

    Gaudet, Cynthia M; Lim, Yow-Pin; Stonestreet, Barbara S; Threlkeld, Steven W

    2016-04-01

    Neonatal cerebral hypoxia-ischemia (HI) commonly results in cognitive and sensory impairments. Early behavioral experience has been suggested to improve cognitive and sensory outcomes in children and animal models with perinatal neuropathology. In parallel, we previously showed that treatment with immunomodulator Inter-alpha Inhibitor Proteins (IAIPs) improves cellular and behavioral outcomes in neonatal HI injured rats. The purpose of the current study was to evaluate the influences of early experience and typical maturation in combination with IAIPs treatment on spatial working and reference memory after neonatal HI injury. A second aim was to determine the effects of these variables on hippocampal CA1 neuronal morphology. Subjects were divided into two groups that differed with respect to the time when exposed to eight arm radial water maze testing: Group one was tested as juveniles (early experience, Postnatal day (P) 36-61) and adults (P88-113), and Group two was tested in adulthood only (P88-113; without early experience). Three treatment conditions were included in each experience group (HI+Vehicle, HI+IAIPs, and Sham subjects). Incorrect arm entries (errors) were compared between treatment and experience groups across three error types (reference memory (RM), working memory incorrect (WMI), working memory correct (WMC)). Early experience led to improved working memory performance regardless of treatment. Combining IAIPs intervention with early experience provided a long-term behavioral advantage on the WMI component of the task in HI animals. Anatomically, early experience led to a decrease in the average number of basal dendrites per CA1 pyramidal neuron for IAIP treated subjects and a significant reduction in basal dendritic length in control subjects, highlighting the importance of pruning in typical early life learning. Our results support the hypothesis that early behavioral experience combined with IAIPs improve outcome on a relativity demanding

  20. LMP-420, a small-molecule inhibitor of TNF-alpha, reduces replication of HIV-1 and Mycobacterium tuberculosis in human cells

    Directory of Open Access Journals (Sweden)

    Tangsinmankong Nutthapong

    2006-03-01

    Full Text Available Abstract Background Co-infections of human immunodeficiency virus (HIV and Mycobacterium tuberculosis (M. Tb are steadily increasing and represent a major health crisis in many developing countries. Both pathogens individually stimulate tumor necrosis factor-alpha (TNF release from infected cells and TNF, in turn, enhances the replication of each. A recent report on a Phase I clinical trial suggested that etanercept (soluble TNF receptor might be beneficial in treating HIV/M. Tb co-infected patients. We sought to determine if a small molecule inhibitor of TNF synthesis and activity could block replication of either organism and thus be a potential adjunct to existing drugs targeting these agents. Results LMP-420, a novel anti-inflammatory agent that inhibits TNF, was tested for HIV-1 inhibition both alone and in combination with AZT (3' -azido-3-deoxythymidine. LMP-420 alone was tested against M. Tb. HIV-1 infected human peripheral blood mononuclear cells (PBMC or M. Tb-infected human alveolar macrophages (AM were treated with a single dose of LMP-420 and viral or bacterial replication determined after 7 or 5 days respectively. Viral replication was determined from supernatant p24 levels measured by ELISA. M. Tb replication was determined by bacterial culture of macrophage lysates. LMP-420 alone inhibited HIV replication over 7 days with an IC50 of ~300 nM. Combination of LMP-420 with AZT doubled the level of HIV inhibition observed with AZT alone. LMP-420 alone inhibited the replication of virulent M. Tb by >80%, more than that observed with anti-TNF antibody alone. Conclusion Inhibition of TNF with inexpensive, small-molecule, orally-active drugs may represent a useful strategy for enhancing the activity of currently-available antiviral and anti-M. Tb agents, particularly in those areas where co-infections with these pathogens act to synergistically enhance each other.

  1. Phase I Dose Escalation Study of Sodium Stibogluconate (SSG, a Protein Tyrosine Phosphatase Inhibitor, Combined with Interferon Alpha for Patients with Solid Tumors

    Directory of Open Access Journals (Sweden)

    Aung Naing, James M. Reuben, Luis H. Camacho, Hui Gao, Bang-Ning Lee, Evan N. Cohen, Claire Verschraegen, Saneese Stephen, Joann Aaron, David Hong, Jennifer Wheler, Razelle Kurzrock

    2011-01-01

    Full Text Available Purpose: Sodium stibogluconate (SSG, a small molecule inhibitor of protein tyrosine phosphatases, combined with IFN-alpha-2b (IFN-α inhibited solid tumor cell line growth in vitro. We conducted a phase I clinical trial with SSG plus IFN-α in advanced cancer patients to assess tolerance, maximum tolerated dose (MTD and immune system effects.Experimental Design: SSG was administered intravenously alone for five days of week 1, cycle 1 (21 days per cycle and together with IFN-α 2b s (3 million units sc TIW in week 2, and after a rest during week 3, on a 2-week on/1-week off cycle. SSG dose levels were 400, 600, 900, 1125, and 1350 mg/m2.Results: Twenty-four patients were studied. Common toxicities included asymptomatic elevated serum lipase, thrombocytopenia, fatigue, fever, chills and anemia. The dose-limiting toxicities (DLT were hypokalemia, thrombocytopenia, fatigue, pancreatitis and skin rash. The MTD was 900 mg/m2 SSG and IFN-α, 3 million units TIW. At this dose, patients had a significantly lower number of regulatory T cells (TR Cells (p = 0.012, myeloid dendritic cells (mDC (p = 0.028; higher percentage of natural killer (NK cells that synthesized perforin (p = 0.046 and of plasmacytoid dendritic cells (pDC that secreted IFN-α (p = 0.018 in response to activation through toll-like receptor (TLR 7 and TLR 8 by CL097, the highly water-soluble derivative of the imidazoquinoline compound R848.Conclusions: SSG in combination with IFN-α 2b was well tolerated and augmented cellular immune parameters.

  2. Alpha-1-antitrypsin is produced by human neutrophil granulocytes and their precursors and liberated during granule exocytosis

    DEFF Research Database (Denmark)

    Clemmensen, Stine N; Jacobsen, Lars C; Rørvig, Sara;

    2011-01-01

    stimulation. A1AT is produced at all stages of myeloid maturation in the bone marrow. The production increases as neutrophils enter circulation and increases further upon migration to tissues as observed in skin windows and when blood neutrophils are incubated with granulocyte colony-stimulating factor......Alpha-1-antitrypsin (A1AT) is an important inhibitor of neutrophil proteases including elastase, cathepsin G, and proteinase 3. Transcription profiling data suggest that A1AT is expressed by human neutrophil granulocytes during all developmental stages. A1AT has hitherto only been found associated...... with azurophile granules in neutrophils indicative of A1AT expression being restricted to the promyelocyte stage. We examined the localization and production of A1AT in healthy donor neutrophils and found A1AT to be a constituent of all granule subtypes and to be released from neutrophils following...

  3. C/EBP beta regulation of the tumor necrosis factor alpha gene.

    OpenAIRE

    Pope, R. M.; Leutz, A; Ness, S A

    1994-01-01

    Activated macrophages contribute to chronic inflammation by the secretion of cytokines and proteinases. Tumor necrosis factor alpha (TNF alpha) is particularly important in this process because of its ability to regulate other inflammatory mediators in an autocrine and paracrine fashion. The mechanism(s) responsible for the cell type-specific regulation of TNF alpha is not known. We present data to show that the expression of TNF alpha is regulated by the transcription factor C/EBP beta (NF-I...

  4. Alpha1 and Alpha2 Integrins Mediate Invasive Activity of Mouse Mammary Carcinoma Cells through Regulation of Stromelysin-1 Expression

    Energy Technology Data Exchange (ETDEWEB)

    Lochter, Andre; Navre, Marc; Werb, Zena; Bissell, Mina J

    1998-06-29

    Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits {alpha}6 and {beta}1, but not against {alpha}1 and {alpha}2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against {beta}1, but not against a6 or {alpha}2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against {alpha}1 integrins impaired only cell adhesion to type IV collagen. Antibodies against {alpha}1, {alpha}2, {alpha}6, and {beta}1, but not {alpha}5, integrin subunits reduced invasion of a reconstituted basement membrane. Integrins {alpha}1 and {alpha}2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against {alpha}1 and {alpha}2, but not {alpha}6 and {beta}1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against {alpha}1 and {alpha}2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-{alpha}6 antibodies. Our data indicate that {alpha}1 and {alpha}2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas {alpha}6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.

  5. 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.

  6. A protein structural approach to the solution of biological problems: alpha 1-antitrypsin as a recent example.

    Science.gov (United States)

    Lomas, D A; Carrell, R W

    1993-09-01

    alpha 1-Antitrypsin is a circulating serine proteinase inhibitor that protects the lungs against proteolysis by the enzyme neutrophil elastase. Most northern Europeans have only the normal M form, but some 4% are heterozygotes for the Z deficiency mutant. This mutant is characterized by the substitution of a positively charged lysine residue for a negatively charged glutamic acid at position 342 and results in normal gene translation but reduced protein secretion into the plasma. The plasma levels of antitrypsin in homozygotes are only 15% of normal, the other 85% being retained in the endoplasmic reticulum of the hepatocyte. This review describes the effect of the Z mutation on the structure and function of antitrypsin and illustrates the importance of understanding protein structure in solving the mechanism of Z antitrypsin retention within the liver. We demonstrate that antitrypsin accumulation in the liver results from a unique interaction between antitrypsin molecules. The Z mutation perturbs the gap between the third and fifth strands of the A sheet, allowing the reactive center loop of one molecule to insert into the A sheet of a second. This loop-sheet polymerization results in the formation of chains of protein which form insoluble inclusions in the endoplasmic reticulum, resulting in hepatocellular damage and cirrhosis. In addition, the Z mutation results in a distortion of the circular dichroic spectrum, a rearrangement of the reactive center loop with respect to the A sheet, and a reduction in association rate constant with the cognate proteinase neutrophil elastase. PMID:8214081

  7. 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

  8. Novel prediction method of beer foam stability using protein Z, barley dimeric alpha-amylase inhibitor-1 (BDAI-1) and yeast thioredoxin.

    Science.gov (United States)

    Iimure, Takashi; Takoi, Kiyoshi; Kaneko, Takafumi; Kihara, Makoto; Hayashi, Katsuhiro; Ito, Kazutoshi; Sato, Kazuhiro; Takeda, Kazuyoshi

    2008-09-24

    Foam stability is an important quality trait of beer. Our previous results of two-dimensional gel electrophoresis (2DE) analyses of beer proteins implied a relationship between barley dimeric alpha-amylase inhibitor-1 (BDAI-1) and beer foam stability as judged by the NIBEM-T analyzer. To develop a novel prediction method of beer foam stability under different conditions of barley cultivar and malt modification, multiple linear regression analysis was applied. The spot intensities of major beer proteins on 2DE gel were quantified and used as explanatory variables. The foam stabilities of 25 beer samples each brewed from malt with different malt modification in one of the three cultivars (cultivars A, B, and C) were explained by the spot intensities of BDAI-1 at the 5% significance level ( r = 0.421). Furthermore, two other major protein spots (b0 and b5) were observed on the 2DE gels of Japanese commercial beer samples with different foam stability. Then, multiple regression for foam stability was calculated using these three spot intensities as explanatory variables. As a result, 72.1% of the beer foam stability in 25 beer samples was explained by a novel multiple regression equation calculated using spot b0 and BDAI-1 as positive explanatory variables and spot b5 as a negative variable. To verify the validity of the multiple regression equation and the explanatory variables, the beer foam stability in practical beer samples was analyzed. As a result, 81.5% of the beer foam stability in 10 Japanese commercial beer samples was also explained by using spot b0 and BDAI-1 as positive explanatory variables and spot b5 as a negative variable. Mass spectrometry analyses followed by database searches revealed that protein spots b0 and b5 were identified as protein Z originated from barley and thioredoxin originated from yeast, respectively. These results confirm that BDAI-1 and protein Z are foam-positive factors and identify yeast thioredoxin as a possible novel foam

  9. A Monoclonal Antibody (MCPR3-7) Interfering with the Activity of Proteinase 3 by an Allosteric Mechanism*

    Science.gov (United States)

    Hinkofer, Lisa C.; Seidel, Susanne A. I.; Korkmaz, Brice; Silva, Francisco; Hummel, Amber M.; Braun, Dieter; Jenne, Dieter E.; Specks, Ulrich

    2013-01-01

    Proteinase 3 (PR3) is an abundant serine protease of neutrophil granules and a major target of autoantibodies (PR3 anti-neutrophil cytoplasmic antibodies) in granulomatosis with polyangiitis. Some of the PR3 synthesized by promyelocytes in the bone marrow escapes the targeting to granules and occurs on the plasma membrane of naive and primed neutrophils. This membrane-associated PR3 antigen may represent pro-PR3, mature PR3, or both forms. To discriminate between mature PR3 and its inactive zymogen, which have different conformations, we generated and identified a monoclonal antibody called MCPR3-7. It bound much better to pro-PR3 than to mature PR3. This monoclonal antibody greatly reduced the catalytic activity of mature PR3 toward extended peptide substrates. Using diverse techniques and multiple recombinant PR3 variants, we characterized its binding properties and found that MCPR3-7 preferentially bound to the so-called activation domain of the zymogen and changed the conformation of mature PR3, resulting in impaired catalysis and inactivation by α1-proteinase inhibitor (α1-antitrypsin). Noncovalent as well as covalent complexation between PR3 and α1-proteinase inhibitor was delayed in the presence of MCPR3-7, but cleavage of certain thioester and paranitroanilide substrates with small residues in the P1 position was not inhibited. We conclude that MCPR3-7 reduces PR3 activity by an allosteric mechanism affecting the S1′ pocket and further prime side interactions with substrates. In addition, MCPR3-7 prevents binding of PR3 to cellular membranes. Inhibitory antibodies targeting the activation domain of PR3 could be exploited as highly selective inhibitors of PR3, scavengers, and clearers of the PR3 autoantigen in granulomatosis with polyangiitis. PMID:23902773

  10. 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

  11. Development of a high-throughput screening-compatible assay to identify inhibitors of the CK2alpha/CK2beta interaction

    DEFF Research Database (Denmark)

    Hochscherf, Jennifer; Lindenblatt, Dirk; Steinkrueger, Michaela; Yoo, Eungyoung; Ulucan, Oezlem; Herzig, Stefan; Issinger, Olaf-Georg; Helms, Volkhard; Goetz, Claudia; Neundorf, Ines; Niefind, Karsten; Pietsch, Markus

    crystal structure of the Pc/CK2alpha1-335 complex. The dissociation constants obtained in the fluorescence anisotropy assay for binding of all compounds to human CK2alpha1-335 were validated by isothermal titration calorimetry. I-Pc was identified as the tightest binding ligand with a KD value of 240n...

  12. Proteinase 3 carries small unusual carbohydrates and associates with αlpha-defensins

    DEFF Research Database (Denmark)

    Zoega, Morten; Ravnsborg, Tina; Højrup, Peter; Houen, Gunnar; Schou, Christian

    2012-01-01

    The neutrophil granulocyte is an important first line of defense against intruding pathogens and it contains a range of granules armed with antibacterial peptides and proteins. Proteinase 3 (PR3) is one among several serine proteases of the azurophilic granules in neutrophil granulocytes. Here, we...... characterize the glycosylation of PR3 and its association with antimicrobial human neutrophil peptides (HNPs, α-defensins) and the effect of these on the mechanism of inhibition of the major plasma inhibitor of PR3, α1-antitrypsin. The glycosylation of purified, mature PR3 showed some heterogeneity with...... carbohydrates at Asn 102 and 147 carrying unusual small moieties indicating heavy processing. Mass spectrometric analysis and immuno blotting revealed strong association of highly purified PR3 with α-defensins and oligomers hereof. Irreversible inhibition of PR3 by α1-antitrypsin did not affect its association...

  13. Tesofensine, a novel triple monoamine reuptake inhibitor, induces appetite suppression by indirect stimulation of alpha1 adrenoceptor and dopamine D1 receptor pathways in the diet-induced obese rat

    DEFF Research Database (Denmark)

    Axel, Anne Marie Dixen; Mikkelsen, Jens D; Hansen, Henrik H

    2010-01-01

    Tesofensine is a novel monoamine reuptake inhibitor that inhibits both norepinephrine, 5-HT, and dopamine (DA) reuptake function. Tesofensine is currently in clinical development for the treatment of obesity, however, the pharmacological basis for its strong effect in obesity management is not cl......Tesofensine is a novel monoamine reuptake inhibitor that inhibits both norepinephrine, 5-HT, and dopamine (DA) reuptake function. Tesofensine is currently in clinical development for the treatment of obesity, however, the pharmacological basis for its strong effect in obesity management...... antagonist), or ritanserin (0.03 mg/kg, 5-HT(2A/C) receptor antagonist). Hence, the mechanism underlying the suppression of feeding by tesofensine in the obese rat is dependent on the drug's ability to indirectly stimulate alpha(1) adrenoceptor and DA D(1) receptor function....

  14. The role of proteases, endoplasmic reticulum stress and SERPINA1 heterozygosity in lung disease and alpha-1 anti-trypsin deficiency.

    LENUS (Irish Health Repository)

    Greene, Catherine M

    2012-02-01

    The serine proteinase inhibitor alpha-1 anti-trypsin (AAT) provides an antiprotease protective screen throughout the body. Mutations in the AAT gene (SERPINA1) that lead to deficiency in AAT are associated with chronic obstructive pulmonary diseases. The Z mutation encodes a misfolded variant of AAT that is not secreted effectively and accumulates intracellularly in the endoplasmic reticulum of hepatocytes and other AAT-producing cells. Until recently, it was thought that loss of antiprotease function was the major cause of ZAAT-related lung disease. However, the contribution of gain-of-function effects is now being recognized. Here we describe how both loss- and gain-of-function effects can contribute to ZAAT-related lung disease. In addition, we explore how SERPINA1 heterozygosity could contribute to smoking-induced chronic obstructive pulmonary diseases and consider the consequences.

  15. Multiple pathways for vacuolar sorting of yeast proteinase A

    DEFF Research Database (Denmark)

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

    1996-01-01

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

  16. 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

    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 and......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...... tumour necrosis factor (TNF) transcripts were amplified successfully from heart, but not thyroid, kidney or liver tissue, of a patient who died following rejection of a transplanted heart, and IL-1alpha, but not IL-6 or TNF, transcripts from lung tissue of a six month old baby who died of viral pneumonia...

  17. 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 ...

  18. 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

  19. Suspecting and Testing for Alpha-1 Antitrypsin Deficiency-An Allergist's and/or Immunologist's Perspective.

    Science.gov (United States)

    Craig, Timothy J

    2015-01-01

    Alpha-1 antitrypsin deficiency (AATD) is a hereditary, monogenic disorder with no unique clinical features. AATD can be difficult to diagnose as patients commonly present with respiratory symptoms often mistaken for other respiratory syndromes such as asthma or smoking-related chronic obstructive pulmonary disease. In addition, symptoms related to AATD may also affect other organs, including the liver, vasculature, and skin. The severity of AATD varies between individuals, and in severe cases, the irreversible lung damage can develop into emphysema. Early diagnosis is critical to enable the implementation of lifestyle changes and therapeutic options that can slow further deterioration of pulmonary tissue. Once AATD is suspected, a range of tests are available (serum alpha-1 proteinase inhibitor [A1-PI] level measurement, phenotyping, genotyping, gene sequencing) for confirming AATD. Currently, intravenous infusion of A1-PI is the only therapy that directly addresses the underlying cause of AATD, and has demonstrated efficacy in a recent randomized, placebo-controlled trial. This review discusses the etiology, testing, and management of AATD from the allergist's and/or immunologist's perspective. It aims to raise awareness of the condition among physicians who care for people with obstructive lung disorders and are therefore likely to see patients with obstructive lung disease that may, in fact, prove to be AATD. PMID:26032475

  20. 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.

  1. 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

  2. Response of digestive cysteine proteinases from the Colorado potato beetle (Leptinotarsa decemlineata) and the black vine weevil (Otiorynchus sulcatus) to a recombinant form of human stefin A.

    Science.gov (United States)

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

    1996-01-01

    The effects of the cystatins, human stefin A (HSA) and oryzacystatin I (OCI) on digestive cysteine proteinases of the Colorado potato beetle (CPB), Leptinotarsa decemlineata, and the black vine weevil (BVW), Otiorynchus sulcatus, were assessed using complementary inhibition assays, cystatin-affinity chromatography, and recombinant forms of the two inhibitors. For both insects, either HSA and OCI used in excess (10 or 20 microM) caused partial and stable inhibition of total proteolytic (azocaseinase) activity, but unlike for OCI the HSA-mediated inhibitions were significantly increased when the inhibitor was used in large excess (100 microM). As demonstrated by complementary inhibition assays, this two-step inhibition of the insect proteases by HSA was due to the differential inactivation of two distinct cysteine proteinase populations in either insect extracts, the rapidly (strongly) inhibited population corresponding to the OCI-sensitive fraction. After removing the cystatin-sensitive proteinases from CPB and BVW midgut extracts using OCI- (or HSA-) affinity chromatography, the effects of the insect "non-target" proteases on the structural integrity of the two cystatins were assessed. While OCI remained essentially stable, HSA was subjected to hydrolysis without the accumulation of detectable stable intermediates, suggesting the presence of multiple exposed cleavage sites sensitive to the action of the insect proteases on this cystatin. This apparent susceptibility of HSA to proteolytic cleavage may partially explain its low efficiency to inactivate the insect OCI-insensitive cysteine proteinases when not used in large excess. It could also have major implications when planning the use of cystatin-expressing transgenic plants for the control of coleopteran pests. PMID:8920105

  3. 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.

  4. p21-activated kinase group II small compound inhibitor GNE-2861 perturbs estrogen receptor alpha signaling and restores tamoxifen-sensitivity in breast cancer cells

    OpenAIRE

    Zhuang, Ting; Zhu, Jian; Li, Zhilun; Lorent, Julie; Zhao, Chunyan; Dahlman-Wright, Karin; Strömblad, Staffan

    2015-01-01

    Estrogen receptor alpha (ERα) is highly expressed in most breast cancers. Consequently, ERα modulators, such as tamoxifen, are successful in breast cancer treatment, although tamoxifen resistance is commonly observed. While tamoxifen resistance may be caused by altered ERα signaling, the molecular mechanisms regulating ERα signaling and tamoxifen resistance are not entirely clear. Here, we found that PAK4 expression was consistently correlated to poor patient outcome in endocrine treated and ...

  5. The effects of TNF-alpha and inhibitors of arachidonic acid metabolism on human colon HT-29 cells depend on differentiation status

    Czech Academy of Sciences Publication Activity Database

    Kovaříková, Martina; Hofmanová, Jiřina; Souček, Karel; Kozubík, Alois

    2004-01-01

    Roč. 72, č. 1 (2004), s. 23-31. ISSN 0301-4681 R&D Projects: GA ČR GA525/01/0419; GA ČR GP524/02/P051; GA AV ČR IBS5004009 Institutional research plan: CEZ:AV0Z5004920 Keywords : colon cancer * cell differentiation * TNF-alpha Subject RIV: BO - Biophysics Impact factor: 4.481, year: 2004

  6. The role of proteinase enzymes in the process of conversion of muscle to meat

    OpenAIRE

    Dümen Emek

    2006-01-01

    Post mortem meat tenderization is a complex mechanism and unfortunately it has not been fully identified scientifically. It is known that endogenous proteinases have an important role in this mechanism. Detailed studies are being performed about the destructive effects of lysosomal proteinases and calcium dependent proteinases on the myofibrils and these are most common topics that are being investigated about meat tenderization processes by the scientists. The aim of this paper is to review ...

  7. 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.

  8. Expression of extracellular acid proteinase by proteolytic Candida spp. during experimental infection of oral mucosa.

    OpenAIRE

    Borg, M; Rüchel, R.

    1988-01-01

    We traced an acid proteinase from Candida spp. in the initial stages of the pathogenesis of the mycosis. On infection of human buccal mucosa, proteinase antigens were detected by immuno-scanning electron microscopy on the surface of adhering blastoconidia and invading filamentous cells of C. albicans serotype A. Proteinase antigens were also present on blastoconidia of C. albicans serotype B, but were missing on filamentous cells of this serotype. Proteolytic isolates of C. tropicalis behaved...

  9. 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

  10. A murine ortholog of the human serpin SCCA2 maps to chromosome 1 and inhibits chymotrypsin-like serine proteinases.

    Science.gov (United States)

    Bartuski, A J; Kamachi, Y; Schick, C; Massa, H; Trask, B J; Silverman, G A

    1998-12-01

    Squamous cell carcinoma antigens (SCCA) 1 and 2 are inhibitory members of the high-molecular-weight serine proteinase inhibitor (serpin) family. The biological functions of SCCA1 and 2 are unknown. One approach to determining the function of human proteins is to study orthologs in other species, such as the mouse. The purpose of this study was to determine whether orthologs to human SCCA1 or 2 exist in the mouse. We report the identification and characterization of a novel serpin, sqn5 (now designated Scca2). Comparative amino acid sequence analysis suggests that Scca2 is a member of the ov-serpin subfamily of serpins with highest homology to SCCA1 and SCCA2. Fluorescence in situ hybridization revealed that the Scca2 mapped near Bcl2 on mouse chromosome 1. This region is syntenic with the human locus for SCCA1 and SCCA2 on 18q21.3. The tissue expression patterns as determined by RT-PCR showed a restricted distribution. Scca2 was detected in the lung, thymus, skin, and uterus, as are SCCA1 and SCCA2. Unlike the SCCAs, however, Scca2 was detected also in the gastrointestinal tract. Enzyme-inhibition assays using a GST-SCCA2 fusion protein revealed that SCCA2 inhibited chymotrypsin-like serine proteinases, but not papain-like cysteine proteinases. SCCA2 inhibited CTSG at 1:1 stoichiometry and with a second-order rate constant of kass = 1.7 x 10(5) M-1 s-1. SCCA2 also inhibited human mast cell chymase but the stoichiometry was 2:1, and the second-order rate constant was kass = 0.9 x 10(4) M-1 s-1. This inhibitory profile is identical to that observed for human SCCA2. Based on these findings, Scca2 appears to be the murine ortholog of human SCCA2. PMID:9828132

  11. The role of proteinase enzymes in the process of conversion of muscle to meat

    Directory of Open Access Journals (Sweden)

    Dümen Emek

    2006-01-01

    Full Text Available Post mortem meat tenderization is a complex mechanism and unfortunately it has not been fully identified scientifically. It is known that endogenous proteinases have an important role in this mechanism. Detailed studies are being performed about the destructive effects of lysosomal proteinases and calcium dependent proteinases on the myofibrils and these are most common topics that are being investigated about meat tenderization processes by the scientists. The aim of this paper is to review the role of proteinase enzymes in the process of conversion of muscle to meat. .

  12. Combined effect of an antifeedant alpha-amylase inhibitor and a predator Cheyletus malaccensis in controlling the stored-product mite Acarus siro

    Czech Academy of Sciences Publication Activity Database

    Hubert, J.; Hýblová, Jana; Münzbergová, Zuzana; Pekár, S.; Křížková, I.; Marešová, Lucie; Stejskal, V.; Mareš, Michael

    2007-01-01

    Roč. 32, č. 1 (2007), s. 41-49. ISSN 0307-6962 R&D Projects: GA MŠk(CZ) 1P04OC842.20; GA MŠk(CZ) 1P04OC853.003; GA AV ČR IAA400550617; GA ČR GP203/02/P081 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z60050516 Keywords : alpha-amylase * Acarus siro * Cheyletus malaccensis * digestion protease Subject RIV: CC - Organic Chemistry Impact factor: 1.410, year: 2007

  13. Self-associating poly(ethylene oxide)-b-poly(alpha-cholesteryl carboxylate-epsilon-caprolactone) block copolymer for the solubilization of STAT-3 inhibitor cucurbitacin I.

    Science.gov (United States)

    Mahmud, Abdullah; Patel, Sarthak; Molavi, Ommoleila; Choi, Phillip; Samuel, John; Lavasanifar, Afsaneh

    2009-03-01

    An increase in the degree of chemical compatibility between drug and polymeric structure in the core has been shown to raise the encapsulation efficiency and lower the rate of drug release from polymeric micelles. In this study, to achieve an optimized polymeric micellar delivery system for the solubilization and controlled delivery of cucurbitacin I (CuI), the Flory-Huggins interaction parameter (chi(sc)) between CuI and poly(epsilon-caprolactone) (PCL), poly(alpha-benzylcarboxylate-epsilon-caprolactone) (PBCL) and poly(alpha-cholesteryl carboxylate-epsilon-caprolactone) (PChCL) structures was calculated by group contribution method (GCM) as an indication for the degree of chemical compatibility between different micellar core structures and CuI. The results pointed to a better compatibility between CuI and PChCL core rationalizing the synthesis of self-associating methoxy poly(ethylene oxide)-b-poly(alpha-cholesteryl carboxylate-epsilon-caprolactone) block copolymer (MePEO-b-PChCL). Novel block copolymer of MePEO-b-PChCL was synthesized through, first, preparation of substituted monomer, that is, alpha-cholesteryl carboxylate-epsilon-caprolactone, and further ring opening polymerization of this monomer by methoxy PEO (5000 g mol(-1)) using stannous octoate as catalyst. Synthesized block copolymers were characterized for their molecular weight and polydispersity by (1)H NMR and gel permeation chromatography. Self-assembled MePEO-b-PChCL micelles were characterized for their size, morphology, critical micellar concentration (CMC), capacity for the physical encapsulation of CuI, and mode of CuI release in comparison to MePEO-b-PCL and MePEO-b-PBCL micelles. Overall, the experimental order for the level of CuI encapsulation in different polymeric micellar formulations was consistent with what was predicted by the Flory-Huggins interaction parameter. Although MePEO-b-PChCL micelles exhibited the highest level of CuI loading, this structure did not show any significant

  14. Coefficient Alpha

    OpenAIRE

    Panayiotis Panayides

    2013-01-01

    Heavy reliance on Cronbach’s alpha has been standard practice in many validation studies. However, there seem to be two misconceptions about the interpretation of alpha. First, alpha is mistakenly considered as an indication of unidimensionality and second, that the higher the value of alpha the better. The aim of this study is to clarify these misconceptions with the use of real data from the educational setting. Results showed that high alpha values can be obtained in multidimensional scale...

  15. 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...

  16. 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

  17. [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

  18. 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...

  19. Link between allergic asthma and airway mucosal infection suggested by proteinase-secreting household fungi.

    Science.gov (United States)

    Porter, P; Susarla, S C; Polikepahad, S; Qian, Y; Hampton, J; Kiss, A; Vaidya, S; Sur, S; Ongeri, V; Yang, T; Delclos, G L; Abramson, S; Kheradmand, F; Corry, D B

    2009-11-01

    Active fungal proteinases are powerful allergens that induce experimental allergic lung disease strongly resembling atopic asthma, but the precise relationship between proteinases and asthma remains unknown. Here, we analyzed dust collected from the homes of asthmatic children for the presence and sources of active proteinases to further explore the relationship between active proteinases, atopy, and asthma. Active proteinases were present in all houses and many were derived from fungi, especially Aspergillus niger. Proteinase-active dust extracts were alone insufficient to initiate asthma-like disease in mice, but conidia of A. niger readily established a contained airway mucosal infection, allergic lung disease, and atopy to an innocuous bystander antigen. Proteinase produced by A. niger enhanced fungal clearance from lung and was required for robust allergic disease. Interleukin 13 (IL-13) and IL-5 were required for optimal clearance of lung fungal infection and eosinophils showed potent anti-fungal activity in vitro. Thus, asthma and atopy may both represent a protective response against contained airway infection due to ubiquitous proteinase-producing fungi. PMID:19710638

  20. EPAS1/HIF-2 alpha-mediated downregulation of tissue factor pathway inhibitor leads to a pro-thrombotic potential in endothelial cells.

    Science.gov (United States)

    Stavik, Benedicte; Espada, Sandra; Cui, Xue Yan; Iversen, Nina; Holm, Sverre; Mowinkel, Marie-Christine; Halvorsen, Bente; Skretting, Grethe; Sandset, Per Morten

    2016-04-01

    Neovascularization and hemorrhaging are evident in advanced atherosclerotic plaques due to hypoxic conditions, and mediate the accumulation of metabolic substrates, inflammatory cells, lipids, and other blood born factors inside the plaque. Tissue factor (TF) pathway inhibitor (TFPI) is mainly expressed by endothelial cells and is the endogenous inhibitor of the coagulation activator TF, which together with the downstream product thrombin can drive plaque progression and atherogenesis. We aimed to investigate the effect of hypoxic conditions on endothelial cell expression and activity of TFPI and TF that are important in coagulation initiation. Hypoxia was induced in primary human umbilical vein endothelial cells using chemicals or 1% oxygen tension, and mRNA and protein expressions were measured using qRT-PCR, ELISA, and Western blot analysis. Microscopy of fluorescence-labeled cells was used to visualize cell-associated TFPI. Cell-surface factor Xa (FXa) activity was measured using a two-stage chromogenic substrate method. We found that hypoxia reduced the TFPI mRNA and protein levels and increased the TF mRNA expression in a dose-dependent manner. The effect on TFPI was apparent on all the protein pools of TFPI, i.e., secreted TFPI, cell-surface associated TFPI, and intracellular TFPI, and seemed to be dependent upon hypoxia inducible factor-2α (HIF-2α). An increase in FXa activity was also observed on the endothelial cell surface, reflecting an increase in pro-thrombotic potential of the cells. Our findings indicate that hypoxic conditions may enhance the pro-coagulant activity of endothelial cells, which may promote atherogenesis in addition to clinical events and thus the severity of atherosclerotic disorders. PMID:26826018

  1. 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

  2. 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

  3. 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

  4. KAPASITAS ANTIOKSIDAN DAN INHIBITOR ALFA GLUKOSIDASE EKSTRAK UMBI BAWANG DAYAK [Antioxidant and Alpha-Glucosidase Inhibitory Properties of Bawang Dayak Bulb Extracts

    Directory of Open Access Journals (Sweden)

    Andi Early Febrinda*

    2013-12-01

    Full Text Available Bawang dayak (Eleutherine palmifolia is an indigenous plant in Borneo traditionally used by Dayak tribes to treat any kind of degenerative deseases including diabetes mellitus. The purpose of this research was to measure antioxidant and antidiabetic capacities of water and ethanolic extracts of bawang dayak bulb. Parameters evaluated in this research were phytochemical screening, total phenolics, flavonoid content, DPPH free-radical scavenging activity, and alpha glucosidase inhibiting (AGI activity. The result showed that the total phenolics and flavonoid content in bawang dayak ethanolic extract (217.71 mg GAE/g and 65.35 mg QE/g were higher than that of the water extract (139.93 mg GAE/g and 16.95 mg QE/g. The ethanolic extract also had higher antioxidant and AGI activities (IC50 112 and 241 ppm than that of the water extract (IC50 526 and 505 ppm. In addition, the IC50 values for AGI in bawang dayak ethanolic extract was lower than acarbose which is known as a commercial antidiabetic agent.

  5. Survival and Effectiveness of Tumour Necrosis Factor-alpha Inhibitors in the Treatment of Plaque Psoriasis under Daily Life Conditions: Report from the Psoriasis Registry Austria.

    Science.gov (United States)

    Inzinger, Martin; Wippel-Slupetzky, Katharina; Weger, Wolfgang; Richter, Leo; Mlynek, Alexander; Fleischander, Barbara; Scheurecker, Christine; Sandor, Nikolaus; Mairhofer, Daniela; Sator, Paul G; Moser-Oberthaler, Sabine; Häring, Nina; Viznerova, Petra; Painsi, Clemens; Tanew, Adrian; Ponholzer, Peter; Tatarski, Rafaella; Brenner, Wilhelm; Stingl, Georg; Salmhofer, Wolfgang; Rappersberger, Klemens; Klein, Georg; Saxinger, Werner; Auböck, Josef; Kölli, Claudia; Trautinger, Franz; Steiner, Andreas; Ratzinger, Gudrun; Strohal, Robert; Riedl, Elisabeth; Lange-Asschenfeldt, Bernhard; Pehamberger, Hubert; Volc-Platzer, Beatrix; Selhofer, Sylvia; Legat, Franz Josef; Müllegger, Robert; Reider, Norbert; Schmuth, Matthias; Hintner, Helmut; Hofer, Angelika; Gruber-Wackernagel, Alexandra; Aberer, Werner; Quehenberger, Franz; Wolf, Peter

    2016-03-01

    This retrospective multicentre analysis from the Psoriasis Registry Austria (PsoRA) was conducted to determine drug effectiveness and survival of anti-tumour necrosis factor alpha (anti-TNF-α) agents in patients with moderate-to-severe chronic plaque psoriasis over a 9-year period. Data on 1,019 treatment cycles with adalimumab (n = 460), etanercept (n = 501), and/or infliximab (n = 58) administered to 827 patients (272 women, 555 men) were available for analysis. Compared with etanercept, adalimumab and infliximab showed superior short-term effectiveness. Intention-to-treat-calculated median drug survivals for adalimumab (1,264 days) and etanercept (1,438 days) were similar to each other (p = 0.74), but significantly superior to that of infliximab (477 days) (p = 7.0e-07 vs. adalimumab and p=2.2e-07 vs. etanercept, respectively). Their drug survival rates at 36 months were 51.6%, 56.0%, and 22.6%, respectively. Survival rates correlated significantly with effectiveness for adalimumab and etanercept, but not for infliximab. PMID:26271044

  6. DEVELOPMENT OF ALOPECIA DURING TREATMENT WITH A TUMOR NECROSIS FACTOR-ALPHA INHIBITOR IN A FEMALE PATIENT WITH PSORIATIC ARTHRITS: A CLINICAL CASE

    Directory of Open Access Journals (Sweden)

    R. G. Mukhina

    2016-01-01

    Full Text Available Objective: to describe a case of the total development of alopecia in a female patient with psoriatic arthritis during treatment with a tumor necrosis factor-αlpha (TNF-α inhibitor. Materials and methods. Patient I., aged 36 years has been followed up at the Kazan’ Center of Rheumatic Diseases and Osteoporosis since 1998. At approximately the same time, the patient noted the appearance of skin eruptions behind the ears, on the skin of the scalp. She was examined by a dermatologist who diagnosed psoriasis. In 2005, she was admitted to Kazan’ Rheumatology Center, City Clinical Hospital Seven, for the development of obvious synovitis of the knee joint and for the inefficiency of therapy with nonsteroidal anti-inflammatory drugs and diagnosed with psoriatic arthritis. During the prescribed therapy with methotrexate 10 mg/week, evident menstrual irregularities were observed in the patient who stopped using the drug herself. The second pregnancy occurred in 2008. Articular syndrome progression and eruptive psoriasis were recorded in the lactation period. After lactation cessation in 2009, she was hospitalized again. Her examination revealed high laboratory activity (erythrocyte sedimentation rate, as high as 40 mm/hr; magnetic resonance imaging of the knee joints showed the signs of bilateral synovitis; lumbar spine radiography exhibited grade II sacroiliitis. Leflunomide 20 mg/day was recommended as a basic drug. In 2012, the patient used leflunomide, her condition worsened; joint pain progressed; new joints were involved into the process, and cutaneous manifestations were aggravated. To verify a diagnosis and to choose therapy, the patient was referred to a consultation at the Moscow Research Institute of Rheumatology. Results. In connection with the high activity of the disease and with no response to the performed therapy, it was recommended to initiate therapy with biologics, such as infliximab, the drug of choice. Seven infliximab

  7. PKC-alpha inhibitor MT477 slows tumor growth with minimal toxicity in in vivo model of non-Ras-mutated cancer via induction of apoptosis.

    Science.gov (United States)

    Jasinski, Piotr; Zwolak, Pawel; Terai, Kaoru; Borja-Cacho, Daniel; Dudek, Arkadiusz Z

    2011-02-01

    MT477 is a novel thiopyrano[2,3-c]quinoline with anti-cancer activity. The purpose of the present study was to evaluate different doses and treatment schedules of MT477 in an in vivo xenograft model of non-Ras-mutated cancer, as well as determine its biological effects and mechanism of action via the four conventional PKC isoforms: α, βI, βII, and γ. Here, we show that MT477 inhibits the activity of PKC-α and its downstream targets, ERK1/2 and Akt, before it has an effect on Ras activity. MT477 treatment of cultured H226 cells induced apoptosis and increased focal cell adhesion and formation of actin stress fibers. H226 tumor size in mice continuously treated with intraperitoneal MT477 (1 mg/kg) was 62.1 ± 15.3% smaller than the average tumor size in control mice. Blood serum chemistry revealed minimal toxicity in mice. Taken together, these results support the conclusion that MT477 acts as a direct PKC-α inhibitor in non-Ras mutated cancer, with maximum effectiveness when given in a continuous treatment schedule. PMID:19795097

  8. 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

  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. Partial characterization of hepatopancreatic and extracellular digestive proteinases of wild and cultivated Octopus maya

    OpenAIRE

    Martinez, Romain; R. Santos; A Alvarez; Cuzon, Gerard; L. Arena; M. Mascaro; Pascual, C; Rosas, C

    2011-01-01

    Proteinases from hepatopancreas (HP) and gastric juice (GJ) from wild and cultured red octopus (Octopus maya) were characterized. Hepatopancreas assays revealed optimal activity at pH 4, 9-10 and 10 for wild and pH 3, 8, and 9, for cultured octopuses, for total proteinases, trypsin and chymotrypsin, respectively. In the gastric juice, maximum activity was recorded at pH 6, 8, and 7 for total proteinases, trypsin, and chymotrypsin, respectively for both wild and cultured octopus. A reduction o...

  11. 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...

  12. Trypsin inhibitor from tamarindus indica L. seeds reduces weight gain and food consumption and increases plasmatic cholecystokinin levels

    OpenAIRE

    Joycellane Alline do Nascimento Campos Ribeiro; Alexandre Coellho Serquiz; Priscila Fabíola dos Santos Silva; Patrícia Batista Barra Medeiros Barbosa; Tarcísio Bruno Montenegro Sampaio; Raimundo Fernandes de Araújo Junior; Adeliana Silva de Oliveira; Richele Janaina Araújo Machado; Bruna Leal Lima Maciel; Adriana Ferreira Uchôa; Elizeu Antunes dos Santos; Ana Heloneida de Araújo Morais

    2015-01-01

    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 cond...

  13. Trypsin inhibitor from tamarindus indica L. seeds reduces weight gain and food consumption and increases plasmatic cholecystokinin levels

    OpenAIRE

    do Nascimento Campos Ribeiro, Joycellane Alline; Serquiz, Alexandre Coellho; dos Santos Silva, Priscila Fabíola; Barbosa, Patrícia Batista Barra Medeiros; Sampaio, Tarcísio Bruno Montenegro; de Araújo, Raimundo Fernandes; Oliveira, Adeliana Silva de; Machado, Richele Janaina Araújo; Maciel, Bruna Leal Lima; Uchôa, Adriana Ferreira; dos Santos, Elizeu Antunes; de Araújo Morais, Ana Heloneida

    2015-01-01

    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 cond...

  14. Androgen receptor inactivation contributes to antitumor efficacy of 17{alpha}-hydroxylase/17,20-lyase inhibitor 3beta-hydroxy-17-(1H-benzimidazole-1-yl)androsta-5,16-diene in prostate cancer.

    Science.gov (United States)

    Vasaitis, Tadas; Belosay, Aashvini; Schayowitz, Adam; Khandelwal, Aakanksha; Chopra, Pankaj; Gediya, Lalji K; Guo, Zhiyong; Fang, Hong-Bin; Njar, Vincent C O; Brodie, Angela M H

    2008-08-01

    We previously reported that our novel compound 3beta-hydroxy-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (VN/124-1) is a potent 17alpha-hydroxylase/17,20-lyase (CYP17) inhibitor/antiandrogen and strongly inhibits the formation and proliferation of human prostate cancer LAPC4 tumor xenografts in severe combined immunodeficient mice. In this study, we report that VN/124-1 and other novel CYP17 inhibitors also cause down-regulation of androgen receptor (AR) protein expression in vitro and in vivo. This mechanism of action seems to contribute to their antitumor efficacy. We compared the in vivo antitumor efficacy of VN/124-1 with that of castration and a clinically used antiandrogen, Casodex, and show that VN/124-1 is more potent than castration in the LAPC4 xenograft model. Treatment with VN/124-1 (0.13 mmol/kg twice daily) was also very effective in preventing the formation of LAPC4 tumors (6.94 versus 2410.28 mm(3) in control group). VN/124-1 (0.13 mmol/kg twice daily) and VN/124-1 (0.13 mmol/kg twice daily) + castration induced regression of LAPC4 tumor xenografts by 26.55% and 60.67%, respectively. Treatments with Casodex (0.13 mmol/kg twice daily) or castration caused significant tumor suppression compared with control. Furthermore, treatment with VN/124-1 caused marked down-regulation of AR protein expression, in contrast to treatments with Casodex or castration that caused significant up-regulation of AR protein expression. The results suggest that VN/124-1 acts by several mechanisms (CYP17 inhibition, competitive inhibition, and down-regulation of the AR). These actions contribute to inhibition of the formation of LAPC4 tumors and cause regression of growth of established tumors. VN/124-1 is more efficacious than castration in the LAPC4 xenograft model, suggesting that the compound has potential for the treatment of prostate cancer. PMID:18723482

  15. Porphyromonas gingivalis Cysteine Proteinase Inhibition by κ-Casein Peptides ▿

    OpenAIRE

    Toh, Elena C. Y.; Dashper, Stuart G.; Huq, N. Laila; Attard, Troy J.; O'Brien-Simpson, Neil M.; Chen, Yu-Yen; Cross, Keith J.; Stanton, David P.; Paolini, Rita A.; Eric C. Reynolds

    2010-01-01

    Porphyromonas gingivalis is a major pathogen associated with chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. The Arg-specific (RgpA/B) and Lys-specific (Kgp) cysteine proteinases of P. gingivalis are major virulence factors for the bacterium. In this study κ-casein(109-137) was identified in a chymosin digest of casein as an inhibiting peptide of the P. gingivalis proteinases. The peptide was synthesized and shown to inhibit proteolytic activity associat...

  16. 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...

  17. 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...

  18. A cysteine proteinase in the penetration glands of the cercariae of Cotylurus cornutus (Trematoda, Strigeidae)

    OpenAIRE

    Moczoń, Tadeusz

    2010-01-01

    A cysteine proteinase from the penetration glands of Cotylurus cornutus cercariae was examined with histochemical and biochemical methods. The enzyme hydrolyzed gelatin, azocoll, azocasein, azoalbumin, N-blocked-l-arginine-4-methoxy-2-naphthylamide, and N-blocked-p-nitroanilide, but did not degrade elastin. The metal ion complexane ethylenediamine tetraacetate and the thiol-reducing compound dithioerythritol enhanced the proteinase activity, whereas the thiol-blocking compounds p-hydroxymercu...

  19. Proteinases of Proteus spp.: purification, properties, and detection in urine of infected patients.

    OpenAIRE

    Loomes, L M; Senior, B. W.; Kerr, M A

    1992-01-01

    The proteinases secreted by pathogenic strains of Proteus mirabilis, P. vulgaris biotype 2, P. vulgaris biotype 3, and P. penneri were purified with almost 100% recovery by affinity chromatography on phenyl-Sepharose followed by anion-exchange chromatography. The proteinase purified from the urinary tract pathogen P. mirabilis, which we had previously shown to degrade immunoglobulins A and G, appeared as a composite of a single band and a double band (53 and 50 kDa, respectively) on sodium do...

  20. In Vivo Analysis of Secreted Aspartyl Proteinase Expression in Human Oral Candidiasis

    OpenAIRE

    Naglik, Julian R.; Newport, George; White, Theodore C.; Fernandes-Naglik, Lynette L.; Greenspan, John S.; Greenspan, Deborah; Sweet, Simon P.; Challacombe, Stephen J; Agabian, Nina

    1999-01-01

    Secreted aspartyl proteinases are putative virulence factors in Candida infections. Candida albicans possesses at least nine members of a SAP gene family, all of which have been sequenced. Although the expression of the SAP genes has been extensively characterized under laboratory growth conditions, no studies have analyzed in detail the in vivo expression of these proteinases in human oral colonization and infection. We have developed a reliable and sensitive procedure to detect C. albicans ...

  1. 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...

  2. 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...

  3. 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...

  4. 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.

  5. Proteinase inhibitors in the salivary glands and saliva of the cockroach Nauphoeta cinerea

    Czech Academy of Sciences Publication Activity Database

    Vinokurov, Konstantin; Taranushenko, Yuliya; Kodrík, Dalibor; Elpidina, E. N.; Sehnal, František

    Wroclaw : Wroclaw University, 2007. s. 37-37. [International Conference on Arthropods: Chemical, Physiological and Environmental Aspects /5./. 16.09.2007-21.09.2007, Bialka Tatrzanska] R&D Projects: GA ČR(CZ) GA522/06/1591; GA MŠk 1M06030 Institutional research plan: CEZ:AV0Z50070508 Keywords : Nauphoeta cinerea Subject RIV: ED - Physiology

  6. Secretory leukocyte proteinase inhibitor-competent DNA deposits are potent stimulators of plasmacytoid dendritic cells

    DEFF Research Database (Denmark)

    Skrzeczynska-Moncznik, Joanna; Wlodarczyk, Agnieszka; Zabieglo, Katarzyna; Kapinska-Mrowiecka, Monika; Marewicz, Ewa; Dubin, Adam; Potempa, Jan; Cichy, Joanna

    2012-01-01

    plasmacytoid dendritic cells (pDCs). As the main source of IFN type I (IFNI), pDCs are crucial contributors to inflammatory and likely wound-healing responses associated with psoriasis. The mechanisms responsible for activation of pDCs in psoriatic skin are therefore of substantial interest. We demonstrate...

  7. Low efficiency processing of an insecticidal Nicotiana proteinase inhibitor precursor in Beta vulgaris hairy roots

    Science.gov (United States)

    Assimilation of dietary proteins is critical to insect survival; therefore, inhibition of digestive proteolytic enzymes presents itself as an effective strategy for control of insect pests. To specifically target proteases of several insect pests of sugar beet, Beta vulgaris, we used PCR and gene s...

  8. 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

  9. Gamma irradiation or hydrocortisone treatment of rats increases the proteinase activity associated with histones of thymus nuclei

    International Nuclear Information System (INIS)

    An increase in the activity of histone-associated rat thymus nucleus proteinases specific for histones H2A, H2B and H1 was shown after γ irradiation or hydrocortisone treatment of animals. Histone H1-specific proteinase activity is dependent on DNA and increases in the presence of denatured DNA, whereas proteinases specific for core histones are inhibited in the presence of denatured DNA. The increase in the activity of histone-associated proteinases depends on the radiation dose and the time after irradiation or hydrocortisone injection. In the presence of dithiothreitol and sodium dodecyl sulfate, these proteinases dissociate from histones. It was found by gel electrophoresis that several proteinases of various molecular masses are closely associated with histones obtained from thymus nuclei of irradiated or hydrocortisone-treated rats. 43 refs., 7 figs

  10. 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...

  11. "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.

  12. Alpha fetoprotein

    Science.gov (United States)

    Fetal alpha globulin; AFP ... Greater than normal levels of AFP may be due to: Cancer in testes , ovaries, biliary (liver secretion) tract, stomach, or pancreas Cirrhosis of the liver Liver cancer ...

  13. Corticosteroid-binding globulin cleavage is paradoxically reduced in alpha-1 antitrypsin deficiency: Implications for cortisol homeostasis.

    Science.gov (United States)

    Nenke, Marni A; Holmes, Mark; Rankin, Wayne; Lewis, John G; Torpy, David J

    2016-01-15

    High-affinity corticosteroid-binding globulin (haCBG) is cleaved by neutrophil elastase (NE) resulting in permanent transition to the low cortisol-binding affinity form (laCBG), thereby increasing cortisol availability at inflammatory sites. Alpha-1 antitrypsin (AAT) is the major inhibitor of NE. AAT deficiency (AATD) predisposes patients to early-onset emphysema due to increased proteolytic destruction from the inherent proteinase-antiproteinase imbalance. We hypothesized that AATD may result in increased CBG cleavage in vivo. We collected demographic data and blood samples from 10 patients with AATD and 28 healthy controls measuring total CBG and haCBG levels by parallel in-house ELISAs, as well as AAT, total and free cortisol levels. haCBG was higher (median [range]); 329 [210-551] vs. 250 [175-365] nmol/L; PAAT levels (P<0.05, R=-0.64). Paradoxically, proteolytic cleavage of CBG was reduced in AATD, despite the recognized increase in NE activity. This implies that NE activity is not the mechanism for systemic CBG cleavage in basal, low inflammatory conditions. Relatively low levels of laCBG may have implications for cortisol action in AATD. PMID:26522656

  14. TNF-Alpha Inhibitors for Chronic Urticaria

    DEFF Research Database (Denmark)

    Sand, Freja Lærke; Thomsen, Simon Francis

    2013-01-01

    Patients with severe chronic urticaria may not respond to antihistamines, and other systemic treatment options may either be ineffective or associated with unacceptable side effects. We present data on efficacy and safety of adalimumab and etanercept in 20 adult patients with chronic urticaria...... be effective and relatively safe treatment options in a significant proportion of patients with chronic urticaria who do not respond sufficiently to high-dose antihistamines or in whom standard immunosuppressive drugs are ineffective or associated with unacceptable side effects....

  15. TNF-alpha inhibitors: Current indications

    Directory of Open Access Journals (Sweden)

    Sharma Rashmi

    2007-01-01

    Full Text Available Advances in the DNA hybrid technology led to the development of various biologicals that specifically target TNF-α. There are currently three anti- TNF- α drugs available- etanercept, infliximab and adalimumab. Etanercept is approved by FDA for rheumatoid arthritis (RA in 2000 followed by its approval for ankylosing spondylitis, psoriasis and psoriatic arthritis. Infliximab and adalimumab are approved by FDA in 2002 for RA. Infliximab is also approved for ankylosing spondylitis, psoriasis, psoriatic arthritis, crohn′s disease and ulcerative colitis and adalimumab for psoriatic arthritis and ankylosing spondylitis. Other conditions like bronchial asthma, diabetes mellitus, malignancies, septic shock, behcet′s disease, bullous dermatitis, neutrophilic dermatitis, toxic epidermal necrolysis, systemic vasculitis, pyoderma gangrenosum, pustular dermatitis, alcoholic hepatitis, cerebral malaria, hemolytic uremic syndrome, pre-eclampsia, allograft rejection, uveitis, otitis media, snakebite, erythema nodosum, myelodysplastic syndromes, graft versus host disease, dermatomyositis and polymyositis are the potential targets for anti-TNF- α therapy. There are resent reports of serious infections like tuberculosis with the use of anti-TNF therapy. In developing country like India these agents should be used with strict pharmaco-vigilance and chemo-prophylaxis for tuberculosis.

  16. Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced type I interferon transcription by decreasing interferon regulatory factor 3/7 in protein levels

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Dang; Fang, Liurong; Luo, Rui; Ye, Rui; Fang, Ying; Xie, Lilan; Chen, Huanchun [Division of Animal Infectious Diseases, State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); Xiao, Shaobo, E-mail: shaoboxiao@yahoo.com [Division of Animal Infectious Diseases, State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China)

    2010-08-13

    Research highlights: {yields} FMDV L{sup pro} inhibits poly(I:C)-induced IFN-{alpha}1/{beta} mRNA expression. {yields} L{sup pro} inhibits MDA5-mediated activation of the IFN-{alpha}1/{beta} promoter. {yields} L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes. {yields} L{sup pro} inhibits IFN-{alpha}1/{beta} promoter activation by decreasing IRF-3/7 in protein levels. {yields} The ability to process eIF-4G of L{sup pro} is not necessary to inhibit IFN-{alpha}1/{beta} activation. -- Abstract: The leader proteinase (L{sup pro}) of foot-and-mouth disease virus (FMDV) has been identified as an interferon-{beta} (IFN-{beta}) antagonist that disrupts the integrity of transcription factor nuclear factor {kappa}B (NF-{kappa}B). In this study, we showed that the reduction of double stranded RNA (dsRNA)-induced IFN-{alpha}1/{beta} expression caused by L{sup pro} was also associated with a decrease of interferon regulatory factor 3/7 (IRF-3/7) in protein levels, two critical transcription factors for activation of IFN-{alpha}/{beta}. Furthermore, overexpression of L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes including 2',5'-OAS, ISG54, IP-10, and RANTES. Screening L{sup pro} mutants indicated that the ability to process eIF-4G of L{sup pro} is not required for suppressing dsRNA-induced activation of the IFN-{alpha}1/{beta} promoter and decreasing IRF-3/7 expression. Taken together, our results demonstrate that, in addition to disrupting NF-{kappa}B, L{sup pro} also decreases IRF-3/7 expression to suppress dsRNA-induced type I IFN production, suggesting multiple strategies used by FMDV to counteract the immune response to viral infection.

  17. Double disruption of the proteinase genes, tppA and pepE, increases the production level of human lysozyme by Aspergillus oryzae.

    Science.gov (United States)

    Jin, Feng Jie; Watanabe, Taisuke; Juvvadi, Praveen Rao; Maruyama, Jun-ichi; Arioka, Manabu; Kitamoto, Katsuhiko

    2007-10-01

    In this study, we investigated the effects of proteinase gene disruption on heterologous protein production by Aspergillus oryzae. The human lysozyme (HLY) was selected for recombinant production as a model for the heterologous protein. A tandem HLY construct fused with alpha-amylase (AmyB) was expressed by A. oryzae in which the Kex2 cleavage site was inserted at the upstream of HLY. HLY was successfully processed from AmyB and produced in the medium. We performed a systematic disruption analysis of five proteinase genes (pepA, pepE, alpA, tppA, and palB) in the HLY-producing strain with the adeA selectable marker. Comparative analysis indicated that disruption of the tppA gene encoding a tripeptidyl peptidase resulted in the highest increase (36%) in the HLY production. We further deleted the tppA gene in the pepE or palB disruptant with another selectable marker, argB. Consequently, a double disruption of the tppA and pepE genes led to a 63% increase in the HLY production compared to the control strain. This is the first study to report that the double disruption of the tppA and pepE genes improved the production level of a heterologous protein by filamentous fungi. PMID:17622525

  18. PAR-2, IL-4R, TGF-beta and TNF-alpha in bronchoalveolar lavage distinguishes extrinsic allergic alveolitis from sarcoidosis

    Czech Academy of Sciences Publication Activity Database

    Matěj, R.; Smětáková, M.; Vašáková, M.; Nováková, J.; Šterclová, M.; Kukal, J.; Olejár, Tomáš

    2014-01-01

    Roč. 8, č. 2 (2014), s. 533-538. ISSN 1792-0981 Institutional support: RVO:67985823 Keywords : sarcoidosis * extrinsic allergic alveolitis * interleukin 4 receptor * transforming growth factor beta * tumor necrosis factor alpha * proteinase activated receptor 2 Subject RIV: EC - Immunology Impact factor: 1.269, year: 2014

  19. Effects of alpha-amylase on in vitro growth of Legionella pneumophila.

    OpenAIRE

    Bortner, C A; Miller, R D; Arnold, R.R.

    1983-01-01

    Sterile parotid saliva inhibited growth of Legionella pneumophila on solid media, and the salivary component involved in this inhibition has been shown to be amylase. Disk diffusion and well plate assays were used to study possible mechanisms for this effect. The amylolytic activity of saliva copurified with inhibitory activity, and both activities were sensitive to proteinase K digestion and heat treatment. In addition, purified alpha-amylase from several sources (bacteria, fungi, porcine pa...

  20. 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.

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

    International Nuclear Information System (INIS)

    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 'proC'-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.

  2. Wheat Subtilisin/Chymotrypsin Inhibitor (WSCI) as a scaffold for novel serine protease inhibitors with a given specificity.

    Science.gov (United States)

    Tedeschi, Francesca; Di Maro, Antimo; Facchiano, Angelo; Costantini, Susan; Chambery, Angela; Bruni, Natalia; Capuzzi, Valeria; Ficca, Anna Grazia; Poerio, Elia

    2012-10-30

    WSCI (Wheat Subtilisin/Chymotrypsin Inhibitor) is a small protein belonging to the Potato inhibitor I family exhibiting a high content of essential amino acid. In addition to bacterial subtilisins and mammalian chymotrypsins, WSCI inhibits chymotrypsin-like activities isolated from digestive traits of a number of insect larvae. In vivo, as suggested for many plant proteinase inhibitors, WSCI seems to play a role of natural defence against attacks of pests and pathogens. The functional region of WSCI, containing the inhibitor reactive site (Met48-Glu49), corresponds to an extended flexible loop (Val42-Asp53) whose architecture is somehow stabilized by a number of secondary interactions established with a small β-sheet located underneath. The aim of this study was to employ a WSCI molecule as a stable scaffold to obtain recombinant inhibitors with new acquired anti-proteinase activity or, alternatively, inactive WSCI variants. A gene sequence coding for the native WSCI, along with genes coding for muteins with different specficities, could be exploited to obtain transformed non-food use plants with improved insect resistance. On the other hand, the genetic transformation of cereal plants over-expressing inactive WSCI muteins could represent a possible strategy to improve the nutritional quality of cereal-based foods, without risk of interference with human or animal digestive enzymes. Here, we described the characterization of four muteins containing single/multiple amino acid substitutions at the WSCI reactive site and/or at its proximity. Modalities of interaction of these muteins with proteinases (subtilisin, trypsin and chymotrypsin) were investigated by time course hydrolysis and molecular simulations studies. PMID:23090387

  3. $\\alpha_s$ review (2016)

    CERN Document Server

    d'Enterria, David

    2016-01-01

    The current world-average of the strong coupling at the Z pole mass, $\\alpha_s(m^2_{Z}) = 0.1181 \\pm 0.0013$, is obtained from a comparison of perturbative QCD calculations computed, at least, at next-to-next-to-leading-order accuracy, to a set of 6 groups of experimental observables: (i) lattice QCD "data", (ii) $\\tau$ hadronic decays, (iii) proton structure functions, (iv) event shapes and jet rates in $e^+e^-$ collisions, (v) Z boson hadronic decays, and (vi) top-quark cross sections in p-p collisions. In addition, at least 8 other $\\alpha_s$ extractions, usually with a lower level of theoretical and/or experimental precision today, have been proposed: pion, $\\Upsilon$, W hadronic decays; soft and hard fragmentation functions; jets cross sections in pp, e-p and $\\gamma$-p collisions; and photon F$_2$ structure function in $\\gamma\\,\\gamma$ collisions. These 14 $\\alpha_s$ determinations are reviewed, and the perspectives of reduction of their present uncertainties are discussed.

  4. 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 ...

  5. The anthelmintic efficacy of natural plant cysteine proteinases against Hymenolepis microstoma in vivo.

    Science.gov (United States)

    Mansur, F; Luoga, W; Buttle, D J; Duce, I R; Lowe, A; Behnke, J M

    2015-09-01

    Little is known about the efficacy of cysteine proteinases (CP) as anthelmintics for cestode infections in vivo. Hymenolepis microstoma is a natural parasite of house mice, and provides a convenient model system for the assessment of novel drugs for anthelmintic activity against cestodes. The experiments described in this paper indicate that treatment of H. microstoma infections in mice with the supernatant of papaya latex (PLS), containing active cysteine proteinases, is only minimally efficacious. The statistically significant effects seen on worm burden and biomass showed little evidence of dose dependency, were temporary and the role of cysteine proteinases as the active principles in PLS was not confirmed by specific inhibition with E-64. Worm fecundity was not affected by treatment at the doses used. We conclude also that this in vivo host-parasite system is not sensitive enough to be used reliably for the detection of cestocidal activity of compounds being screened as potential, novel anthelmintics. PMID:25226116

  6. Cleavage of fibrinogen by proteinases elicits allergic responses through Toll-like receptor 4.

    Science.gov (United States)

    Millien, Valentine Ongeri; Lu, Wen; Shaw, Joanne; Yuan, Xiaoyi; Mak, Garbo; Roberts, Luz; Song, Li-Zhen; Knight, J Morgan; Creighton, Chad J; Luong, Amber; Kheradmand, Farrah; Corry, David B

    2013-08-16

    Proteinases and the innate immune receptor Toll-like receptor 4 (TLR4) are essential for expression of allergic inflammation and diseases such as asthma. A mechanism that links these inflammatory mediators is essential for explaining the fundamental basis of allergic disease but has been elusive. Here, we demonstrate that TLR4 is activated by airway proteinase activity to initiate both allergic airway disease and antifungal immunity. These outcomes were induced by proteinase cleavage of the clotting protein fibrinogen, yielding fibrinogen cleavage products that acted as TLR4 ligands on airway epithelial cells and macrophages. Thus, allergic airway inflammation represents an antifungal defensive strategy that is driven by fibrinogen cleavage and TLR4 activation. These findings clarify the molecular basis of allergic disease and suggest new therapeutic strategies. PMID:23950537

  7. 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.

  8. 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.

  9. Chemistry in a microenvironment of low pH, generated with the aid of an immobilized proteinase.

    Science.gov (United States)

    Silver, M S; Haskell, J H

    1990-05-31

    alpha-Chymotrypsin, when immobilized in a collodion membrane, exhibits high activity and remarkable stability. When the immobilized proteinase is exposed to 15 mM ethyl N-acetyl-L-tyrosinate in dilute pH 8.5 buffer it generates a microenvironment which, indicator studies suggest, has an effective pH of approximately 4. The presence of this locally highly acidic region produces a marked increase in the rate of hydrolysis of BzPheal = Ala dissolved in the buffer solution (BzPheal = Ala is the acylhydrazide obtained from the reaction between N-benzoyl-L-phenylalaninal and N-acetyl-L-alanine hydrazide). The observed rate is 10-times greater than in comparable control experiments incorporating a concentrated buffer solution, in which a pH-gradient does not form. The enhanced hydrolysis rate is quantitatively explained if it is attributed to the approximately 20 microliters of pH 4 solution within the membrane. Other experimental data are also consistent with this hypothesis. PMID:2354198

  10. The aspartic proteinase family of three Phytophthora species

    Directory of Open Access Journals (Sweden)

    ten Have Arjen

    2011-05-01

    Full Text Available Abstract Background Phytophthora species are oomycete plant pathogens with such major social and economic impact that genome sequences have been determined for Phytophthora infestans, P. sojae and P. ramorum. Pepsin-like aspartic proteinases (APs are produced in a wide variety of species (from bacteria to humans and contain conserved motifs and landmark residues. APs fulfil critical roles in infectious organisms and their host cells. Annotation of Phytophthora APs would provide invaluable information for studies into their roles in the physiology of Phytophthora species and interactions with their hosts. Results Genomes of Phytophthora infestans, P. sojae and P. ramorum contain 11-12 genes encoding APs. Nine of the original gene models in the P. infestans database and several in P. sojae and P. ramorum (three and four, respectively were erroneous. Gene models were corrected on the basis of EST data, consistent positioning of introns between orthologues and conservation of hallmark motifs. Phylogenetic analysis resolved the Phytophthora APs into 5 clades. Of the 12 sub-families, several contained an unconventional architecture, as they either lacked a signal peptide or a propart region. Remarkably, almost all APs are predicted to be membrane-bound. Conclusions One of the twelve Phytophthora APs is an unprecedented fusion protein with a putative G-protein coupled receptor as the C-terminal partner. The others appear to be related to well-documented enzymes from other species, including a vacuolar enzyme that is encoded in every fungal genome sequenced to date. Unexpectedly, however, the oomycetes were found to have both active and probably-inactive forms of an AP similar to vertebrate BACE, the enzyme responsible for initiating the processing cascade that generates the Aβ peptide central to Alzheimer's Disease. The oomycetes also encode enzymes similar to plasmepsin V, a membrane-bound AP that cleaves effector proteins of the malaria parasite

  11. Involvement of mast cells and proteinase-activated receptor 2 in oxaliplatin-induced mechanical allodynia in mice.

    Science.gov (United States)

    Sakamoto, Ayumi; Andoh, Tsugunobu; Kuraishi, Yasushi

    2016-03-01

    The chemotherapeutic agent oxaliplatin induces neuropathic pain, a dose-limiting side effect, but the underlying mechanisms are not fully understood. Here, we show the potential involvement of cutaneous mast cells in oxaliplatin-induced mechanical allodynia in mice. A single intraperitoneal injection of oxaliplatin induced mechanical allodynia, which peaked on day 10 after injection. Oxaliplatin-induced mechanical allodynia was almost completely prevented by congenital mast cell deficiency. The numbers of total and degranulated mast cells was significantly increased in the skin after oxaliplatin administration. Repetitive topical application of the mast cell stabilizer azelastine hydrochloride inhibited mechanical allodynia and the degranulation of mast cells without affecting the number of mast cells in oxaliplatin-treated mice. The serine protease inhibitor camostat mesilate and the proteinase-activated receptor 2 (PAR2) antagonist FSLLRY-NH2 significantly inhibited oxaliplatin-induced mechanical allodynia. However, it was not inhibited by the H1 histamine receptor antagonist terfenadine. Single oxaliplatin administration increased the activity of cutaneous serine proteases, which was attenuated by camostat and mast cell deficiency. Depletion of the capsaicin-sensitive primary afferents by neonatal capsaicin treatment almost completely prevented oxaliplatin-induced mechanical allodynia, the increase in the number of mast cells, and the activity of cutaneous serine proteases. These results suggest that serine protease(s) released from mast cells and PAR2 are involved in oxaliplatin-induced mechanical allodynia. Therefore, oxaliplatin may indirectly affect the functions of mast cells through its action on capsaicin-sensitive primary afferents. PMID:26804251

  12. The $\\alpha-\\alpha$ fishbone potential revisited

    CERN Document Server

    Day, J P; Elhanafy, M; Smith, E; Woodhouse, R; Papp, Z

    2011-01-01

    The fishbone potential of composite particles simulates the Pauli effect by nonlocal terms. We determine the $\\alpha-\\alpha$ fishbone potential by simultaneously fitting to two-$\\alpha$ resonance energies, experimental phase shifts and three-$\\alpha$ binding energies. We found that essentially a simple gaussian can provide a good description of two-$\\alpha$ and three-$\\alpha$ experimental data without invoking three-body potentials.

  13. Ca2+-dependent proteolytic activity in crab claw muscle: effects of inhibitors and specificity for myofibrillar proteins

    International Nuclear Information System (INIS)

    The claw closer muscle of the Bermuda land crab, Gecarcinus lateralis, undergoes a sequential atrophy and restoration during each molting cycle. The role of Ca2+-dependent proteinases in the turn-over of myofibrillar protein in normal anecdysial (intermolt) claw muscle is described. Crab Ca2+-dependent proteinase degrades the myofibrillar proteins actin, myosin heavy and light chains, paramyosin, tropomyosin, and troponin-T and -I. Ca2+-dependent proteinase activity in whole homogenates and 90,000 x g supernatant fractions from muscle homogenates has been characterized with respect to Ca2+ requirement, substrate specificity, and effects of proteinase inhibitors. The enzyme is inhibited by antipain, leupeptin, E-64, and iodoacetamide; it is insensitive to pepstatin A. The specificity of crab Ca2+-dependent proteinase was examined with native myosin with normal ATPase activity as well as with radioiodinated myosin and radioiodinated hemolymph proteins. Hydrolysis of 125I-myosin occurs in two phases, both Ca2+-dependent: (1) heavy chain (M/sub r/ = 200,000) is cleaved into four large fragments (M/sub r/ = 160,000, 110,000, 73,000, 60,000) and numerous smaller fragments; light chain (M/sub r/ = 18,000) is cleaved to a 15,000-Da fragment; (2) the fragments produced in the first phase are hydrolyzed to acid-soluble material. Although radioiodinated native hemolymph proteins are not susceptible to the Ca2+-dependent proteinase, those denatured by carboxymethylation are degraded. These data suggest that crab Ca2+-dependent proteinase is involved in turnover of myofibrillar protein in normal muscle and muscle undergoing proecdysial atrophy

  14. Alpha One Foundation

    Science.gov (United States)

    ... Tested Find Support Find Doctor What Is Alpha-1? Alpha-1 Antitrypsin Deficiency (Alpha-1) is a ... results for inhaled augmentation More News Our Number One Goal: Find a cure for Alpha-1. Website ...

  15. Alpha-1 Antitrypsin Test

    Science.gov (United States)

    ... helpful? Also known as: Alpha 1 -antitrypsin; A1AT; AAT Formal name: Alpha 1 Antitrypsin; α1-antitrypsin Related ... know? How is it used? Alpha-1 antitrypsin (AAT) testing is used to help diagnose alpha-1 ...

  16. Alpha spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Krueger, Felix; Wilsenach, Heinrich; Zuber, Kai [IKTP TU-Dresden, Dresden (Germany)

    2014-07-01

    Alpha decays from long living isotopes are one of the limiting backgrounds for experiments searching for rare decays with stringent background constrains, such as neutrinoless double beta decay experiments. It is thus very important to accurately measure the half-lives of these decays, in order to properly model their background contribution. Therefore, it is important to be able to measure half-lives from alpha decays of the order of 1 x 10{sup 15} yr. A measurement of such a long lived decay imposes, however, a series of challenges, where the correct discrimination between background and true signal is critical. There is also a more general interest in such long living half-life measurements, as their value depends crucially on the underlying nuclear model. This work proposes a setup to measure long lived alpha decays, based on the design of the Frisch-Grid ionisation chamber. It is shown that the proposed design provides a good separation of signal and background events. It is also demonstrated that, with pulse shape analysis, it is possible to constrain the source position of the decay, further improving the quality of the data. A discussion of the characterisation of the detector is also presented as well as some results obtained with calibration sources.

  17. Alpha spectroscopy

    International Nuclear Information System (INIS)

    Alpha decays from long living isotopes are one of the limiting backgrounds for experiments searching for rare decays with stringent background constrains, such as neutrinoless double beta decay experiments. It is thus very important to accurately measure the half-lives of these decays, in order to properly model their background contribution. Therefore, it is important to be able to measure half-lives from alpha decays of the order of 1 x 1015 yr. A measurement of such a long lived decay imposes, however, a series of challenges, where the correct discrimination between background and true signal is critical. There is also a more general interest in such long living half-life measurements, as their value depends crucially on the underlying nuclear model. This work proposes a setup to measure long lived alpha decays, based on the design of the Frisch-Grid ionisation chamber. It is shown that the proposed design provides a good separation of signal and background events. It is also demonstrated that, with pulse shape analysis, it is possible to constrain the source position of the decay, further improving the quality of the data. A discussion of the characterisation of the detector is also presented as well as some results obtained with calibration sources.

  18. 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

  19. Allicin from garlic strongly inhibits cysteine proteinases and cytopathic effects of Entamoeba histolytica.

    OpenAIRE

    Ankri, S; Miron, T; Rabinkov, A; Wilchek, M; Mirelman, D

    1997-01-01

    The ability of Entamoeba histolytica trophozoites to destroy monolayers of baby hamster kidney cells is inhibited by allicin, one of the active principles of garlic. Cysteine proteinases, an important contributor to amebic virulence, as well as alcohol dehydrogenase, are strongly inhibited by allicin.

  20. 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.

  1. Secreted aspartate proteinases, a virulence factor of Candida spp.: Occurrence among clinical isolates

    Czech Academy of Sciences Publication Activity Database

    Hamal, P.; Dostál, Jiří; Raclavský, V.; Krylová, M.; Pichová, Iva; Hrušková-Heidingsfeldová, Olga

    2004-01-01

    Roč. 49, č. 4 (2004), s. 491-496. ISSN 0015-5632 R&D Projects: GA MZd NI6485 Institutional research plan: CEZ:AV0Z4055905 Keywords : Candida spp. * aspartate proteinases * RAPD typing Subject RIV: CE - Biochemistry Impact factor: 1.034, year: 2004

  2. Serine proteinase of Renibacterium salmoninarum digests a major autologous extracellular and cell-surface protein.

    Science.gov (United States)

    Rockey, D D; Turaga, P S; Wiens, G D; Cook, B A; Kaattari, S L

    1991-10-01

    Renibacterium salmoninarum is a pathogen of salmonid fish that produces large amounts of extracellular protein (ECP) during growth. A proteolytic activity present in ECP at elevated temperatures digested the majority of the proteins in ECP. This digestion was also associated with the loss of ECP immunosuppressive function. In vitro activity of the proteinase in ECP was temperature dependent: it was not detected in an 18-h digest at 4 and 17 degrees C but became readily apparent at 37 degrees C. Proteinase activity was detected at bacterial physiological temperatures (17 degrees C) in reactions incubated for several days. Under these conditions, digestion of partially purified p57, a major constituent of ECP and a major cell-surface protein, yielded a spectrum of breakdown products similar in molecular weight and antigenicity to those in ECP. This pattern of digestion suggests that most of the immunologically related constituents of ECP are p57 and its breakdown products. The proteolytic activity was sensitive to phenylmethylsulfonyl fluoride, methanol, and ethanol and to 10-min incubation at temperatures above 65 degrees C. Electrophoretic analysis of the proteinase on polyacrylamide gels containing proteinase substrates indicated the native form to be 100 kDa or greater. The enzyme was active against selected unrelated substrates only when coincubated with a denaturant (0.1% lauryl sulfate) and (or) a reducing agent (20 mM dithiothreitol). PMID:1777853

  3. Fasciola gigantica cathepsin L proteinase-based synthetic peptide for immunodiagnosis and prevention of sheep fasciolosis

    Czech Academy of Sciences Publication Activity Database

    Ježek, Jan; El Ridi, R.; Salah, M.; Wagih, A.; Aziz, H. W.; Tallima, H.; El Shafie, M. H.; Khalek, T. A.; Ammou, F. F. A.; Strongylis, C.; Moussis, V.; Tsikaris, V.

    2008-01-01

    Roč. 90, č. 3 (2008), s. 349-357. ISSN 0006-3525 Institutional research plan: CEZ:AV0Z40550506 Keywords : cathepsin L proteinase * peptides * sequential oligopeptide carriers * synthetic peptide vaccine * Fasciiola gigantica Subject RIV: CC - Organic Chemistry Impact factor: 2.823, year: 2008

  4. 肿瘤坏死因子α抑制剂单药疗法致感染风险的Meta分析%Risk of infection in monotherapy with tumor necrosis factor alpha inhibitor: a Meta-analysis

    Institute of Scientific and Technical Information of China (English)

    李文思; 吕迁洲; 陈璋璋

    2015-01-01

    Objective To evaluate the risk of infection in monotherapy of tumor necrosis factor alpha (TNF-α) inhibitor.Methods The database of Pubed, Embase, Cochrane library, and Web with Science were searched from the inception to November, 2014.The literatures of randomized controlled trials in English which included reports that only used TNF-α inhibitor (the test group) and placebo or positive controlled drug (the control group) were selected.The methodological quality of the literatures which enrolled into the study were assessed by Jadad scale (inferior quality: < 3 points, high quality: 3-5 points).The software RevMan 5.2 was used for Meta-analysis.The infection rate and the severe infection rate were expressed by relative risk (RR), Peto odds ratio (Peto OR)and 95 % confidence interval (CI).Results A total of 33 trials presented by 32 reports, and 11 819 patients (7 408 cases in the test group using adalimumab or golimumab or infliximab or etanercept, respectively, and 4 411 cases in the control group using placebo, positive control drug such as methotrexate or salazosulfapyridine, respectively) were enrolled into the Meta-analysis.The Jadad scores of the 33 trials were all ≥ 3 points.The results of the Meta-analysis showed that the overall total incidence of infection in patients who used TNF-α inhibitors only was higher than that in the patients who used placebo [33.03% (1 702/5 153) vs.29.53% (873/2 956), RR =1.17, 95% CI: 1.09-1.25, P <0.000 01].There was no significant difference in the overall incidence of infection between the test group and the positive controlled drug group [50.1% (362/723)vs.48.3% (320/662), RR =1.10, 95% CI: 0.90-1.34, P =0.36].There were no significant differences in the incidence of severe infection between the test group and the placebo control group, the positive controlled drug group [1.4% (73/5 067)vs.1.7% (48/2 902), Peto OR =0.90, 95% CI: 0.61-1.32, P =0.46;2.4% (34/1 410) vs.2.8% (28/976), RR =1

  5. 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

  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

    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.

  8. The Role of alpha-Glucosidase in Germinating Barley Grains

    DEFF Research Database (Denmark)

    Stanley, Duncan; Rejzek, Martin; Næsted, Henrik;

    2011-01-01

    chemical-genetic and reverse-genetic approaches. We identified iminosugar inhibitors of a recombinant form of an alpha-glucosidase previously discovered in barley endosperm (ALPHA-GLUCOSIDASE97 [HvAGL97]), and applied four of them to germinating grains. All four decreased the Glc-to-maltose ratio in the...

  9. Human small cell lung cancer NYH cells selected for resistance to the bisdioxopiperazine topoisomerase II catalytic inhibitor ICRF-187 demonstrate a functional R162Q mutation in the Walker A consensus ATP binding domain of the alpha isoform

    DEFF Research Database (Denmark)

    Wessel, I; Jensen, L H; Jensen, P B;

    1999-01-01

    Bisdioxopiperazine drugs such as ICRF-187 are catalytic inhibitors of DNA topoisomerase II, with at least two effects on the enzyme: namely, locking it in a closed-clamp form and inhibiting its ATPase activity. This is in contrast to topoisomerase II poisons as etoposide and amsacrine (m-AMSA), w...

  10. 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...

  11. Relationship between Candida albicans producing proteinase (CAPP) and its environmental pH--comparison with a case of trichophyton mentagrophytes.

    OpenAIRE

    Ko, I. J.; Kim, C. W.; Houh, W.; Tsuboi, R; Matsuda, K; Ogawa, H.

    1987-01-01

    Candida albicans produced a karatinolytic proteinase (KPase) or C. albicans producing proteinase (CAPP), a proposed new term for this enzyme, and Trichophyton mentagrophytes also produced KPase when cultivated in liquid medium containing human stratum corneum (HSC) as the nitrogen source, but were unable to do so when cultivated in sabouraud dextrose broth. Purified KPase from the culture supernatants of C. albicans had a molecular weight of 42,000 and an optimum pH at 4.0. The KPase was foun...

  12. 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...

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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...

  19. 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...

  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. 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...

  2. 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

  3. 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 ...

  4. Luminal trypsin may regulate enterocytes through proteinase-activated receptor 2

    OpenAIRE

    Kong, Wuyi; McConalogue, Karen; Khitin, Lev M.; Hollenberg, Morley D; Payan, Donald G.; Böhm, Stephan K.; Nigel W. Bunnett

    1997-01-01

    Proteinase-activated receptor 2 (PAR-2) is a recently characterized G-protein coupled receptor that is cleaved and activated by pancreatic trypsin. Trypsin is usually considered a digestive enzyme in the intestinal lumen. We examined the hypothesis that trypsin, at concentrations normally present in the lumen of the small intestine, is also a signaling molecule that specifically regulates enterocytes by activating PAR-2. PAR-2 mRNA was highly expressed in the mucosa of the small intestine and...

  5. High-affinity binding of two molecules of cysteine proteinases to low-molecular-weight kininogen.

    OpenAIRE

    Turk, B.; Stoka, V.; Björk, I.; Boudier, C.; Johansson, G.; Dolenc, I.; Colic, A.; Bieth, J. G.; Turk, V.

    1995-01-01

    Human low-molecular-weight kininogen (LK) was shown by fluorescence titration to bind two molecules of cathepsins L and S and papain with high affinity. By contrast, binding of a second molecule of cathepsin H was much weaker. The 2:1 binding stoichiometry was confirmed by titration monitored by loss of enzyme activity and by sedimentation velocity experiments. The kinetics of binding of cathepsins L and S and papain showed the two proteinase binding sites to have association rate constants k...

  6. In vitro differential activity of phospholipases and acid proteinases of clinical isolates of Candida

    Directory of Open Access Journals (Sweden)

    Aurean D'Eça Júnior

    2011-06-01

    Full Text Available INTRODUCTION: Candida yeasts are commensals; however, if the balance of normal flora is disrupted or the immune defenses are compromised, Candida species can cause disease manifestations. Several attributes contribute to the virulence and pathogenicity of Candida, including the production of extracellular hydrolytic enzymes, particularly phospholipase and proteinase. This study aimed to investigate the in vitro activity of phospholipases and acid proteinases in clinical isolates of Candida spp. METHODS: Eighty-two isolates from hospitalized patients collected from various sites of origin were analyzed. Phospholipase production was performed in egg yolk medium and the production of proteinase was verified in a medium containing bovine serum albumin. The study was performed in triplicate. RESULTS: Fifty-six (68.3% of isolates tested were phospholipase positive and 16 (44.4% were positive for proteinase activity. C. tropicalis was the species with the highest number of positive isolates for phospholipase (91.7%. Statistically significant differences were observed in relation to production of phospholipases among species (p<0,0001 and among the strains from different sites of origin (p=0.014. Regarding the production of acid protease, the isolates of C. parapsilosis tested presented a larger number of producers (69.2%. Among the species analyzed, the percentage of protease producing isolates did not differ statistically (χ2=1.9 p=0.5901 (χ2=1.9 p=0.5901. CONCLUSIONS: The majority of C. non-albicans and all C. albicans isolates were great producers of hydrolytic enzymes and, consequently, might be able to cause infection under favorable conditions.

  7. In vitro evaluation of proteinase, phospholipase and haemolysin activities of Candida species isolated from clinical specimens

    OpenAIRE

    Sachin C.D; Ruchi K; Santosh S.

    2012-01-01

    Background: Virulence attributes of Candida species include adherence to host tissues, morphological changes and secretion of extracellular hydrolytic enzymes. These enzymes play pivotal roles in pathogenicity of candida infection. Aim: The present study aimed to determine phospholipase, proteinase and haemolysin activities in Candida species isolated from various clinical samples. Material and Method: A total of 110 Candida species isolated from various clinical specimens were identified up ...

  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. [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

  10. Aggregation properties of whey protein hydrolysates generated with Bacillus licheniformis proteinase activities.

    Science.gov (United States)

    Spellman, David; Kenny, Patricia; O'Cuinn, Gerard; FitzGerald, Richard J

    2005-02-23

    Hydrolysis of whey protein concentrate (WPC) with Alcalase 2.4 L, a Bacillus licheniformis proteinase preparation, induces gelation. The aggregation behavior of WPC hydrolysates generated with Alcalase and Prolyve 1000, a Bacillus licheniformis proteinase that did not induce gelation, were studied by turbidity and particle size analysis. With the use of synthetic peptide substrates, it was shown that Alcalase contains a glutamyl endopeptidase (GE) activity not present in Prolyve. Comparison of the aggregation behavior of WPC hydrolysates generated with Alcalase, Prolyve, and combinations of Prolyve with a GE activity isolated from Alcalase showed that GE was responsible for the observed enzyme-induced peptide aggregation in Alcalase hydrolysates. Hydrolysates generated with Prolyve, having a degree of hydrolysis (DH) of 11.8% and 10.4% of peptide material greater than 10 kDa, could be induced to aggregate by the addition of GE. These results emphasize the contribution of enzyme specificity to the physicochemical and functional characteristics of proteinase hydrolysates of WPC. PMID:15713050

  11. The anthelmintic efficacy of natural plant cysteine proteinases against the rat tapeworm Hymenolepis diminuta in vivo.

    Science.gov (United States)

    Mansur, F; Luoga, W; Buttle, D J; Duce, I R; Lowe, A; Behnke, J M

    2016-05-01

    Hymenolepis diminuta is a natural parasite of the common brown rat Rattus norvegicus, and provides a convenient model system for the assessment of the anthelmintic activity of novel drugs against cestodes. The experiments described in this paper indicate that treatment of rats infected with H. diminuta with a supernatant extract of papaya latex, containing a mixture of four cysteine proteinases, was moderately efficacious, resulting in a significant, but relatively small, reduction in worm burden and biomass. However, faecal egg output was not affected by treatment. In our experiments these effects were only partially dose-dependent, although specific inhibition by E-64 confirmed the role of cysteine proteinases as the active principles in papaya latex affecting worm growth but not statistically reducing worm burden. Data collected for a further 7 days after treatment indicated that the effects of papaya latex supernatant on worm loss and on worm growth were not enhanced. Our findings provide a starting point for further refinement in formulation and delivery, or assessment of alternative natural plant-derived cysteine proteinases in efforts to develop these naturally occurring enzymes into broad-spectrum anthelmintics, with efficacy against cestodes as well as nematodes. PMID:25761568

  12. In vitro anthelmintic effects of cysteine proteinases from plants against intestinal helminths of rodents.

    Science.gov (United States)

    Stepek, Gillian; Lowe, Ann E; Buttle, David J; Duce, Ian R; Behnke, Jerzy M

    2007-12-01

    Infections with gastrointestinal (GI) nematodes are amongst the most prevalent worldwide, especially in tropical climates. Control of these infections is primarily through treatment with anthelmintic drugs, but the rapid development of resistance to all the currently available classes of anthelmintic means that alternative treatments are urgently required. Cysteine proteinases from plants such as papaya, pineapple and fig are known to be substantially effective against three rodent GI nematodes, Heligmosomoides polygyrus, Trichuris muris and Protospirura muricola, both in vitro and in vivo. Here, based on in vitro motility assays and scanning electron microscopy, we extend these earlier reports, demonstrating the potency of this anthelmintic effect of plant cysteine proteinases against two GI helminths from different taxonomic groups - the canine hookworm, Ancylostoma ceylanicum, and the rodent cestode, Rodentolepis microstoma. In the case of hookworms, a mechanism of action targeting the surface layers of the cuticle indistinguishable from that reported earlier appears to be involved, and in the case of cestodes, the surface of the tegumental layers was also the principal location of damage. Hence, plant cysteine proteinases have a broad spectrum of activity against intestinal helminths (both nematodes and cestodes), a quality that reinforces their suitability for development as a much-needed novel treatment against GI helminths of humans and livestock. PMID:18005461

  13. 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.

  14. 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

  15. The high-resolution crystal structure of phosphatidylinositol 4-kinase II beta and the crystal structure of phosphatidylinositol 4-kinase II alpha containing a nucleoside analogue provide a structural basis for isoform-specific inhibitor design

    Czech Academy of Sciences Publication Activity Database

    Klíma, Martin; Bäumlová, Adriana; Chalupská, Dominika; Hřebabecký, Hubert; Dejmek, Milan; Nencka, Radim; Bouřa, Evžen

    2015-01-01

    Roč. 71, č. 7 (2015), s. 1555-1563. ISSN 1399-0047 R&D Projects: GA ČR GJ15-21030Y; GA ČR GA15-09310S; GA MŠk LO1302 EU Projects: European Commission(XE) 333916 - STARPI4K Institutional support: RVO:61388963 Keywords : phosphatidyl inositol * kinase * crystal structure * ATP * inhibitor Subject RIV: CE - Biochemistry Impact factor: 2.674, year: 2014

  16. Participation of deoxyribonucleic acid polymerase alpha in amplification of ribosomal deoxyribonucleic acid in Xenopus laevis.

    OpenAIRE

    Zimmermann, W.; Weissbach, A

    1981-01-01

    Aphidicolin, a known inhibitor of eucaryotic deoxyribonucleic acid (DNA) polymerase alpha, efficiently inhibited amplification of ribosomal DNA during oogenesis in Xenopus laevis. DNA polymerase alpha, but not DNA polymerase gamma, as isolated from ovaries, was sensitive to aphidicolin. DNA polymerase beta was not detectable in Xenopus ovary extracts. Therefore, DNA polymerase alpha plays a major role in ribosomal ribonucleic acid gene amplification.

  17. 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...

  18. 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

  19. [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. PMID:27169244

  20. Predictive role of HER2/neu, topoisomerase-II-alpha, and tissue inhibitor of metalloproteinases (TIMP-1) for response to adjuvant taxane-based chemotherapy in patients with intermediate-risk breast cancer

    DEFF Research Database (Denmark)

    Erber, Ramona; Gluz, Oleg; Brünner, Nils;

    2015-01-01

    this study, we investigate a large cohort of patients with intermediate-risk BC treated within the WSG EC-DOC Trial for the predictive impact of topoisomerase-II-alpha, HER2/neu, and TIMP-1. Tumor tissue was available in a representative cohort of 772 cases of the WSG EC-DOC Trial collective which......Taxane-anthracycline-based adjuvant chemotherapy is standard of care in patients with node-positive breast cancer (BC) but is also associated with severe side effects and significant costs. It is yet unclear, which biomarkers would predict benefit from taxanes and/or general chemoresistance. In...... status, high TIMP-1 and low topoisomerase-II-alpha protein expression. Significant prognostic factors in multivariate analysis were EC-Doc therapy (HR = 0.61; 95 %CI 0.38-0.986), age <50 years old (HR = 1.682; 95 %CI 1.025-2.579), centrally assessed grade 3 (HR = 4.657; 95 %CI 1.809-11.989), and high Ki...

  1. Pattern secretion of matrix Metalloproteinases and their biological tissue inhibitors by human glomerular mesangial cells in culture

    OpenAIRE

    "Hosseini R; Hampel G; Jung K

    2001-01-01

    The glomerular mesangial cells (GMC) play a central role in the synthesis and turnover of the glomerular mesangial matrix. The breakdown of the matrix likely depends on the balance between of a variety of proteinases including matrix metalloproteinases and their biological inhibitors secreted by the GMC, and any disturbance in the balance may result in appearance of various pathological states such as glomerulosclerosis. We therefore studied pattern secretion of matrix metalloproteinases (MMP...

  2. Synergistic apoptosis induction in leukemic cells by the phosphatase inhibitor salubrinal and proteasome inhibitors.

    Directory of Open Access Journals (Sweden)

    Hannes C A Drexler

    Full Text Available BACKGROUND: Cells adapt to endoplasmic reticulum (ER-stress by arresting global protein synthesis while simultaneously activating specific transcription factors and their downstream targets. These processes are mediated in part by the phosphorylation-dependent inactivation of the translation initiation factor eIF2alpha. Following restoration of homeostasis protein synthesis is resumed when the serine/threonine-protein phosphatase PP1 dephosphorylates and reactivates eIF2alpha. Proteasome inhibitors, used to treat multiple myeloma patients evoke ER-stress and apoptosis by blocking the ER-associated degradation of misfolded proteins (ERAD, however, the role of eIF2alpha phosphorylation in leukemic cells under conditions of proteasome inhibitor-mediated ER stress is currently unclear. METHODOLOGY AND PRINCIPAL FINDINGS: Bcr-Abl-positive and negative leukemic cell lines were used to investigate the functional implications of PP1-related phosphatase activities on eIF2alpha phosphorylation in proteasome inhibitor-mediated ER stress and apoptosis. Rather unexpectedly, salubrinal, a recently identified PP1 inhibitor capable to protect against ER stress in various model systems, strongly synergized with proteasome inhibitors to augment apoptotic death of different leukemic cell lines. Salubrinal treatment did not affect the phosphorlyation status of eIF2alpha. Furthermore, the proapoptotic effect of salubrinal occurred independently from the chemical nature of the proteasome inhibitor, was recapitulated by a second unrelated phosphatase inhibitor and was unaffected by overexpression of a dominant negative eIF2alpha S51A variant that can not be phosphorylated. Salubrinal further aggravated ER-stress and proteotoxicity inflicted by the proteasome inhibitors on the leukemic cells since characteristic ER stress responses, such as ATF4 and CHOP synthesis, XBP1 splicing, activation of MAP kinases and eventually apoptosis were efficiently abrogated by the

  3. Corrosion inhibitors

    International Nuclear Information System (INIS)

    In this paper, we briefly describe the characteristics, cost and electrochemical nature of the corrosion phenomena as well as some of the technologies that are currently employed to minimize its effect. The main subject of the paper however, deals with the description, classification and mechanism of protection of the so-called corrosion inhibitors. Examples of the use of these substances in different aggressive environments are also presented as means to show that these compounds, or their combination, can in fact be used as excellent and relatively cheap technologies to control the corrosion of some metals. In the last part of the paper, the most commonly used techniques to evaluate the efficiency and performance of corrosion inhibitors are presented as well as some criteria to make a careful and proper selection of a corrosion inhibitor technology in a given situation. (Author) 151 refs

  4. Synthetic peptides and fluorogenic substrates related to the reactive site sequence of Kunitz-type inhibitors isolated from Bauhinia: interaction with human plasma kallikrein.

    Science.gov (United States)

    Oliva, M L; Santomauro-Vaz, E M; Andrade, S A; Juliano, M A; Pott, V J; Sampaio, M U; Sampaio, C A

    2001-01-01

    We have previously described Kunitz-type serine proteinase inhibitors purified from Bauhinia seeds. Human plasma kallikrein shows different susceptibility to those inhibitors. In this communication, we describe the interaction of human plasma kallikrein with fluorogenic and non-fluorogenic peptides based on the Bauhinia inhibitors' reactive site. The hydrolysis of the substrate based on the B. variegata inhibitor reactive site sequence, Abz-VVISALPRSVFIQ-EDDnp (Km 1.42 microM, kcat 0.06 s(-1), and kcat/Km 4.23 x 10(4) M(-1) s(-1)), is more favorable than that of Abz-VMIAALPRTMFIQ-EDDnp, related to the B. ungulata sequence (Km 0.43 microM, kcat 0.00017 s(-1), and kcat/Km 3.9 x 10(2) M(-1) s(-1)). Human plasma kallikrein does not hydrolyze the substrates Abz-RPGLPVRFESPL-EDDnp and Abz-FESPLRINIIKE-EDDnp based on the B. bauhinioides inhibitor reactive site sequence, the most effective inhibitor of the enzyme. These peptides are competitive inhibitors with Ki values in the nM range. The synthetic peptide containing 19 amino acids based on the B. bauhinioides inhibitor reactive site (RPGLPVRFESPL) is poorly cleaved by kallikrein. The given substrates are highly specific for trypsin and chymotrypsin hydrolysis. Other serine proteinases such as factor Xa, factor XII, thrombin and plasmin do not hydrolyze B. bauhinioides inhibitor related substrates. PMID:11258660

  5. Curcumin, a Potential Inhibitor of Up-regulation of TNF-alpha and IL-6 Induced by Palmitate in 3T3-L1 Adipocytes through NF-kappaB and JNK Pathway

    Institute of Scientific and Technical Information of China (English)

    SHAO-LING WANG; YING EI; YING WEN; YAN-FENG CHEN; LI-XIN NA; SONG-TAO LI; CHANG-HAO SUN

    2009-01-01

    Objective To investigate the attenuating effect of curcumin, an anti-inflammatory compound derived from dietary spice turmeric (Curcuma longa) on the pro-inflammatory insulin-resistant state in 3T3-L1 adipocytes. Methods Glucose uptake rate was determined with the [3H] 2-deoxyglucose uptake method. Expressions of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured by quantitative RT-PCR analysis and ELISA. Nuclear transcription factor kappaB p65 (NF-κB p65) and mitogen-activated protein kinase (MAPKs) were detected by Western blot assay. Results The basal glucose uptake was not altered, and curcumin increased the insulin-stimulated glucose uptake in 3T3-L1 cells. Curcumin suppressed the transcription and secretion of TNF-α and IL-6 induced by palmitate in a concentration-dependent manner. Palmitate induced nuclear translocation of NF-kB. The activities of Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase1/2 (ERK1/2) and p38MAPK decreased in the presence of curcumin. Moreover, pretreatment with SP600125 (inhibitor of JNK) instead of PD98059 or SB203580 (inhibitor of ERK 1/2 or p38MAPK, respectively) decreased the up-regulation of TNF-α induced by palmitate. Conclusion Curcumin reverses palmitate-induced insulin resistance state in 3T3-L1 adipocytes through the NF-kB and JNK pathway.

  6. Traditionally used plants in diabetes therapy: phytotherapeutics as inhibitors of alpha-amylase activity Plantas tradicionalmente utilizadas na terapia da diabetes: fitomedicamentos como inibidores da atividade alfa-amilase

    Directory of Open Access Journals (Sweden)

    Ingrid Funke

    2006-03-01

    Full Text Available Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycaemia. There are many and diverse therapeutic strategies in the management of Type 2 diabetes. The inhibition of alpha-amylase activity is only one possibility to lower postprandial blood glucose levels. In our in-vitro studies we could demonstrate that different plants, mostly traditionally used in common diabetic therapy in Africa or Europe, are able to inhibit alpha-amylase, which is responsible for the breakdown of oligosaccharides into monosaccharides which are absorbed. An inhibition of alpha-amylase activity of 90% was seen with the extract of the leaves of Tamarindus indica. To quantify inhibtion rates, acarbose was used (IC50: 23.2 µM. Highest inhibition level of acarbose in our testmodel was about 85%. Additionally tests with pure polyphenolic compounds might explain the biological activity of the selected plants.Diabetes mellitus é uma desordem metabólica caracterizada pela hiperglicemia crônica. Existem diversas estratégias terapêuticas no tratamento da diabetes Tipo 2. A inibição da atividade da a-amilase é apenas uma possibilidade de reduzir os níveis de glicose posprandiais. Nos nossos estudos in vitro pudemos demonstrar que diferentes plantas, especialmente as tradicionalmente usadas em terapia comum de diabetes na África ou Europa, são capazes de inibir a a-amilase, a qual é responsável pela quebra dos oligossacarídeos em monossacarídeos, os quais são absorvidos. Uma inibição da atividade da a-amilase da ordem de 90% foi observada com o extrato das folhas de Tamarindus indica. Para quantificar os graus de inibição, acarbose foi usada (IC50: 23,2 mM. O maior grau de inibição de acarbose no nosso modelo de teste foi de cerca de 85%. Adicionalmente testes com compostos polifenólicos puros poderão explicar a atividade biológica das plantas selecionadas.

  7. 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

  8. A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: Activation of proteinase-activated receptor 1 and epidermal growth factor receptor

    International Nuclear Information System (INIS)

    Biological functions of tissue kallikrein (TK, KLK1) are mainly mediated by kinin generation and subsequent kinin B2 receptor activation. In this study, we investigated the potential role of TK and its signaling pathways in cultured human keratinocyte migration and in a rat skin wound healing model. Herein, we show that TK promoted cell migration and proliferation in a concentration- and time-dependent manner. Inactive TK or kinin had no significant effect on cell migration. Interestingly, cell migration induced by active TK was not blocked by icatibant or L-NAME, indicating an event independent of kinin B2 receptor and nitric oxide formation. TK's stimulatory effect on cell migration was inhibited by small interfering RNA for proteinase-activated receptor 1 (PAR1), and by PAR1 inhibitor. TK-induced migration was associated with increased phosphorylation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), which was blocked by inhibition of protein kinase C (PKC), Src, EGFR and ERK. TK-induced cell migration and EGFR phosphorylation were blocked by metalloproteinase (MMP) inhibitor, heparin, and antibodies against EGFR external domain, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR). Local application of TK promoted skin wound healing in rats, whereas icatibant and EGFR inhibitor blocked TK's effect. Skin wound healing was further delayed by aprotinin and neutralizing TK antibody. This study demonstrates a novel role of TK in skin wound healing and uncovers new signaling pathways mediated by TK in promoting keratinocyte migration through activation of the PAR1-PKC-Src-MMP pathway and HB-EGF/AR shedding-dependent EGFR transactivation.

  9. 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.

  10. 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

  11. 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.

  12. Ab initio alpha-alpha scattering.

    Science.gov (United States)

    Elhatisari, Serdar; Lee, Dean; Rupak, Gautam; Epelbaum, Evgeny; Krebs, Hermann; Lähde, Timo A; Luu, Thomas; Meißner, Ulf-G

    2015-12-01

    Processes such as the scattering of alpha particles ((4)He), the triple-alpha reaction, and alpha capture play a major role in stellar nucleosynthesis. In particular, alpha capture on carbon determines the ratio of carbon to oxygen during helium burning, and affects subsequent carbon, neon, oxygen, and silicon burning stages. It also substantially affects models of thermonuclear type Ia supernovae, owing to carbon detonation in accreting carbon-oxygen white-dwarf stars. In these reactions, the accurate calculation of the elastic scattering of alpha particles and alpha-like nuclei--nuclei with even and equal numbers of protons and neutrons--is important for understanding background and resonant scattering contributions. First-principles calculations of processes involving alpha particles and alpha-like nuclei have so far been impractical, owing to the exponential growth of the number of computational operations with the number of particles. Here we describe an ab initio calculation of alpha-alpha scattering that uses lattice Monte Carlo simulations. We use lattice effective field theory to describe the low-energy interactions of protons and neutrons, and apply a technique called the 'adiabatic projection method' to reduce the eight-body system to a two-cluster system. We take advantage of the computational efficiency and the more favourable scaling with system size of auxiliary-field Monte Carlo simulations to compute an ab initio effective Hamiltonian for the two clusters. We find promising agreement between lattice results and experimental phase shifts for s-wave and d-wave scattering. The approximately quadratic scaling of computational operations with particle number suggests that it should be possible to compute alpha scattering and capture on carbon and oxygen in the near future. The methods described here can be applied to ultracold atomic few-body systems as well as to hadronic systems using lattice quantum chromodynamics to describe the interactions of

  13. Ab initio alpha-alpha scattering

    Science.gov (United States)

    Elhatisari, Serdar; Lee, Dean; Rupak, Gautam; Epelbaum, Evgeny; Krebs, Hermann; Lähde, Timo A.; Luu, Thomas; Meißner, Ulf-G.

    2015-12-01

    Processes such as the scattering of alpha particles (4He), the triple-alpha reaction, and alpha capture play a major role in stellar nucleosynthesis. In particular, alpha capture on carbon determines the ratio of carbon to oxygen during helium burning, and affects subsequent carbon, neon, oxygen, and silicon burning stages. It also substantially affects models of thermonuclear type Ia supernovae, owing to carbon detonation in accreting carbon-oxygen white-dwarf stars. In these reactions, the accurate calculation of the elastic scattering of alpha particles and alpha-like nuclei—nuclei with even and equal numbers of protons and neutrons—is important for understanding background and resonant scattering contributions. First-principles calculations of processes involving alpha particles and alpha-like nuclei have so far been impractical, owing to the exponential growth of the number of computational operations with the number of particles. Here we describe an ab initio calculation of alpha-alpha scattering that uses lattice Monte Carlo simulations. We use lattice effective field theory to describe the low-energy interactions of protons and neutrons, and apply a technique called the ‘adiabatic projection method’ to reduce the eight-body system to a two-cluster system. We take advantage of the computational efficiency and the more favourable scaling with system size of auxiliary-field Monte Carlo simulations to compute an ab initio effective Hamiltonian for the two clusters. We find promising agreement between lattice results and experimental phase shifts for s-wave and d-wave scattering. The approximately quadratic scaling of computational operations with particle number suggests that it should be possible to compute alpha scattering and capture on carbon and oxygen in the near future. The methods described here can be applied to ultracold atomic few-body systems as well as to hadronic systems using lattice quantum chromodynamics to describe the interactions of

  14. 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, ...

  15. Fibrinogen degradation by two neutral granulocyte proteinases. Influence of calcium on the generation of fibrinogen degradation products with anticlotting properties.

    Science.gov (United States)

    Bingenhkeimer, C; Gramse, M; Egbring, R; Havemann, K

    1981-07-01

    Degradation of human fibrinogen by elastase-like proteinase, chymotrypsin-like proteinase and plasmin, was done in the presence and absence of calcium ions, respectively. The resulting fibrinogen degradation products were tested for their coagulant and anti-coagulant properties. The results show that 1. fibrinogenolysis is delayed in the presence of calcium ions. Higher enzyme concentrations are required to get unclottable split products when calcium ions are present. 2. The fibrinogen fragments obtained in the presence of calcium are different in their molecular weights and anticoagulant activities compared to those obtained in the absence of calcium ions. This effect of calcium is most striking during fibrinogen cleavage by chymotrypsin-like proteinase. Elastase and plasmin-induced fibrinogenolysis was substantially influenced by calcium only at a late degradation stage. PMID:6456216

  16. 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.

  17. S-glutathionylated serine proteinase inhibitors as plasma biomarkers in assessing response to redox-modulating drugs.

    Science.gov (United States)

    Grek, Christina L; Townsend, Danyelle M; Uys, Joachim D; Manevich, Yefim; Coker, Woodrow J; Pazoles, Christopher J; Tew, Kenneth D

    2012-05-01

    Many cancer drugs impact cancer cell redox regulatory mechanisms and disrupt redox homeostasis. Pharmacodynamic biomarkers that measure therapeutic efficacy or toxicity could improve patient management. Using immunoblot analyses and mass spectrometry, we identified that serpins A1 and A3 were S-glutathionylated in a dose- and time-dependent manner following treatment of mice with drugs that alter reactive oxygen or nitrogen species. Tandem mass spectrometry analyses identified Cys(256) of serpin A1 and Cys(263) of serpin A3 as the S-glutathionylated residues. In human plasma from cancer patients, there were higher levels of unmodified serpin A1 and A3, but following treatments with redox active drugs, relative S-glutathionylation of these serpins was higher in plasma from normal individuals. There is potential for S-glutathionylated serpins A1 and A3 to act as pharmacodynamic biomarkers for evaluation of patient response to drugs that target redox pathways. PMID:22406622

  18. 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

    The seed-feeding beetle Callosobruchus maculatus is an important cowpea pest (Vigna unguiculata) as well as an interesting model to study insect digestive physiology. The larvae of C. maculatus rely on cysteine and aspartic peptidases to digest proteins in their diet. In this work, the global...

  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...

  20. Peptidomimetic inhibitors of extracellular aspartic proteinases of .I.Candida albicans./I. and .I.Candida tropicalis./I..

    Czech Academy of Sciences Publication Activity Database

    Brožková, Kateřina; Křížová, Ivana; Pavlíčková, Libuše; Hradilek, Martin; Fusek, Martin; Ruml, T.; Souček, Milan; Pichová, Iva

    1999-01-01

    Roč. 64, č. 1 (1999), s. 130-137. ISSN 0010-0765 R&D Projects: GA ČR GA203/95/1028; GA ČR GA303/98/1612 Institutional research plan: CEZ:AV0Z4055905 Subject RIV: CE - Biochemistry Impact factor: 0.717, year: 1999

  1. Differential elicitation of two processing proteases control the processing pattern of the trypsin proteinase inhibitor precursor in Nicotiana attenuata

    Czech Academy of Sciences Publication Activity Database

    Horn, Martin; Patankar, A. G.; Zavala, J. A.; Wu, J.; Marešová, Lucie; Vůjtěchová, Milana; Mareš, Michael; Baldwin, I. T.

    2005-01-01

    Roč. 139, - (2005), s. 375-388. ISSN 0032-0889 R&D Projects: GA ČR(CZ) GA522/04/1286; GA AV ČR(CZ) IAA4055303 Institutional research plan: CEZ:AV0Z40550506 Keywords : posttranslational modifications * defense * differential fragmentation Subject RIV: CE - Biochemistry Impact factor: 6.114, year: 2005

  2. Potency Comparison of Peptidomimetic Inhibitors Against HIV-1 and HIV-2 Proteinases: Design of Equipotent Lead Compounds

    Czech Academy of Sciences Publication Activity Database

    Weber, Jan; Majer, Pavel; Litera, Jaroslav; Urban, Jan; Souček, Milan; Vondrášek, Jiří; Konvalinka, Jan; Novek, Petr; Sedláček, Juraj; Štrop, Petr

    1997-01-01

    Roč. 341, č. 1 (1997), s. 62-69. ISSN 0003-9861 R&D Projects: GA ČR GA303/96/1235; GA AV ČR IAA4055503 Grant ostatní: Internation Research Scholar's HHMI 75195-540801 Source of funding: US Impact factor: 2.649, year: 1997

  3. 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. PMID:26892536

  4. Faddeev calculation of 3 alpha and alpha alpha Lambda systems using alpha alpha resonating-group method kernel

    CERN Document Server

    Fujiwara, Y; Kohno, M; Suzuki, Y; Baye, D; Sparenberg, J M

    2004-01-01

    We carry out Faddeev calculations of three-alpha (3 alpha) and two-alpha plus Lambda (alpha alpha Lambda) systems, using two-cluster resonating-group method kernels. The input includes an effective two-nucleon force for the alpha alpha resonating-group method and a new effective Lambda N force for the Lambda alpha interaction. The latter force is a simple two-range Gaussian potential for each spin-singlet and triplet state, generated from the phase-shift behavior of the quark-model hyperon-nucleon interaction, fss2, by using an inversion method based on supersymmetric quantum mechanics. Owing to the exact treatment of the Pauli-forbidden states between the clusters, the present three-cluster Faddeev formalism can describe the mutually related, alpha alpha, 3 alpha and alpha alpha Lambda systems, in terms of a unique set of the baryon-baryon interactions. For the three-range Minnesota force which describes the alpha alpha phase shifts quite accurately, the ground-state and excitation energies of 9Be Lambda are...

  5. 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...

  6. 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.

  7. Essential role of nuclear factor (NF)-kappaB-inducing kinase and inhibitor of kappaB (IkappaB) kinase alpha in NF-kappaB activation through lymphotoxin beta receptor, but not through tumor necrosis factor receptor I.

    Science.gov (United States)

    Matsushima, A; Kaisho, T; Rennert, P D; Nakano, H; Kurosawa, K; Uchida, D; Takeda, K; Akira, S; Matsumoto, M

    2001-03-01

    Both nuclear factor (NF)-kappaB-inducing kinase (NIK) and inhibitor of kappaB (IkappaB) kinase (IKK) have been implicated as essential components for NF-kappaB activation in response to many external stimuli. However, the exact roles of NIK and IKKalpha in cytokine signaling still remain controversial. With the use of in vivo mouse models, rather than with enforced gene-expression systems, we have investigated the role of NIK and IKKalpha in signaling through the type I tumor necrosis factor (TNF) receptor (TNFR-I) and the lymphotoxin beta receptor (LTbetaR), a receptor essential for lymphoid organogenesis. TNF stimulation induced similar levels of phosphorylation and degradation of IkappaBalpha in embryonic fibroblasts from either wild-type or NIK-mutant mice. In contrast, LTbetaR stimulation induced NF-kappaB activation in wild-type mice, but the response was impaired in embryonic fibroblasts from NIK-mutant and IKKalpha-deficient mice. Consistent with the essential role of IKKalpha in LTbetaR signaling, we found that development of Peyer's patches was defective in IKKalpha-deficient mice. These results demonstrate that both NIK and IKKalpha are essential for the induction of NF-kappaB through LTbetaR, whereas the NIK-IKKalpha pathway is dispensable in TNFR-I signaling. PMID:11238593

  8. 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

  9. 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. PMID:21656364

  10. 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.

  11. Candida tropicalis Biofilms: Biomass, Metabolic Activity and Secreted Aspartyl Proteinase Production.

    Science.gov (United States)

    Negri, Melyssa; Silva, Sónia; Capoci, Isis Regina Grenier; Azeredo, Joana; Henriques, Mariana

    2016-04-01

    According to epidemiological data, Candida tropicalis has been related to urinary tract infections and haematological malignancy. Several virulence factors seem to be responsible for C. tropicalis infections, for example: their ability to adhere and to form biofilms onto different indwelling medical devices; their capacity to adhere, invade and damage host human tissues due to enzymes production such as proteinases. The main aim of this work was to study the behaviour of C. tropicalis biofilms of different ages (24-120 h) formed in artificial urine (AU) and their ability to express aspartyl proteinase (SAPT) genes. The reference strain C. tropicalis ATCC 750 and two C. tropicalis isolates from urine were used. Biofilms were evaluated in terms of culturable cells by colony-forming units enumeration; total biofilm biomass was evaluated using the crystal violet staining method; metabolic activity was evaluated by XTT assay; and SAPT gene expression was determined by real-time PCR. All strains of C. tropicalis were able to form biofilms in AU, although with differences between strains. Candida tropicalis biofilms showed a decrease in terms of the number of culturable cells from 48 to 72 h. Generally, SAPT3 was highly expressed. C. tropicalis strains assayed were able to form biofilms in the presence of AU although in a strain- and time-dependent way, and SAPT genes are expressed during C. tropicalis biofilm formation. PMID:26572148

  12. Age-dependent changes in extracellular proteins, aminopeptidase and proteinase activities in Frankia isolate BR.

    Science.gov (United States)

    Müller, A; Benoist, P; Diem, H G; Schwencke, J

    1991-12-01

    To investigate protein secretion by the nitrogen-fixing actinomycete Frankia isolate BR, we designed a rapid DEAE adsorption, salt elution and Biogel P6DG desalination method to concentrate protein from the growth medium. Secreted proteins reached a maximum concentration (5.6 gm l-1) in the medium at growth arrest. Analysis by SDS-PAGE detected up to 63 extracellular polypeptides when Frankia cells were grown under stirred conditions in BAP medium supplemented with phosphatidylcholine and MES buffer and 65 proteins in stirred BAP media alone. The pattern of extracellular polypeptides changed during growth. Several extracellular proteolytic activities were detected and compared with intracellular ones. The substrate specificity of the extracellular and intracellular aminopeptidase activities were the same. Also, the electrophoretic migration patterns of secreted and intracellular aminopeptidases could not be distinguished. Secretion of the proline-specific aminopeptidase FAP proteinase (PF) were secreted: 10 had the same electrophoretic mobility as their intracellular counterparts after SDS-gelatine-PAGE while five (PF - 39.5, PF - 38.5, PF - 36.5, PF - 25.5 and PF - 20.5 kDa) had a different electrophoretic mobility and, therefore, appeared to be exclusively extracellular. At least seven extracellular proteinases appeared to increase coordinately in activity shortly before growth arrest. PMID:15101385

  13. Exposure of hydrophobic moieties promotes the selective degradation of hydrogen peroxide-modified hemoglobin by the multicatalytic proteinase complex, proteasome.

    Science.gov (United States)

    Giulivi, C; Pacifici, R E; Davies, K J

    1994-06-01

    The physiologically relevant stress of a flux of H2O2 increased hemoglobin (Hb) degradation in red blood cells (RBC) and increased the proteolytic susceptibility of Hb in vitro. After exposure to low H2O2 flux rates (6-32 microM/min) Hb exhibited increased exposure of hydrophobic (Trp, Met) and basic (Lys) amino acid R groups, increased hydrophobicity, and increased proteolytic susceptibility during subsequent incubation with RBC extracts, a partially purified preparation called Fraction II (which retains all of the proteolytic activities of RBC extracts), or the purified 670-kDa RBC multicatalytic proteinase complex proteasome. Hydrophobicity was measured by butyl-Sepharose hydrophobic interaction chromatography, by the free energy of transfer from water to ethanol, and by heat denaturation assays. Proteolytic susceptibility was measured by release of free alanine, by fluorescamine-reactive free amino groups, and by release of acid-soluble radioactivity from radiolabeled Hb. Low H2O2 flux rates also caused significant charge changes in Hb (isoelectric focusing gels) and extensive noncovalent aggregation (presumably due to increased hydrophobic interactions) but only limited covalent cross-linking (comparison of sodium dodecyl sulfate-polyacylamide gel electrophoresis (SDS-PAGE) and nondenaturing PAGE). Exposure to higher H2O2 flux rates (56-120 microM/min) caused progressive oxidative destruction of exposed hydrophobic amino acids, decreased hydrophobicity as judged by butyl-Sepharose chromatography and heat denaturation assays, increased hydrophilicity as judged by measurements of the free energy of transfer (delta G') from water to ethanol, and decreased proteolytic susceptibility during incubation with RBC extracts, Fraction II, or purified proteasome. High H2O2 flux rates also caused further charge changes and the extensive formation of covalently cross-linked Hb molecules. Linear regression analyses revealed correlations of 0.8-0.99 for the relationship

  14. Review of alpha_s determinations

    CERN Document Server

    Pich, Antonio

    2013-01-01

    The present knowledge on the strong coupling is briefly summarized. The most precise determinations of alpha_s, at different energies, are reviewed and compared at the Z mass scale, using the predicted QCD running. The impressive agreement achieved between experimental measurements and theoretical predictions constitutes a beautiful and very significant test of Asymptotic Freedom, establishing QCD as the fundamental theory of the strong interaction. The world average value of the strong coupling is found to be alpha_s(M_Z^2)= 0.1186 \\pm 0.0007.

  15. Involvement of capsaicin-sensitive afferent nerves in the proteinase-activated receptor 2-mediated vasodilatation in the rat dura mater.

    Science.gov (United States)

    Dux, M; Rosta, J; Sántha, P; Jancsó, G

    2009-07-01

    Neurogenic inflammation of the dura mater encephali has been suggested to contribute to the mechanisms of meningeal nociception and blood flow regulation. Recent findings demonstrated that the rat dura mater is innervated by trigeminal capsaicin-sensitive peptidergic nociceptive afferent nerves which mediate meningeal vascular responses through activation of the transient receptor potential vanilloid type 1 (TRPV1) receptor. The present work explored the functional significance of the capsaicin-sensitive subpopulation of dural afferent nerves via their contribution to the meningeal vascular responses evoked through activation of the proteinase-activated receptor 2 (PAR-2). The vascular responses of the dura mater were studied by laser Doppler flowmetry in a rat open cranial window preparation. Topical applications of trypsin, a PAR-2-activator, or Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-NH(2)), a selective PAR-2 agonist peptide, resulted in dose-dependent increases in meningeal blood flow. The SLIGRL-NH(2)-induced vasodilatation was significantly reduced following capsaicin-sensitive afferent nerve defunctionalization by prior systemic capsaicin treatment and by pretreatment of the dura mater with the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8-37). Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME) an unspecific inhibitor of nitric oxide (NO) production, but not 1-(2-trifluoromethylphenyl) imidazole (TRIM), a neuronal NO synthase inhibitor, also inhibited the vasodilator response to SLIGRL-NH(2). The vasodilator responses elicited by very low concentrations of capsaicin (10 nM) were significantly enhanced by prior application of SLIGRL-NH(2). The present findings demonstrate that activation of the PAR-2 localized on capsaicin-sensitive trigeminal nociceptive afferent nerves induces vasodilatation in the dural vascular bed by mechanisms involving NO and CGRP release. The results indicate that the PAR-2-mediated activation and

  16. Mutants of common bean alpha-amylase inhibitor-2 as an approach to investigate binding specificity to alpha-amylases Mutantes do inibidor-2 de alfa-amilase do feijão-comum para investigação da especificidade de ligação a alfa-amilases

    Directory of Open Access Journals (Sweden)

    Maria Cristina Mattar da Silva

    2004-03-01

    Full Text Available Despite the presence of a family of defense proteins, Phaseolus vulgaris can be attacked by bruchid insects resulting in serious damage to stored grains. The two distinct active forms of a-amylase inhibitors, a-AI1 and a-AI2, in P. vulgaris show different specificity toward a-amylases. Zabrotes subfasciatus a-amylase is inhibited by a-AI2 but not by a-AI1. In contrast, porcine a-amylase is inhibited by a-AI1 but not by a-AI2. The objective of this work was to understand the molecular basis of the specificity of two inhibitors in P. vulgaris (a-AI1 and a-AI2 in relation to a-amylases. Mutants of a-AI2 were made and expressed in tobacco plants. The results showed that all the a-AI2 mutant inhibitors lost their activity against the insect a-amylases but none exhibited activity toward the mammalian a-amylase. The replacement of His33 of a-AI2 with the a-AI1-like sequence Ser-Tyr-Asn abolished inhibition of Z. subfasciatus a-amylase. From structural modeling, the conclusion is that the size and complexity of the amylase-inhibitor interface explain why mutation of the N-terminal loop and resultant abolition of Z. subfasciatus a-amylase inhibition are not accompanied by gain of inhibitory activity against porcine a-amylase.Apesar de possuir uma família de proteínas de defesa, o feijão-comum (Phaseolus vulgaris L. pode ser atacado por insetos bruquídeos causando sérios danos aos grãos armazenados. O P. vulgaris possui duas formas ativas de inibidores de a-amilases, denominadas a-AI1 e a-AI2, que apresentam diferentes especificidades em relação às a-amilases. A a-amilase de Zabrotes subfasciatus é inibida por a-AI2 mas não por a-AI1. Em contraste, a a-amilase pancreática de porco é inibida por a-AI1 mas não é por a-AI2. O objetivo deste trabalho foi entender as bases moleculares da especificidade desses inibidores em relação às a-amilases. Para tanto, foram construídos mutantes do a-AI2, os quais foram expressados em plantas de fumo

  17. Review of alpha_s determinations

    OpenAIRE

    Pich, Antonio

    2013-01-01

    The present knowledge on the strong coupling is briefly summarized. The most precise determinations of alpha_s, at different energies, are reviewed and compared at the Z mass scale, using the predicted QCD running. The impressive agreement achieved between experimental measurements and theoretical predictions constitutes a beautiful and very significant test of Asymptotic Freedom, establishing QCD as the fundamental theory of the strong interaction. The world average value of the strong coupl...

  18. World Summary of $\\alpha_s$ (2015)

    CERN Document Server

    Bethke, Siegfried; Salam, Gavin P

    2015-01-01

    This is a preliminary update of the measurements of α s and the determination of the world average value of α s (M Z 2 ) presented in the 2013/2014 edition of the Review of Particle Properties [1]. A number of studies which became available since late 2013 provide new results for each of the (previously 5, now) 6 subclasses of measurements for which pre-average values of $\\alpha_s (M_Z^2)$ are determined.

  19. 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

  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...

  1. 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

  2. The Contribution of Proteinase-Activated Receptors to Intracellular Signaling, Transcellular Transport and Autophagy in Alzheimer´s Disease

    Czech Academy of Sciences Publication Activity Database

    Matěj, R.; Rohan, Z.; Holada, K.; Olejár, Tomáš

    2015-01-01

    Roč. 12, č. 1 (2015), s. 2-12. ISSN 1567-2050 Institutional support: RVO:67985823 Keywords : Alzheimer ´s Disease * autophagy * proteinase-activated receptors Subject RIV: EA - Cell Biology Impact factor: 3.889, year: 2014

  3. 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...

  4. Proteinases of betaretroviruses bind single-stranded nucleic acids through a novel interaction module, the G-patch

    Czech Academy of Sciences Publication Activity Database

    Švec, Martin; Bauerová, Helena; Pichová, Iva; Konvalinka, Jan; Stříšovský, Kvido

    Praha : JPM, 2004 - (Hunter, E.; Ruml, T.; Pichová, I.; Rumlová, M.; Sakalian, M.). s. 75 ISBN 80-86313-13-1. [The Retrovirus Assembly Meeting. 02.10.2004-06.10.2004, Praha] Keywords : proteinases * betaretroviruses Subject RIV: CE - Biochemistry

  5. 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...

  6. Crystallographic and computational studies on 4-phenyl-N-(beta-D-glucopyranosyl)-1H-1,2,3-triazole-1-acetamide, an inhibitor of glycogen phosphorylase: comparison with alpha-D-glucose, N-acetyl-beta-D-glucopyranosylamine and N-benzoyl-N'-beta-D-glucopyranosyl urea binding.

    Science.gov (United States)

    Alexacou, Kyra-Melinda; Hayes, Joseph M; Tiraidis, Costas; Zographos, Spyros E; Leonidas, Demetres D; Chrysina, Evangelia D; Archontis, Georgios; Oikonomakos, Nikos G; Paul, Jashuva V; Varghese, Babu; Loganathan, Duraikkannu

    2008-05-15

    4-Phenyl-N-(beta-D-glucopyranosyl)-1H-1,2,3-triazole-1-acetamide (glucosyltriazolylacetamide) has been studied in kinetic and crystallographic experiments with glycogen phosphorylase b (GPb), in an effort to utilize its potential as a lead for the design of potent antihyperglycaemic agents. Docking and molecular dynamics (MD) calculations have been used to monitor more closely the binding modes in operation and compare the results with experiment. Kinetic experiments in the direction of glycogen synthesis showed that glucosyltriazolylacetamide is a better inhibitor (K(i) = 0.18 mM) than the parent compound alpha-D-glucose (K(i) = 1.7 mM) or beta-D-glucose (K(i) = 7.4 mM) but less potent inhibitor than the lead compound N-acetyl-beta-D-glucopyranosylamine (K(i) = 32 microM). To elucidate the molecular basis underlying the inhibition of the newly identified compound, we determined the structure of GPb in complex with glucosyltriazolylacetamide at 100 K to 1.88 A resolution, and the structure of the compound in the free form. Glucosyltriazolylacetamide is accommodated in the catalytic site of the enzyme and the glucopyranose interacts in a manner similar to that observed in the GPb-alpha-D-glucose complex, while the substituent group in the beta-position of the C1 atom makes additional hydrogen bonding and van der Waals interactions to the protein. A bifurcated donor type hydrogen bonding involving O3H, N3, and N4 is seen as an important structural motif strengthening the binding of glucosyltriazolylacetamide with GP which necessitated change in the torsion about C8-N2 bond by about 62 degrees going from its free to the complex form with GPb. On binding to GP, glucosyltriazolylacetamide induces significant conformational changes in the vicinity of this site. Specifically, the 280s loop (residues 282-288) shifts 0.7 to 3.1 A (CA atoms) to accommodate glucosyltriazolylacetamide. These conformational changes do not lead to increased contacts between the inhibitor and the

  7. Introduction of α-hydroxymethyamino acid residues in substrate specificity P1 position of trypsin inhibitor SFTI-1 from sunflower seeds retains its activity

    International Nuclear Information System (INIS)

    In many complexes formed by serine proteinases and their inhibitors, the hydroxyl group provided by water molecule or by the inhibitor Ser residue is located close to the inhibitor P1-P1' reactive site. In order to investigate the role of this group, we synthesized analogues of trypsin inhibitor SFTI-1 isolated from the seeds of sunflower modified in P1 by α-hydroxymethylserine (HmSer) and both enantiomers of α-hydroxymethylvaline (HmVal). All the synthesized analogues inhibited bovine β-trypsin and human leukocyte elastase. SFTI-1 analogues with HmVal and HmSer appear to be potent inhibitors of bovine β-trypsin, whereas [Val5]SFTI-1 is practically inactive. Also trypsin inhibitory activity of [Ser5]SFTI-1 is significantly lower. Since the electrostatic interaction between protonated ε-NH2 group of the inhibitor P1 position and β-carboxylate of trypsin Asp189 is the main driving force for interaction of both molecules, the results obtained are very interesting. We believe that these SFTI-1 analogues belong to a novel class of serine proteinase inhibitors

  8. Reassessment of acarbose as a transition state analogue inhibitor of cyclodextrin glycosyltransferase

    OpenAIRE

    Mosi, Renee; Sham, Howard; Uitdehaag, Joost C.M.; Ruiterkamp, Richard; Dijkstra, Bauke W.; Withers, Stephen G.

    1998-01-01

    The binding of several different active site mutants of Bacillus circulans cyclodextrin,glycosyltransferase to the inhibitor acarbose has been investigated through measurement of Ki values. The mutations represent several key amino acid positions, most of which are believed to play important roles in governing the product specificity of cyclodextrin glycosyltransferase. Michaelis-Menten parameters for the substrates alpha-maltotriosyl fluoride (alpha G3F) and alpha-glucosyl fluoride (alpha GF...

  9. 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 ...

  10. 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.

  11. PDE-5 inhibitors: clinical points.

    Science.gov (United States)

    Doumas, Michael; Lazaridis, Antonios; Katsiki, Niki; Athyros, Vasilios

    2015-01-01

    Erectile dysfunction is usually of vascular origin and is frequently encountered in men with cardiovascular disease. The introduction of phosphodiesterase-5 inhibitors has revolutionized the management of patients with erectile dysfunction. Currently available phosphodiesterase-5 inhibitors have distinct pharmacokinetic and pharmacodynamic properties, thus permitting for tailoring sexual therapy according to patient characteristics and needs. Phosphodiesterase-5 inhibitors possess vasorelaxing properties and exert systemic hemodynamic effects, which need to be taken into account when other cardiovascular drugs are co-administered. Special caution is needed with alpha-blockers, while the co-administration with nitrates is contra-indicated due to the risk of life-threatening hypotension. This review presents the advent of sexual therapy, describes the mechanism of action and the specific characteristics of commercially available phosphodiesterase-5 inhibitors, summarizes the efficacy and safety of these drugs with special emphasis on the cardiovascular system, and discusses the clinical criteria used for the selection of each drug for the individual patient. PMID:25392015

  12. 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

  13. 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

  14. 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)

  15. Protein degradation in Euglena gracilis: Purification and characterization of the major proteinase

    International Nuclear Information System (INIS)

    Protolysis in a crude extract of Euglena gracilis was characterized by autolysis and the hydrolysis of 125I-labeled bovine serum albumin (125I-BSA). Both procedures showed similar properties: stimulation by dithiothreitol, inhibition by leupeptin, and the same pH optima. Hydrolysis of 125I-BSA increased with growth stage and with the depletion of nutrient in the medium. The major proteolytic enzyme was purified to near homogeneity from extracts of dark-grown, stationary-phase Euglena gracilis by acid treatment, and by chromatography on CM-cellulose, DEAE-cellulose, Sephadex G-75, and hydroxyapatite using 125I-BSA as substrate. The molecular weight of the proteinase was 30,000 when determined by gel filtration on Sephadex G-75 and 15,000 when estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme therefore appears to be composed of two subunits

  16. 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.

  17. Lyman Alpha Control

    CERN Document Server

    Nielsen, Daniel Stefaniak

    2015-01-01

    This document gives an overview of how to operate the Lyman Alpha Control application written in LabVIEW along with things to watch out for. Overview of the LabVIEW code itself as well as the physical wiring of and connections from/to the NI PCI-6229 DAQ box is also included. The Lyman Alpha Control application is the interface between the ALPHA sequencer and the HighFinesse Wavelength Meter as well as the Lyman Alpha laser setup. The application measures the wavelength of the output light from the Lyman Alpha cavity through the Wavelength Meter. The application can use the Wavelength Meter’s PID capabilities to stabilize the Lyman Alpha laser output as well as switch between up to three frequencies.

  18. New ALPHA-2 magnet

    CERN Multimedia

    Anaïs Schaeffer

    2012-01-01

    On 21 June, members of the ALPHA collaboration celebrated the handover of the first solenoid designed for the ALPHA-2 experiment. The magnet has since been successfully installed and is working well.   Khalid Mansoor, Sumera Yamin and Jeffrey Hangst in front of the new ALPHA-2 solenoid. “This was the first of three identical solenoids that will be installed between now and September, as the rest of the ALPHA-2 device is installed and commissioned,” explains ALPHA spokesperson Jeffrey Hangst. “These magnets are designed to allow us to transfer particles - antiprotons, electrons and positrons - between various parts of the new ALPHA-2 device by controlling the transverse size of the particle bunch that is being transferred.” Sumera Yamin and Khalid Mansoor, two Pakistani scientists from the National Centre for Physics in Islamabad, came to CERN in February specifically to design and manufacture these magnets. “We had the chance to work on act...

  19. Chronic obstructive pulmonary disease in alpha1-antitrypsin PI MZ heterozygotes

    DEFF Research Database (Denmark)

    Hersh, C P; Dahl, Morten; Ly, N P; Berkey, C S; Nordestgaard, B G; Silverman, E K

    2004-01-01

    Severe alpha(1)-antitrypsin deficiency, usually related to homozygosity for the protease inhibitor (PI) Z allele, is a proven genetic risk factor for chronic obstructive pulmonary disease (COPD). The risk of COPD in PI MZ heterozygous individuals is controversial.......Severe alpha(1)-antitrypsin deficiency, usually related to homozygosity for the protease inhibitor (PI) Z allele, is a proven genetic risk factor for chronic obstructive pulmonary disease (COPD). The risk of COPD in PI MZ heterozygous individuals is controversial....

  20. Structure of leech derived tryptase inhibitor (LDTI-C) in solution.

    Science.gov (United States)

    Mühlhahn, P; Czisch, M; Morenweiser, R; Habermann, B; Engh, R A; Sommerhoff, C P; Auerswald, E A; Holak, T A

    1994-12-01

    The three-dimensional solution structure of the leech derived tryptase inhibitor form C (LDTI-C), an inhibitor of 46 amino acids which contains 3 disulfide bridges, has been determined using 2D NMR spectroscopy. The 3D structure was determined on the basis of 262 interresidue interproton distance constraints derived from nuclear Overhauser enhancement measurements and 25 phi angles, supplemented by 3 psi and 15 chi 1 angles. The core of LDTI-C is very well defined and consists of a short 3(10)-helix-loop and a short two-stranded antiparallel beta-sheet between residues 13-14 and 20-21. The N-terminus is fixed to the core by two disulfide bridges, while the C-terminus is connected to the beta-sheet via the third disulfide bridge. The binding loop in LDTI exhibits lowest energy conformations belonging to the canonical conformation of serine proteinase inhibitors. PMID:7988692

  1. Alpha Shapes and Proteins

    DEFF Research Database (Denmark)

    Winter, Pawel; Sterner, Henrik; Sterner, Peter

    We provide a unified description of (weighted) alpha shapes, beta shapes and the corresponding simplicialcomplexes. We discuss their applicability to various protein-related problems. We also discuss filtrations of alpha shapes and touch upon related persistence issues.We claim that the full...... potential of alpha-shapes and related geometrical constructs in protein-related problems yet remains to be realized and verified. We suggest parallel algorithms for (weighted) alpha shapes, and we argue that future use of filtrations and kinetic variants for larger proteins will need such implementation....

  2. Targeted Alpha Therapy: From Alpha to Omega

    International Nuclear Information System (INIS)

    This review covers the broad spectrum of Targeted Alpha Therapy (TAT) research in Australia; from in vitro and in vivo studies to clinical trials. The principle of tumour anti-vascular alpha therapy (TAVAT) is discussed in terms of its validation by Monte Carlo calculations of vascular models and the potential role of biological dosimetry is examined. Summmary of this review is as follows: 1. The essence of TAT 2. Therapeutic objectives 3. TAVAT and Monte Carlo microdosimetry 4. Biological dosimetry 5. Preclinical studies 6. Clinical trials 7. What next? 8. Obstacles. (author)

  3. A rare case of alpha 1-antitrypsin deficiency associated with hypogammaglobulinemia and recurrent pulmonary thrombosis

    Directory of Open Access Journals (Sweden)

    Raghav Gupta

    2014-01-01

    Full Text Available Alpha 1-antitrypsin (AAT belongs to the family of serpins (serine protease inhibitors. Loop sheet polymerization is the pathology behind serpinopathies which encompasses AAT, anti-thrombin III and neuroserpin deficiency. To the best of our knowledge, we report the first case of alpha 1-antitrypsin deficiency associated with hypogammaglobulinemia and recurrent pulmonary thrombosis without any concomitant use of drugs.

  4. Alpha2-antiplasmin gene deficiency in mice is associated with enhanced fibrinolytic potential without overt bleeding

    OpenAIRE

    Lijnen, Roger; Okada, K.; Matsuo, O; Collen, Desire; Dewerchin, Mieke

    1999-01-01

    alpha2-antiplasmin (alpha2-AP) is the main physiologic plasmin inhibitor in mammalian plasma. Inactivation of the murine alpha2-AP gene was achieved by replacing, through homologous recombination in embryonic stem cells, a 7-kb genomic sequence encoding the entire murine protein (exon 2 through part of exon 10, including the stop codon) with the neomycin resistance expression cassette. Germline transmission of the mutated allele was confirmed by Southern blot analysis. Mendelian inheritance o...

  5. Alpha-particle diagnostics

    Energy Technology Data Exchange (ETDEWEB)

    Young, K.M.

    1991-01-01

    This paper will focus on the state of development of diagnostics which are expected to provide the information needed for {alpha}- physics studies in the future. Conventional measurement of detailed temporal and spatial profiles of background plasma properties in DT will be essential for such aspects as determining heating effectiveness, shaping of the plasma profiles and effects of MHD, but will not be addressed here. This paper will address (1) the measurement of the neutron source, and hence {alpha}-particle birth profile, (2) measurement of the escaping {alpha}-particles and (3) measurement of the confined {alpha}-particles over their full energy range. There will also be a brief discussion of (4) the concerns about instabilities being generated by {alpha}-particles and the methods necessary for measuring these effects. 51 refs., 10 figs.

  6. Imaging alpha particle detector

    Science.gov (United States)

    Anderson, D.F.

    1980-10-29

    A method and apparatus for detecting and imaging alpha particles sources is described. A dielectric coated high voltage electrode and a tungsten wire grid constitute a diode configuration discharge generator for electrons dislodged from atoms or molecules located in between these electrodes when struck by alpha particles from a source to be quantitatively or qualitatively analyzed. A thin polyester film window allows the alpha particles to pass into the gas enclosure and the combination of the glass electrode, grid and window is light transparent such that the details of the source which is imaged with high resolution and sensitivity by the sparks produced can be observed visually as well. The source can be viewed directly, electronically counted or integrated over time using photographic methods. A significant increase in sensitivity over other alpha particle detectors is observed, and the device has very low sensitivity to gamma or beta emissions which might otherwise appear as noise on the alpha particle signal.

  7. Activity of recombinant trypsin isoforms on human proteinase-activated receptors (PAR): mesotrypsin cannot activate epithelial PAR-1, -2, but weakly activates brain PAR-1

    OpenAIRE

    Grishina, Zoryana; Ostrowska, Ewa; Halangk, Walter; Sahin-Tóth, Miklós; Reiser, Georg

    2005-01-01

    Trypsin-like serine proteinases trigger signal transduction pathways through proteolytic cleavage of proteinase-activated receptors (PARs) in many tissues. Three members, PAR-1, PAR-2 and PAR-4, are trypsin substrates, as trypsinolytic cleavage of the extracellular N terminus produces receptor activation. Here, the ability of the three human pancreatic trypsin isoforms (cationic trypsin, anionic trypsin and mesotrypsin (trypsin IV)) as recombinant proteins was tested on PARs.Using fura 2 [Ca2...

  8. Targeted alpha therapy for cancer

    Science.gov (United States)

    Allen, Barry J.; Raja, Chand; Rizvi, Syed; Li, Yong; Tsui, Wendy; Zhang, David; Song, Emma; Qu, Chang Fa; Kearsley, John; Graham, Peter; Thompson, John

    2004-08-01

    Targeted alpha therapy (TAT) offers the potential to inhibit the growth of micrometastases by selectively killing isolated and preangiogenic clusters of cancer cells. The practicality and efficacy of TAT is tested by in vitro and in vivo studies in melanoma, leukaemia, colorectal, breast and prostate cancers, and by a phase 1 trial of intralesional TAT for melanoma. The alpha-emitting radioisotope used is Bi-213, which is eluted from the Ac-225 generator and chelated to a cancer specific monoclonal antibody (mab) or protein (e.g. plasminogen activator inhibitor-2 PAI2) to form the alpha-conjugate (AC). Stable alpha-ACs have been produced which have been tested for specificity and cytotoxicity in vitro against melanoma (9.2.27 mab), leukaemia (WM60), colorectal (C30.6), breast (PAI2, herceptin), ovarian (PAI2, herceptin, C595), prostate (PAI2, J591) and pancreatic (PAI2, C595) cancers. Subcutaneous inoculation of 1-1.5 million human cancer cells into the flanks of nude mice causes tumours to grow in all mice. Tumour growth is compared for untreated controls, nonspecific AC and specific AC, for local (subcutaneous) and systemic (tail vein or intraperitoneal) injection models. The 213Bi-9.2.27 AC is injected into secondary skin melanomas in stage 4 patients in a dose escalation study to determine the effective tolerance dose, and to measure kinematics to obtain the equivalent dose to organs. In vitro studies show that TAT is one to two orders of magnitude more cytotoxic to targeted cells than non-specific ACs, specific beta emitting conjugates or free isotopes. In vivo local TAT at 2 days post-inoculation completely prevents tumour formation for all cancers tested so far. Intra-lesional TAT can completely regress advanced sc melanoma but is less successful for breast and prostate cancers. Systemic TAT inhibits the growth of sc melanoma xenografts and gives almost complete control of breast and prostate cancer tumour growth. Intralesional doses up to 450 µCi in human

  9. 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

  10. 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. .

  11. 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...

  12. Saccharomyces cerevisiae can secrete Sapp1p proteinase of Candida parapsilosis but cannot use it for efficient nitrogen acquisition

    Czech Academy of Sciences Publication Activity Database

    Vinterová, Zuzana; Bauerová, Václava; Dostál, Jiří; Sychrová, Hana; Hrušková-Heidingsfeldová, Olga; Pichová, Iva

    2013-01-01

    Roč. 51, č. 3 (2013), s. 336-344. ISSN 1225-8873 R&D Projects: GA ČR GA310/09/1945; GA ČR GAP302/12/1151 Institutional support: RVO:61388963 ; RVO:67985823 Keywords : Candida parapsilosis * Saccharomyces cerevisiae * secreted aspartic proteinase * SAPP1 * nitrogen metabolism Subject RIV: EE - Microbiology, Virology; EE - Microbiology, Virology (FGU-C) Impact factor: 1.529, year: 2013

  13. Factors affecting the anthelmintic efficacy of cysteine proteinases against GI nematodes and their formulation for use in ruminants

    OpenAIRE

    Luoga, Wenceslaus

    2013-01-01

    Gastrointestinal (GI) nematodes are important helminth pathogens responsible for severe losses to livestock industries and human health throughout the world. Control of these infections relies primarily on chemotherapy; however there is rapid development of resistance to all available classes of anthelmintic drugs, and therefore new alternative treatments are urgently required. Plant cysteine proteinases (CPs) from papaya latex, pineapple fruit and stem extracts have been demonstrated to b...

  14. 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...

  15. 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...

  16. 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 (...

  17. Evidence for the presence of proteolytically active secreted aspartic proteinase 1 of Candida parapsilosis in the cell wall

    Czech Academy of Sciences Publication Activity Database

    Vinterová, Zuzana; Šanda, Miloslav; Dostál, Jiří; Hrušková-Heidingsfeldová, Olga; Pichová, Iva

    2011-01-01

    Roč. 20, č. 12 (2011), s. 2004-2012. ISSN 0961-8368 R&D Projects: GA MŠk(CZ) LC531; GA ČR GA310/09/1945 Institutional research plan: CEZ:AV0Z40550506 Keywords : Candida parapsilosis * secreted aspartic proteinases * Sapp1p * cell wall * biotin * proteolytic activity Subject RIV: CE - Biochemistry Impact factor: 2.798, year: 2011

  18. 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...

  19. Proteinases of betaretroviruses bind single-stranded nucleic acids through a novel interaction module, the G-patch

    Czech Academy of Sciences Publication Activity Database

    Švec, Martin; Bauerová, Helena; Pichová, Iva; Konvalinka, Jan; Stříšovský, Kvido

    2004-01-01

    Roč. 576, 1/2 (2004), s. 271-276. ISSN 0014-5793 R&D Projects: GA AV ČR IAA4055304; GA MŠk LN00A032 Grant ostatní: 5th Framework(XE) QLK2-CT-2001-02360 Institutional research plan: CEZ:AV0Z4055905 Keywords : retrovirus * aspartic proteinase * maturation Subject RIV: CE - Biochemistry Impact factor: 3.843, year: 2004

  20. 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 ...