Sample records for alpha-1 proteinase inhibitor

  1. Limited proteolysis by macrophage elastase inactivates human alpha 1- proteinase inhibitor



    Inflammatory mouse peritoneal macrophages secrete a metalloproteinase that is not inhibited by alpha 1-proteinase inhibitor. This proteinase, macrophage elastase, recognizes alpha 1-proteinase inhibitor with macrophage elastase does not involve a stable proteinase-inhibitor complex and results in the proteolytic removal of a peptide of apparent molecular weight 4,000-5,000 from the inhibitor. After degradation by macrophage elastase, alpha 1-proteinase inhibitor is no longer able to inhibit h...

  2. Serpin alpha 1proteinase inhibitor probed by intrinsic tryptophan fluorescence spectroscopy.



    Various conformational forms of the archetypal serpin human alpha 1proteinase inhibitor (alpha 1PI), including ordered polymers, active and inactive monomers, and heterogeneous aggregates, have been produced by refolding from mild denaturing conditions. These forms presumably originate by different folding pathways during renaturation, under the influence of the A and C sheets of the molecule. Because alpha 1PI contains only two Trp residues, at positions 194 and 238, it is amenable to fluore...

  3. Non enzymatic glycosylation of alpha-1-proteinase inhibitor of human plasma.

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    Phadke M


    Full Text Available Human plasma contains inhibitors, which control the activity of proteolytic enzymes. Alpha-1-proteinase inhibitor and alpha-2-macroglobulin are two of them present in high concentration in human plasma, which inhibit action of trypsin among other proteinases. The trypsin inhibitory capacity (TIC of human plasma is observed to be decreased in pathological conditions like diabetes mellitus. The mechanisms of decrease in TIC was due to nonenzymatic glycosylation of alpha-1-proteinase inhibitor (A1PI. A1PI was partially purified from normal human plasma by steps involving ammonium sulphate precipitation, DEAE Sepharose CL6B chromatography, Concanavalin A Sepharose Chromatography and Sephadex G-100 Gel filtration. Purified inhibitor was glycosylated in vitro by incubating it with varying glucose concentrations, under nitrogen for different periods of time in reducing conditions. After glycosylation, the molecular weight of inhibitor increased from 52 kDa to 57 KDa because of binding with glucose molecules. The percent free amino groups in the protein decreased with increasing glucose concentration and days of incubation. The TIC of such modified inhibitor decreased significantly. Decrease in TIC was dependent on the glucose concentration and period of incubation used during in-vitro glycosylation of native inhibitor.

  4. Serpin alpha 1proteinase inhibitor probed by intrinsic tryptophan fluorescence spectroscopy. (United States)

    Koloczek, H.; Banbula, A.; Salvesen, G. S.; Potempa, J.


    Various conformational forms of the archetypal serpin human alpha 1proteinase inhibitor (alpha 1PI), including ordered polymers, active and inactive monomers, and heterogeneous aggregates, have been produced by refolding from mild denaturing conditions. These forms presumably originate by different folding pathways during renaturation, under the influence of the A and C sheets of the molecule. Because alpha 1PI contains only two Trp residues, at positions 194 and 238, it is amenable to fluorescence quenching resolved spectra and red-edge excitation measurements of the Trp environment. Thus, it is possible to define the conformation of the various forms based on the observed fluorescent properties of each of the Trp residues measured under a range of conditions. We show that denaturation in GuHCl, or thermal denaturation in Tris, followed by renaturation, leads to the formation of polymers that contain solvent-exposed Trp 238, which we interpret as ordered head-to-tail polymers (A-sheet polymers). However, thermal denaturation in citrate leads to shorter polymers where some of the Trp 238 residues are not solvent accessible, which we interpret as polymers capped by head-to-head interactions via the C sheet. The latter treatment also generates monomers thought to represent a latent form, but in which the environment of Trp 238 is occluded by ionized groups. These data indicate that the folding pathway of alpha 1PI, and presumably other serpins, is sensitive to solvent composition that affects the affinity of the reactive site loop for the A sheet or the C sheet. PMID:8931141

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

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    Chotirmall SH


    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

  6. Expression of alpha 1-proteinase inhibitor in Escherichia coli: effects of single amino acid substitutions in the active site loop on aggregate formation

    NARCIS (Netherlands)

    Schulze, A.J.; Degryse, E.; Speck, D.; Huber, R.; Bischoff, Rainer


    Overproduction of eukaryotic proteins in microorganisms often leads to the formation of insoluble protein aggregates which accumulate as intracellular inclusion bodies. alpha 1-Proteinase inhibitor (alpha 1-PI) when produced as a cytoplasmic protein in Escherichia coli (E. coli) forms inclusion bodi

  7. A feedback regulatory pathway between LDL and alpha-1 proteinase inhibitor in chronic inflammation and infection. (United States)

    Bristow, Cynthia L; Modarresi, Rozbeh; Babayeva, Mariya A; LaBrunda, Michelle; Mukhtarzad, Roya; Trucy, Maylis; Franklin, Aaron; Reeves, Rudy E R; Long, Allegra; Mullen, Michael P; Cortes, Jose; Winston, Ronald


    Dietary lipids are transported via lymph to the liver and transformed to lipoproteins which bind to members of the low density lipoprotein receptor family (LDL-RFMs). Certain LDL-RFMs, e.g., very low density lipoprotein receptor (VLDLR), are also bound by inactivated proteinase inhibitors, the most abundant being α1proteinase inhibitor (α1PI, α1antitrypsin). Inflammation/infection, including HIV-1 infection, is accompanied by low levels of CD4+ T cells and active α1PI and high levels of inactivated α1PI. By inducing LDL-RFMs-mediated cellular locomotion, active α1PI regulates the number of CD4+ T cells. We sought to investigate whether CD4+ T cells and α1PI directly impact lipoprotein levels. At the cellular level, we show that active α1PI is required for VLDLR-mediated uptake of receptor-associated cargo, specifically CD4-bound HIV-1. We show that active α1PI levels linearly correlate with LDL levels in HIV-1 infected individuals (P<0.001) and that therapeutic, weekly infusions of active α1PI elevate the number of CD4+ T cells and HDL levels while lowering LDL levels in patients on antiretroviral therapy with controlled HIV-1. Based on the unusual combination of lipodystrophy and low levels of α1PI and CD4+ T cells in HIV-1 disease, we reveal that LDL and α1PI participate in a feedback regulatory pathway. We demonstrate integral roles for sequentially acting active and inactive α1PI in the uptake and recycling of receptors and cargo aggregated with VLDLR including CD4 and chemokine receptors. Evidence supports a role for α1PI as a primary sentinel to deploy the immune system as a consequence of its role in lipoprotein transport.

  8. Hypersensitivity Vasculitis with Leukocytoclastic Vasculitis Associated with Alpha-1-Proteinase Inhibitor

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    Nicola W. Mwirigi


    Full Text Available Prolastin is a commercially available form of alpha-1-antitrypsin (AAT that is derived from pooled human plasma and used for treatment of severe alpha-1-antitrypsin deficiency (AATD. We describe a patient with AATD who developed presumed hypersensitivity vasculitis (HV following a Prolastin infusion. Hypersensitivity vasculitis (HV, or cutaneous vasculitis, is characterized by inflammation of the small vessels of the skin with resultant ischemia to the distally supplied areas. To our knowledge, this is the first reported case of presumed hypersensitivity vasculitis following Prolastin infusion.

  9. Alpha-1 proteinase inhibitor M358R reduces thrombin generation when displayed on the surface of cells expressing tissue factor. (United States)

    Gierczak, Richard F; Pepler, Laura; Bhagirath, Vinai; Liaw, Patricia C; Sheffield, William P


    The M358R variant of alpha-1-proteinase inhibitor (API) is a potent soluble inhibitor of thrombin. Previously we engineered AR-API M358R, a membrane-bound form of this protein and showed that it inhibited exogenous thrombin when expressed on transfected cells lacking tissue factor (TF). To determine the suitability of AR-API M358R for gene transfer to vascular cells to limit thrombogenicity, we tested the ability of AR-API M358R to inhibit endogenous thrombin generated in plasma via co-expression co-expressing it on the surface of cells expressing TF. Transfected AR-API M358R formed inhibitory complexes with thrombin following exposure of recalcified, defibrinated plasma to TF on T24/83 cells, but discontinuously monitored thrombin generation was unaffected. Similarly, AR-API M358R expression did not reduce continuously monitored thrombin generation by T24/83 cell suspensions exposed to recalcified normal plasma in a Thrombogram-Thrombinoscope-type thrombin generation assay (TGA); in contrast, 1 μM hirudin variant 3 or soluble API M358R abolished thrombin generation. Gene transfer of TF to HEK 293 conferred the ability to support TF-dependent thrombin generation on HEK 293 cells. Co-transfection of HEK 293 cells with a 9:1 excess of DNA encoding AR-API M358R to that encoding TF reduced peak thrombin generation approximately 3-fold compared to controls. These in vitro results suggest that surface display of API M358R inhibits thrombin generation when the tethered serpin is expressed in excess of TF, and suggest its potential to limit thrombosis in appropriate vascular beds in animal models.

  10. Expression screening of bacterial libraries of recombinant alpha-1 proteinase inhibitor variants for candidates with thrombin inhibitory capacity. (United States)

    Bhakta, Varsha; Gierczak, Richard F; Sheffield, William P


    Exhaustive mutagenesis studies of the reactive centre loop (RCL), a key structural component of proteins belonging to the serpin superfamily of protease inhibitors, are complicated by the size of the RCL, serpin conformational complexity, and, for most serpins, the lack of a serpin-dependent phenotype of expressing cells. Here, we describe a thrombin capture assay that distinguished thrombin-inhibitory recombinant human alpha-1 proteinase inhibitor (API M358R) from non-inhibitory API variants in Escherichia coli lysates prepared from either single clones or pools. Binding of API proteins in the lysates to thrombin immobilized on microtiter plate wells was quantified via colour generated by a peroxidase-coupled anti-API antibody. Bacterial expression plasmids encoding inhibitory API M358R were mixed 1:99 with plasmids encoding non-inhibitory API T345R/M358R and the resulting library screened in pools of 10. All above-background signals arising from pools or subsequently re-probed single clones were linked to the presence of plasmids encoding API M358R. Screening of a portion of another expression library encoding hypervariable API with all possibilities at codons 352-358 also yielded only novel, thrombin-inhibitory variants. Probing a smaller library expressing all possible codons at Ala347 yielded the wild type, 6 different functional variants, one partially active variant, and two variants with no thrombin-inhibitory activity. API antigen levels varied considerably less among Ala347 variants than activity levels, and comparison of rate constants of inhibition of purified API variants to their corresponding thrombin capture assay lysate values was used to establish the sensitivity and specificity of the assay. The results indicate that the approach is sufficiently robust to correctly identify functional versus non-functional candidates in API expression libraries, and could be of value in systematically probing structure/function relationships not only in the API

  11. Safety and pharmacokinetics of 120 mg/kg versus 60 mg/kg weekly intravenous infusions of alpha-1 proteinase inhibitor in alpha-1 antitrypsin deficiency: a multicenter, randomized, double-blind, crossover study (SPARK). (United States)

    Campos, Michael A; Kueppers, Friedrich; Stocks, James M; Strange, Charlie; Chen, Junliang; Griffin, Rhonda; Wang-Smith, Laurene; Brantly, Mark L


    Augmentation therapy with the approved dose of 60 mg/kg weekly intravenous (IV) alpha-1 proteinase inhibitor (alpha1-PI), achieves a trough serum level of 11 μM in individuals with alpha-1 antitrypsin deficiency (AATD), yet this is still below the level observed in healthy individuals. This study assessed the safety and pharmacokinetic profile of weekly infusions of a 120 mg/kg dose of alpha1-PI in 30 adults with AATD. Subjects with symptomatic, genetically determined (genotypes PI*ZZ, PI*Z(null), PI*(null)(null) or PI*(Z)Mmalton) AATD were randomly assigned to weekly infusions of 60 or 120 mg/kg alpha1-PI (Prolastin-C®) for 8 weeks before crossing over to the alternate dose for 8 weeks. Adverse events (AEs) (including exacerbations), vital signs, pulmonary function tests, and laboratory assessments were recorded. Pharmacokinetic measurements included AUC0-7days, Cmax, trough, tmax, and t1/2, based on serum alpha1-PI concentrations. In total for both treatments, 112 AEs were reported, with exacerbation of COPD being the most frequent, consistent with the subjects' diagnoses. Mean steady-state serum alpha1-PI concentrations following 120 mg/kg weekly IV alpha1-PI were higher than with the 60 mg/kg dose and mean trough concentrations were 27.7 versus 17.3 μM, respectively. Dose proportionality was demonstrated for AUC0-7days and Cmax, with low inter-subject variability. The 120 mg/kg alpha1-PI weekly dose was considered to be safe and well tolerated, and provided more favorable physiologic alpha1-PI serum levels than the currently recommended 60 mg/kg dose. The effect of this dosing regimen on slowing and/or preventing emphysema progression in subjects with AATD warrants further investigation.

  12. Comparison of mammalian and bacterial expression library screening to detect recombinant alpha-1 proteinase inhibitor variants with enhanced thrombin inhibitory capacity. (United States)

    Gierczak, Richard F; Bhakta, Varsha; Xie, Michael; Sheffield, William P


    Serpins are a widely distributed family of serine proteases. A key determinant of their specificity is the reactive centre loop (RCL), a surface motif of ∼20 amino acids in length. Expression libraries of variant serpins could be rapidly probed with proteases to develop novel inhibitors if optimal systems were available. The serpin variant alpha-1 proteinase inhibitor M358R (API M358R) inhibits the coagulation protease thrombin, but at sub-maximal rates compared to other serpins. Here we compared two approaches to isolate functional API variants from serpin expression libraries, using the same small library of API randomized at residue 358 (M358X): flow cytometry of transfected HEK 293 cells expressing membrane-displayed API; and a thrombin capture assay (TCA) performed on pools of bacterial lysates expressing soluble API. No enrichment for specific P1 residues was observed when the RCL codons of the 1% of sorted transfected 293 cells with the highest fluorescent thrombin-binding signals were subcloned and sequenced. In contrast, screening of 16 pools of bacterial API-expressing transformants led to the facile identification of API M358R and M358K as functional variants. Kinetic characterization showed that API M358R inhibited thrombin 17-fold more rapidly than API M358K. Reducing the incubation time with immobilized thrombin improved the sensitivity of TCA to detect supra-active API M358R variants and was used to screen a hypervariable library of API variants expressing 16 different amino acids at residues 352-357. The most active variant isolated, with TLSATP substituted for FLEAI, inhibited thrombin 2.9-fold more rapidly than API M358R. Our results indicate that flow cytometric approaches used in protein engineering of antibodies are not appropriate for serpins, and highlight the utility of the optimized TCA for serpin protein engineering.

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

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    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

  14. 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

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    Sveger Tomas


    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.

  15. Safety and tolerability of an intravenously administered alpha1-proteinase inhibitor at an increased infusion rate: a novel, randomized, placebo-masked, infusion rate-controlled, crossover study in healthy adults

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    Ngo LY


    Full Text Available Leock Y Ngo,1 Adam Haeberle,1 Jacqueline Dyck-Jones,1 David Gelmont,1 Leman Yel11Baxter Healthcare Corporation, Westlake Village, CA, USAPurpose: Alpha1-proteinase inhibitor (A1PI is indicated for chronic augmentation therapy in adults with emphysema due to congenital deficiency of A1PI. An intravenous infusion rate of 0.04 mL/kg/minute is currently recommended for the A1PI product, Glassia®. This randomized, placebo-masked, rate-controlled, crossover study was designed to evaluate the safety and tolerability of A1PI administration at an increased infusion rate.Patients and methods: A total of 30 healthy male and female subjects aged 19–61 years were enrolled. Each subject received simultaneous intravenous infusions of A1PI (Glassia® and placebo (human albumin 2.5% administered through a single infusion site on two separate treatment periods. Subjects were randomized in a 1:1 ratio to receive either test treatment (A1PI 0.2 mL/kg/minute + placebo 0.04 mL/kg/minute, or reference treatment (A1PI 0.04 mL/kg/minute + placebo 0.2 mL/kg/minute on Day 1. On Day 15, subjects received the other treatment regimen in a crossover sequence.Results: A total of 36 adverse events (AEs, regardless of causality, were reported; all were non-serious and of mild intensity, with headaches and dizziness occurring most frequently (12 [33.3%] and three [8.3%] of 36 AEs, respectively. Only seven AEs in six subjects were assessed as related to study treatment: with two AEs reported in two subjects treated with the 0.2 mL/kg/minute rate compared with five AEs in four subjects treated with the 0.04 mL/kg/minute rate.Conclusions: This study demonstrated the safety and tolerability of an A1PI product at an increased infusion rate (0.2 mL/kg/minute resulting in a shorter infusion duration in healthy subjects.Keywords: A1PI, Glassia, administration rate, Alpha-1 antitrypsin, ATT

  16. Proteinase inhibitors in Brazilian leguminosae

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    C. A. M. Sampaio


    Full Text Available Serine proteinase inhitors, in the seeds of several Leguminosae from the Pantanal region (West Brazil, were studied using bovine trypsin, a digestive enzyme, Factor XIIa and human plasma Kallikrein, two blood clotting factors. The inhibitors were purified from Enterolobium contortisiliquum (Mr=23,000, Torresea cearensis (Mr = 13,000, Bauhinia pentandra (Mr = 20,000 and Bauhinia bauhinioides (Mr = 20,000. E. contortisiliquum inhibitor inactivates all three enzymes, whereas the T. cearensis inhibitor inactivates trypsin and Factor XSSa, but does nor affect plasma kallikrein; both Bauhinia inhibitors, on the other hand, inactivate trypsin and plasma kallikrein but only the Bpentandra inhibitor affects Factor XIIa. Ki values were calculated between 10 [raised to the power of] -7 and 10 [raised to the power of] -8 M.


    NARCIS (Netherlands)



    In the sera of patients with Wegener's granulomatosis (WG), C-ANCA can be detected that are directed against proteinase 3 (PR3). We have previously observed that C-ANCA interfere with PR3 proteolytic activity and with complexation of PR3 with its major physiologic inhibitor, alpha1-antitrypsin (alph

  18. Evolutionary mechanisms acting on proteinase inhibitor variability. (United States)

    Christeller, John T


    The interaction of proteinase inhibitors produced, in most cases, by host organisms and the invasive proteinases of pathogens or parasites or the dietary proteinases of predators, results in an evolutionary 'arms race' of rapid and ongoing change in both interacting proteins. The importance of these interactions in pathogenicity and predation is indicated by the high level and diversity of observable evolutionary activity that has been found. At the initial level of evolutionary change, recruitment of other functional protein-folding families has occurred, with the more recent evolution of one class of proteinase inhibitor from another, using the same mechanism and proteinase contact residues. The combination of different inhibitor domains into a single molecule is also observed. The basis from which variation is possible is shown by the high rate of retention of gene duplication events and by the associated process of inhibitory domain multiplication. At this level of reorganization, mutually exclusive splicing is also observed. Finally, the major mechanism by which variation is achieved rapidly is hypervariation of contact residues, an almost ubiquitous feature of proteinase inhibitors. The diversity of evolutionary mechanisms in a single class of proteins is unlikely to be common, because few systems are under similar pressure to create variation. Proteinase inhibitors are therefore a potential model system in which to study basic evolutionary process such as functional diversification.

  19. Proteolytic Cleavage of Various Human Serum Proteinase Inhibitors by Candida albicans Aspartic Proteinase


    Tsushima, Hirofumi; MINE, Hiroko


    The secreted Candida albicans aspartic proteinase (SAP) is presumed to be one of the putative Candida virulence factors, while serum proteinase inhibitors depend on host defense mechanisms. We examined the interaction between SAP and serum proteinase inhibitors, such as C1-inhibitor, α2 plasmin inhibitor, and antithrombin III. SAP progressively inactivated plasmin inhibitory activity of C1-inhibitor and α2 plasmin inhibitor. It also inactivated thrombin inhibitory activity of antithrombin III...

  20. Comparison of concentrations of two proteinase inhibitors, porcine pancreatic elastase inhibitory capacity, and cell profiles in sequential bronchoalveolar lavage samples. (United States)

    Morrison, H M; Kramps, J A; Dijkman, J H; Stockley, R A


    Bronchoalveolar lavage is used to obtain cells and proteins from the lower respiratory tract for diagnosis and research. Uncertainity exists about which site in the lung is sampled by the lavage fluid and what effect different lavage volumes have on recovery of the constituents of lavage fluid. Dilution of alveolar lining fluid by lavage fluid is variable and results are usually expressed as protein ratios to surmount this problem. We have compared cell profiles and the concentrations of two proteinase inhibitors--the low molecular weight bronchial protease inhibitor antileucoprotease and alpha 1 proteinase inhibitor, together with alpha 1 proteinase inhibitor function and its relationship to the cell profile in sequential bronchoalveolar lavage fluid samples from patients undergoing bronchoscopy. There was no difference in total or differential cell counts or albumin or alpha 1 proteinase inhibitor concentrations between the first and second halves of the lavage. Both the concentration of antileucoprotease and the ratio of antileucoprotease to albumin were, however, lower in the second half of the lavage (2p less than 0.01 and 2p less than 0.05 respectively). There was no difference in the function of alpha 1 proteinase inhibitor (assessed by inhibition of porcine pancreatic elastase--PPE) between aliquots (0.28 mole PPE inhibited/mol alpha 1 proteinase inhibitor; range 0-1.19 for the first half and 0.37 mol PPE inhibited/mol alpha 1 proteinase inhibitor; range 0.10-0.80 for the second half). About 60-70% of alpha 1 proteinase inhibitor in each half of the lavage fluid was inactive as an inhibitor. The function of alpha 1 proteinase inhibitor did not differ between bronchitic smokers and ex-smokers. Alpha 1 proteinase inhibitor function was not related to the number of total white cells, macrophages, or neutrophils in the lavage fluid. Contamination of lavage by red blood cells was found to alter the concentration of alpha 1 proteinase inhibitor but not its

  1. Novel proteinase inhibitor promotes resistance to insects (United States)

    A novel Beta vulgaris serine proteinase inhibitor gene (BvSTI) and its protein are identified in response to insect feeding on B. vulgaris seedlings. BvSTI is cloned into an expression vector with constitutive promoter and transformed into Nicotiana benthamiana plants to assess BvSTI’s ability to ...

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

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


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

  3. Domain 15 of the serine proteinase inhibitor LEKTI blocks HIV infection in vitro

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    David Palesch


    Full Text Available Background: Lympho-epithelial Kazal-type-related inhibitor (LEKTI is a 15-domain serine proteinase inhibitor, parts of which have first been isolated from human blood filtrate. It is encoded by the gene SPINK5. In the past, different groups reported antiviral activities of certain serine proteinase inhibitors, such as mucous proteinase inhibitor and alpha1-proteinase inhibitor. The purpose of this study was to test two representative domains of the proteinase inhibitor LEKTI for anti-HIV activities.Methods: LEKTI domains 6 and 15 were recombinantly produced in E.coli. To test their inhibitory activity against HIV infection, the reporter cell line P4-R5 MAGI carrying an HIV-inducible reporter gene was infected by a CCR5-tropic HIV strain in the presence of different inhibitor concentrations. After three days, infection rates were determined by quantifying ß-galactosidase activities using the Galacto-Light Plus™ ß-Galactosidase Reporter Gene Assay.Results: In contrast to LEKTI domain 6, LEKTI domain 15 suppressed HIV-induced reporter gene activities with an IC50 value of approximately 29 µM.Conclusion: LEKTI domain 15 represents an inhibitor of HIV infection. (Med J Indones. 2013;22:131-5. doi: 10.13181/mji.v22i3.580Keywords: HIV, inhibition, LEKTI, P4-R5 MAGI

  4. Granzyme M is a regulatory protease that inactivates proteinase inhibitor 9, an endogenous inhibitor of granzyme B. (United States)

    Mahrus, Sami; Kisiel, Walter; Craik, Charles S


    Granzyme M is a trypsin-fold serine protease that is specifically found in the granules of natural killer cells. This enzyme has been implicated recently in the induction of target cell death by cytotoxic lymphocytes, but unlike granzymes A and B, the molecular mechanism of action of granzyme M is unknown. We have characterized the extended substrate specificity of human granzyme M by using purified recombinant enzyme, several positional scanning libraries of coumarin substrates, and a panel of individual p-nitroanilide and coumarin substrates. In contrast to previous studies conducted using thiobenzyl ester substrates (Smyth, M. J., O'Connor, M. D., Trapani, J. A., Kershaw, M. H., and Brinkworth, R. I. (1996) J. Immunol. 156, 4174-4181), a strong preference for leucine at P1 over methionine was demonstrated. The extended substrate specificity was determined to be lysine = norleucine at P4, broad at P3, proline > alanine at P2, and leucine > norleucine > methionine at P1. The enzyme activity was found to be highly dependent on the length and sequence of substrates, indicative of a regulatory function for human granzyme M. Finally, the interaction between granzyme M and the serpins alpha(1)-antichymotrypsin, alpha(1)-proteinase inhibitor, and proteinase inhibitor 9 was characterized by using a candidate-based approach to identify potential endogenous inhibitors. Proteinase inhibitor 9 was effectively hydrolyzed and inactivated by human granzyme M, raising the possibility that this orphan granzyme bypasses proteinase inhibitor 9 inhibition of granzyme B.

  5. Inhibition of the 20S proteosome by a protein proteinase inhibitor: evidence that a natural serine proteinase inhibitor can inhibit a threonine proteinase. (United States)

    Yabe, Kimihiko; Koide, Takehiko


    The 20S proteasome (20S) is an intracellular threonine proteinase (Mr 750,000) that plays important roles in many cellular regulations. Several synthetic peptide inhibitors and bacteria-derived inhibitors such as lactacystin and epoxomicin have been identified as potent proteasome inhibitors. However, essentially no protein proteinase inhibitor has been characterized. By examining several small size protein proteinase inhibitors, we found that a well-known serine proteinase inhibitor from bovine pancreas, basic pancreatic trypsin inhibitor (BPTI), inhibits the 20S in vitro and ex vivo. Inhibition of the 20S by BPTI was time- and concentration-dependent, and stoichiometric. To inhibit the 20S activity, BPTI needs to enter into the interior of the 20S molecule. The molar ratio of BPTI to the 20S in the complex was estimated as approximately six BPTI to one 20S, thereby two sets of three peptidase activities (trypsin-like, chymotrypsin-like and caspase-like) of the 20S were all inhibited. These results indicate that an entrance hole to the 20S formed by seven alpha-subunits is sufficiently large for BPTI to enter. This report is essentially the initial description of the inhibition of a threonine proteinase by a protein serine proteinase inhibitor, suggesting a common mechanism of inhibition between serine and threonine proteinases by a natural protein proteinase inhibitor.

  6. Proteinase activity in human and murine saliva as a biomarker for proteinase inhibitor efficacy. (United States)

    Fingleton, Barbara; Menon, Ramkumar; Carter, Kathy J; Overstreet, P Dawn; Hachey, David L; Matrisian, Lynn M; McIntyre, J Oliver


    As molecularly targeted agents reach the clinic, there is a need for assays to detect their presence and effectiveness against target molecules in vivo. Proteinase inhibitors are one example of a class of therapeutic agent for which satisfactory methods of identifying successful target modulation in vivo are lacking. This is of particular importance while these drugs are in clinical trials because standard maximum-tolerated dose-finding studies often are not suitable due to lack of toxicity. Saliva represents a readily accessible bodily fluid that can be repeatedly sampled and used for assaying in vivo effects of systemic drugs. Here we show the development of a simple assay that can be used to measure proteinase activity in saliva and proteinase inhibition after systemic treatment with three different proteinase inhibitors. A variety of gelatinolytic activities present in human and murine saliva have been assayed with a fluorescent dye-labeled substrate and assigned to different proteinase categories by inclusion of specific classes of inhibitors. Treatment of mice with either matrix metalloproteinase inhibitors or a urokinase inhibitor for a period as short as 48 hours results in levels of the drugs that can be detected in saliva by mass spectrometry and concomitant decreases in salivary proteinase activity, thus demonstrating that these inhibitors successfully modulate their targets in vivo.

  7. Molecular dynamic and docking interaction study of Heterodera glycines serine proteinase with Vigna mungo proteinase inhibitor. (United States)

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


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

  8. Action of plant proteinase inhibitors on enzymes of physiopathological importance. (United States)

    Oliva, Maria Luiza V; Sampaio, Misako U


    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.

  9. A serine proteinase inhibitor from frog eggs with bacteriostatic activity. (United States)

    Han, Yaoping; Yu, Haining; Yang, Xinbo; Rees, Huw H; Liu, Jingze; Lai, Ren


    By Sephadex G-50 gel filtration, Resource Q anionic exchange and C4 reversed phase liquid high performance liquid chromatography, a proteinase inhibitor protein (Ranaserpin) was identified and purified from the eggs of the odour frog, Rana grahami. The protein displayed a single band adjacent to the molecular weight marker of 14.4 kDa analyzed by SDS-PAGE. The inhibitor protein homogeneity and its molecular weight were confirmed again by MALDI-TOF mass spectrometry analysis. The MALDI-TOF mass spectrum analysis gave this inhibitor protein an m/z of 14422.26 that was matched well with the result from SDS-PAGE. This protein is a serine proteinase inhibitor targeting multiple proteinases including trypsin, elastase, and subtilisin. Ranaserpin inhibited the proteolytic activities of trypsin, elastase, and subtilisin. It has an inhibitory constant (K(i)) of 6.2 x 10(-8) M, 2.7 x 10(-7) M and 2.2 x 10(-8) M for trypsin, elastase, and subtilisin, respectively. This serine proteinase inhibitor exhibited bacteriostatic effect on Gram-positive bacteria Bacillus subtilis (ATCC 6633). It was suggested that ranaserpin might act as a defensive role in resistance to invasion of pests or pathogens. This is the first report of serine proteinase inhibitor and its direct defensive role from amphibian eggs.

  10. Antiviral cytokines induce hepatic expression of the granzyme B inhibitors, proteinase inhibitor 9 and serine proteinase inhibitor 6. (United States)

    Barrie, Mahmoud B; Stout, Heather W; Abougergi, Marwan S; Miller, Bonnie C; Thiele, Dwain L


    Expression of the granzyme B inhibitors, human proteinase inhibitor 9 (PI-9), or the murine orthologue, serine proteinase inhibitor 6 (SPI-6), confers resistance to CTL or NK killing by perforin- and granzyme-dependent effector mechanisms. In light of prior studies indicating that virally infected hepatocytes are selectively resistant to this CTL effector mechanism, the present studies investigated PI-9 and SPI-6 expression in hepatocytes and hepatoma cells in response to adenoviral infection and to cytokines produced during antiviral immune responses. Neither PI-9 nor SPI-6 expression was detected by immunoblotting in uninfected murine or human hepatocytes. Similarly, human Huh-7 hepatoma cells were found to express only very low levels of PI-9 relative to levels detected in perforin- and granzyme-resistant CTL or lymphokine-activated killer cells. Following in vivo adenoviral infection or in vitro culture with IFN-alphabeta or IFN-gamma, SPI-6 expression was induced in murine hepatocytes. Similarly, after culture with IFN-alpha, induction of PI-9 mRNA and protein expression was observed in human hepatocytes and Huh-7 cells. IFN-gamma and TNF-alpha also induced 4- to 10-fold higher levels of PI-9 mRNA expression in Huh-7 cells, whereas levels of mRNA encoding a related serine proteinase inhibitor, proteinase inhibitor 8, were unaffected by culture of Huh-7 cells with IFN-alpha, IFN-gamma, or TNF-alpha. These findings indicate that cytokines that promote antiviral cytopathic responses also regulate expression of the cytoprotective molecules, PI-9 and SPI-6, in hepatocytes that are potential targets of CTL and NK effector mechanisms.

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

    Directory of Open Access Journals (Sweden)

    Maria Luiza V. Oliva


    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.

  12. Identification and characterization of alpha-I-proteinase inhibitor from common carp sarcoplasmic proteins. (United States)

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


    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%.

  13. Silk gland-specific proteinase inhibitor serpin16 from the Bombyx mori shows cysteine proteinase inhibitory activity. (United States)

    Guo, Peng-Chao; Dong, Zhaoming; Xiao, Li; Li, Tao; Zhang, Yan; He, Huawei; Xia, Qingyou; Zhao, Ping


    Serpins (serine proteinase inhibitors) are widely distributed in different species and are well known for their inhibitory activities towards serine proteinases. Here, we report the functional characterization of Bombyx mori serpin16. Expression analysis showed that serpin16 was specifically expressed at high levels in the silk gland at both the transcriptional and translational levels. Moreover, homology modeling and multi-sequence alignment suggested that serpin16 had a canonical serpin fold, but it contained a unique reactive center loop, which was obviously shorter than that of typical serpins. Inhibitory activity analyses revealed that the target proteinase of serpin18 is a cysteine proteinase, rather than a serine proteinase. Furthermore, a Michaelis complex model of serpin16 with its target proteinase was constructed to explain the structural basis of how serpin16 recognizes the cysteine proteinase and its target specificity.


    Directory of Open Access Journals (Sweden)

    Ana Maria Abreu Velez


    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.

  15. Immunomodulation by α(1)-proteinase inhibitor: lack of chemotactic effects of recombinant human α(1)-proteinase inhibitor from yeast on human peripheral blood granulocytes


    Mosheimer, Birgit; Alzner, Reinhard; Wiedermann, Christian J.


    Introduction: Recombinant α(1)-proteinase inhibitor, clinically developed for inhalative augmentation therapy in patients with α(1)-proteinase inhibitor deficiency or cystic fibrosis, may directly contribute to leukocyte accumulation as it may function as a chemoattractant. The migratory effects of yeast-derived human recombinant α(1)-proteinase inhibitor on human peripheral blood neutrophils and eosinophils were therefore tested in vitro. Materials and Methods: Human peripheral blood leukocy...

  16. Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor. (United States)

    Odei-Addo, Frank; Frost, Carminita; Smith, Nanette; Ogawa, Tomohisa; Muramoto, Koji; Oliva, Maria Luiza Vilela; Gráf, László; Naude, Ryno


    One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10 kDa, respectively, and under non-reducing conditions, 26 kDa was observed. The inhibitor was shown to inhibit bovine trypsin (Ki of 3.45 nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor.

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

    DEFF Research Database (Denmark)

    Andersen, J T


    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...... of 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...... or unwilling to undergo surgical resection of the prostate will benefit from such therapy....

  18. Characterization of a novel Kazal-type serine proteinase inhibitor of Arabidopsis thaliana. (United States)

    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


    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.

  19. The granzyme B inhibitor proteinase inhibitor 9 (PI9) is expressed by human mast cells.

    NARCIS (Netherlands)

    Bladergroen, B.A.; Strik, M.C.; Wolbink, A.M.; Wouters, D.; Broekhuizen, R.; Kummer, J.A.; Hack, C.E.


    The activity of granzyme B, a main effector molecule of cytotoxic T lymphocytes (CTL) and natural killer cells, is regulated by the human intracellular serpin proteinase inhibitor 9 (PI9). This inhibitor is particularly expressed by CTL and dendritic cells, in which it serves to protect these cells

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

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


    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, bovine trypsin (EC, papain (EC, 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.

  1. 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.;


    Proteins of the serpin superfamily (similar to43 kDa) from mature cereal grains are in vitro suicide-substrate inhibitors of specific mammalian serine proteinases of the chymotrypsin family. However, unlike the 'standard-mechanism' serine proteinase inhibitors (

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

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


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

  3. Serine proteinase inhibitors in the Compositae: distribution, polymorphism and properties. (United States)

    Konarev, Alexander V; Anisimova, Irina N; Gavrilova, V A; Vachrusheva, T E; Konechnaya, G Yu; Lewis, Mervyn; Shewry, Peter R


    Multiple molecular forms of inhibitors of trypsin (TI) and chymotrypsin (CI), which are typical digestive enzymes of insects, mammals and micro-organisms, and subtilisin (SI), a proteinase of many bacteria and phytopathogenic fungi, were identified in seeds and vegetative organs of the majority of 128 wild and cultivated species representing 65 genera of three of the subfamilies of the Compositae. Inhibitors with M(r) ranging from 7450 to 7800 and combining activities towards subtilisin and trypsin and/or chymotrypsin (T/C/SI) had the widest distribution and may be involved in plant defense mechanisms. They were found in many species of the subfamilies Carduoideae (genera Carthamus, Centaurea, Cirsium), Cichorioideae (Lactuca, Taraxacum) and Asteroideae (Helianthus, Cosmos, Bidens). Partial amino acid sequencing showed that the safflower (Carthamus tinctorius) T/C/SI and Cosmos bipinnatus T/C/SI, T/SI and C/SI belonged to the potato I inhibitor family. The most active, variable and heterogeneous inhibitors were found in species of the tribe Heliantheae, which is placed in the evolutionary advanced subfamily Asteroideae. Seeds of Helianthus species, Eclipta prostrata, Gailardia aristata, Zinnia elegans and Silphium perfoliatum contained various TI with M(r) ranging from 1500 to 14,750, with some also containing SI. H. annuus seeds contain a unique cyclic TI of M(r) 1514 and similar TI were also present in other Helianthus spp. and the related species Tithonia diversifolia. Zinnia elegans contained a TI with M(r) 11,350 which appeared to represent a novel type of inhibitor distantly related to the cereal subgroup of Bowman-Birk inhibitors. TI and T/SI varied widely in H. annuus lines and wild Helianthus species in their presence or absence and composition. Similar T/SI components were found in the cultivated diploid H. annuus and annual diploid species with the B genome but not in perennials with the A genome. Some T/SI, SI and TI were detected in vegetative organs

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

    NARCIS (Netherlands)

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


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

  5. Effect of Recombinant alpha1-Antitrypsin Fc-Fused (AAT-Fc)Protein on the Inhibition of Inflammatory Cytokine Production and Streptozotocin-Induced Diabetes

    NARCIS (Netherlands)

    Lee, S.; Lee, Y.; Hong, K.; Hong, J.; Bae, S.; Choi, J.; Jhun, H.; Kwak, A.; Kim, E.; Jo, S.; Dinarello, C.A.; Kim, S.


    alpha1-Antitrypsin (AAT) is a member of the serine proteinase inhibitor family that impedes the enzymatic activity of serine proteinases, including human neutrophil elastase, cathepsin G and neutrophil proteinase 3. Here, we expressed recombinant AAT by fusing the intact AAT gene to the constant reg

  6. Structure and function of invertebrate Kazal-type serine proteinase inhibitors. (United States)

    Rimphanitchayakit, Vichien; Tassanakajon, Anchalee


    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  8. Stress inducible proteinase inhibitor diversity in Capsicum annuum

    Directory of Open Access Journals (Sweden)

    Mishra Manasi


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

  9. Purification and partial characterization of α1-proteinase inhibitor in the common marmoset (Callithrix jacchus). (United States)

    Parambeth, Joseph Cyrus; Suchodolski, Jan S; Steiner, Jörg M


    Fecal alpha1-proteinase inhibitor (α1-PI) concentration has been to diagnose enteric protein loss in dogs and cats. Chronic lymphocytic enteritis is commonly seen in the marmoset (Callithrix jaccus) and is characterized by hypoalbuminemia. As a prelude to immunoassay development for detecting enteric protein loss, marmoset serum α1-PI was purified using immunoaffinity chromatography and ceramic hydroxyapatite chromatography. Partial characterization was performed by reducing gel electrophoresis and enzyme inhibitory assays. Protein identity was confirmed with peptide mass fingerprinting and N-terminal amino acid sequencing. Molecular mass, relative molecular mass, and isoelectric point for marmoset α1-PI were 54 kDa, 51,677, and 4.8-5.4, respectively. Trypsin, chymotrypsin, and elastase inhibitory activity were observed. N-terminal amino acid sequence for marmoset α1-PI was EDPQGDAAQKMDTSHH. In conclusion, marmoset α1-PI was successfully purified from serum with an overall yield of 12% using a rapid and efficient method. Purified marmoset α1-PI has characteristics similar to those of α1-PI reported for other species.

  10. Cloning of a serine proteinase inhibitor from bovine brain: expression in the brain and characterization of its target proteinases. (United States)

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


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

  11. General up regulation of Spodoptera frugiperda trypsins and chymotrypsins allows its adaptation to soybean proteinase inhibitor. (United States)

    Brioschi, Daniela; Nadalini, Larissa D; Bengtson, Mario H; Sogayar, Mari Cleide; Moura, Daniel S; Silva-Filho, Marcio C


    The existence of a diverse serine proteinase gene family in lepidopteran insects suggests they play a significant role in the insect adaptation to plant proteinase inhibitors. These proteinases have been shown to be involved in the process of proteolytic digestion in insect larvae. We carried out a selective transcriptome study of midguts from Spodoptera frugiperda larvae fed on a diet supplemented with soybean proteinase inhibitor (SPI). Using subtracted cDNA libraries made of gut-expressed transcripts, a total of 2100 partial sequences were obtained, of those 38% were related to digestive process. Two large and diverse groups of chymotrypsins and trypsins were obtained, and some of these proteinase-encoding genes were further characterized by quantitative RT-PCR. The transcription analyses revealed two groups: one group of genes constitutively expressed in the control larvae that is up regulated by introducing SPI to the diet, and a second group that is absent in the control but is induced by the SPI-rich diet. This observation suggests that adaptation of S. frugiperda to SPI involves de novo synthesis and also up regulation of existing enzymes. Proteases from intestines of larvae reared on a diet with SPI showed insensitivity to the inhibitor. The proteases were also insensitive to a broad-spectrum potato proteinase inhibitor preparation. We propose that adaptation of S. frugiperda to SPI follows a "shotgun" approach, based on a general up regulation of a large set of endoproteinases.

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

    Directory of Open Access Journals (Sweden)

    Lepelley Maud


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

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

    Directory of Open Access Journals (Sweden)

    Punt Peter J


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

  14. Serine proteinase inhibitors in seeds of Cycas siamensis and other gymnosperms. (United States)

    Konarev, Alexander V; Lovegrove, Alison; Shewry, Peter R


    Seeds of 32 species selected from two of the four major groups of gymnosperms, the ancient Cycadales and the economically important Coniferales, were analysed for inhibitors (I) of the serine proteinases trypsin (T), chymotrypsin (C), subtilisin (S) and elastase (E) using isoelectric focusing (IEF) combined with gelatin replicas. Subtilisin inhibitors were detected in 17 species, being particularly active in the Cycadales. Several species of the genera Cephalotaxus, Pseudotsuga and Cycas contained inhibitors active against elastase while strong CSTIs and CSIs were also present in Cycas pectinata and C. siamensis. No inhibitors were detected in seeds of Chamaecyparis, Thuja, Abies, Larix, Picea and Pinus spp. Serine proteinase inhibitors were purified from seeds of C. siamensis by affinity chromatography using trypsin and chymotrypsin, IEF and SDS-PAGE. Several CSTI components with M(r) ranging from 4000 to 18,000 were partially sequenced using Edman degradation and mass spectrometry. Most of the sequences were similar to a hypothetical protein encoded by an mRNA from sporophylls of C. rumphii which in turn was similar to Kunitz-type proteinase inhibitors from flowering plants. Analysis of expressed sequence tag (EST) databases confirmed the presence of mRNAs encoding Kunitz-type inhibitors in the Cycadales and Coniferales and also demonstrated their presence in a third major group of gymnosperms, the Ginkgoales. This is the first report of Kunitz-type serine proteinase inhibitors from plants other than Angiosperms.

  15. 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;


    Secretory leukocyte proteinase inhibitor (SLPI) is a well-established inhibitor of serine proteases such as human neutrophil elastase (HNE) and a NF-κB regulatory agent in immune cells. In this paper, we report that SLPI plays a previously uncharacterized role in regulating activation...

  16. Several properties of the partially purified proteinase inhibitor in eggplant exocarp. (United States)

    Kanamori, M; Ibuki, F; Yamada, M; Tashiro, M; Miyoshi, M


    A proteinase inhibitor was isolated and partially purified from the exocarp of eggplant, Solanum melongena L., by means of acetate buffer extraction, heat treatment, salting-out and column chromatography on DEAE-cellulose. This preparation showed inhibitory activities on various proteinases; trypsin [EC] and Pronase were strongly inhibited while alpha-chymotrypsin [EC] and Nagarse were weakly inhibited. The inhibitor was a protein substance, and, therefore, it was gradually inactivated by the long-time incubation with Pronase. The inhibition mode was non-competitive on trypsin and competitive on Pronase on the basis of Lineweaver-Burk plots. The investigations on the inhibition behavior in the co-existence of two kinds of proteinases suggested that the inhibitor was not of multi-headed type.

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

    Directory of Open Access Journals (Sweden)

    Leah Theresa Sigle


    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.

  18. Changes in midgut endopeptidase activity of Spodoptera frugiperda (Lepidoptera: Noctuidae) are responsible for adaptation to soybean proteinase inhibitors. (United States)

    Paulillo, L C; Lopes, A R; Cristofoletti, P T; Parra, J R; Terra, W R; Silva-Filho, M C


    The development of transgenic maize plants expressing soybean proteinase inhibitors could reduce the economic damage of one of the major maize pests in Brazil, the fall armyworm, Spodoptera frugiperda (J.E. Smith, 1797). We examined the influence of soybean proteinase inhibitors on digestive enzyme properties and development of S. frugiperda larvae. The inhibition of trypsin and chymotrypsin activities in vitro by soybean proteinase inhibitors suggested that either Kunitz (SBTI) or Bowman-Birk (SBBI) would have a potential antimetabolic effect when ingested by insect larvae. However, chronic ingestion of semipurified soybean inhibitors did not result in a significant reduction of growth and development of fall armyworm. Therefore, digestive serine proteinase activities (trypsin and chymotrypsin) of fall armyworm larvae were characterized. The results suggest that S. frugiperda was able to physiologically adapt to dietary proteinase inhibitors by altering the complement of proteolytic enzymes in the insect midguts.

  19. LEKTI domain 15 is a functional Kazal-type proteinase inhibitor. (United States)

    Vitzithum, Klaus; Lauber, Thomas; Kreutzmann, Peter; Schulz, Axel; Sommerhoff, Christian P; Rösch, Paul; Marx, Ute C


    The multidomain proteinase inhibitor LEKTI (lympho-epithelial Kazal-type related inhibitor) consists of 15 potential serine proteinase inhibitory domains. In various diseases such as the severe skin disorder Netherton syndrome as well as atopy, defects in the gene encoding LEKTI have been identified that generate premature termination codons of translation, suggesting a specific role of the COOH-terminal part of LEKTI in healthy individuals. We overexpressed and purified a sequence comprising the 15th domain of LEKTI for further characterisation. Here, we present a high yield expression system for recombinant production and efficient purification of LEKTI domain 15 as a highly soluble protein with a uniform disulfide pattern that is identical to that of other known Kazal-type inhibitors. Also, the expected P1P1' site was confirmed. LEKTI domain 15 is a well-structured protein as verified by circular dichroism (CD) spectroscopy and a tight-binding and stable inhibitor of the serine proteinase trypsin. These findings confirm the designation of domain 15 as a proteinase inhibitor of the Kazal family.

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

    Directory of Open Access Journals (Sweden)

    Ann C Smigocki

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

  1. Pest protection conferred by a Beta vulgaris serine proteinase inhibitor gene. (United States)

    Smigocki, Ann C; Ivic-Haymes, Snezana; Li, Haiyan; Savić, Jelena


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

  2. Differential antibiosis against Helicoverpa armigera exerted by distinct inhibitory repeat domains of Capsicum annuum proteinase inhibitors. (United States)

    Joshi, Rakesh S; Gupta, Vidya S; Giri, Ashok P


    Plant defensive serine proteinase inhibitors (PIs) are known to have negative impact on digestive physiology of herbivore insects and thus have a crucial role in plant protection. Here, we have assessed the efficacy and specificity of three previously characterized inhibitory repeat domain (IRD) variants from Capsicum annuum PIs viz., IRD-7, -9 and -12 against gut proteinases from Helicoverpa armigera. Comparative study of in silico binding energy revealed that IRD-9 possesses higher affinity towards H. armigera serine proteinases as compared to IRD-7 and -12. H. armigera fed on artificial diet containing 5 TIU/g of recombinant IRD proteins exhibited differential effects on larval growth, survival rate and other nutritional parameters. Major digestive gut trypsin and chymotrypsin genes were down regulated in the IRD fed larvae, while few of them were up-regulated, this indicate alterations in insect digestive physiology. The results corroborated with proteinase activity assays and zymography. These findings suggest that the sequence variations among PIs reflect in their efficacy against proteinases in vitro and in vivo, which also could be used for developing tailor-made multi-domain inhibitor gene(s).

  3. Human neutrophil defensins and secretory leukocyte proteinase inhibitor in squamous metaplastic epithelium of bronchial airways.

    NARCIS (Netherlands)

    Aarbiou, J.; Schadewijk, A. van; Stolk, J.; Sont, J.K.; Boer, W.I.; Rabe, K.F.; Krieken, J.H.J.M. van; Mauad, T.; Hiemstra, P.S.


    OBJECTIVE: The aim of this study was to analyze a possible contribution of human neutrophil defensins and secretory leukocyte proteinase inhibitor (SLPI) to the induction of airway epithelial changes such as squamous cell metaplasia. MATERIALS AND METHODS: The presence of these molecules and the num

  4. Insect resistance to sugar beet pests mediated by a Beta vulgaris proteinase inhibitor transgene (United States)

    We transformed sugar beet (Beta vulgaris) hairy roots and Nicotiana benthamiana plants with a Beta vulgaris root gene (BvSTI) that codes for a serine proteinase inhibitor. BvSTI is a root gene cloned from the F1016 breeding line that has moderate levels of resistance to the sugar beet root maggot ...

  5. Insect and wound induced GUS gene expression from a Beta vulgaris proteinase inhibitor gene promoter (United States)

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

  6. 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;


    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...

  7. A 3D model of SARS_CoV 3CL proteinase and its inhibitors design by virtual screening

    Institute of Scientific and Technical Information of China (English)

    LUOCheng; CHENJing; LUOHai-Bin; CHENLi-Li; LIGuo-Wei; SUNTao; YUChang-Ying; YUELi-Duo; SHENJian-Hua; JIANGHua-Liang; XIONGBin; GUIChun-Shan; XUXiao-Ying; DUANWen-Hu; SHENJing-Kang; QINLei; SHITi-Liu; LIYi-Xue; CHENKai-Xian; LUOXiao-Min; SHENXu


    AIM:To constructed a three-dimensional (3D) model for the 3C like (3CL) proteinase of SARS coronavirus (SARS_CoV), and to design inhibitors of the 3CL proteinase based on the 3D model. METHODS: Bioinformatics analyses were performed to search the homologous proteins of the SARS_CoV 3CL proteinase from the GenBank and PDB database. A 3D model of the proteinase was constructed by using homology modeling technique. Targeting to the 3D model and its X-ray crystal structure of the main proteinase (Mpro) of transmissible gastroenteritis virus(TGEV), virtual screening was performed employing molecular docking method to identify possible 3CL proteinase inhibitors from small molecular databases. RESULTS:Sequence alignment indicated that the SARS_CoV 3CL proteinase was extremely homologous to TGEV Mpro, especially the substrate-binding pocket (active site). Accordingly, a 3D model for the SARS_CoV 3CL proteinase was constructed based on the crystal structure of TGEV Mpro. The 3D model adopts a similar fold of the TGEV mpro, its structure and binding pocket feature are almost as same as that of TGEV Mpro. The tested virtual screening indicated that 73 available proteinase inhibitors in the MDDR database might dock into both the binding pockets of the TGEV Mpro and the SARS_CoV 3CL proteinase. CONCLUSIONS:Either the 3D model of the SARS_CoV 3CL proteinase or the X-ray crystal stucture of the TGEV Mpro may be used as a starting point for design anti-SARS drugs. Screening the known proteinase inhibitors may be an appreciated shortcut to discover anti-SARS drugs.

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

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


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

  9. Solution Structure of the Squash Aspartic Acid Proteinase Inhibitor (SQAPI) and Mutational Analysis of Pepsin Inhibition (United States)

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


    The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NMR and show that SQAPI shares the same fold as a plant cystatin. The structure is characterized by a four-strand anti-parallel β-sheet gripping an α-helix in an analogous manner to fingers of a hand gripping a tennis racquet. Truncation and site-specific mutagenesis revealed that the unstructured N terminus and the loop connecting β-strands 1 and 2 are important for pepsin inhibition, but the loop connecting strands 3 and 4 is not. Using ambiguous restraints based on the mutagenesis results, SQAPI was then docked computationally to pepsin. The resulting model places the N-terminal strand of SQAPI in the S′ side of the substrate binding cleft, whereas the first SQAPI loop binds on the S side of the cleft. The backbone of SQAPI does not interact with the pepsin catalytic Asp32–Asp215 diad, thus avoiding cleavage. The data show that SQAPI does share homologous structural elements with cystatin and appears to retain a similar protease inhibitory mechanism despite its different target. This strongly supports our hypothesis that SQAPI evolved from an ancestral cystatin. PMID:20538608

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

    Directory of Open Access Journals (Sweden)

    Zeng-Fu Xu


    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.

  11. The characterization of SaPIN2b, a plant trichome-localized proteinase inhibitor from Solanum americanum. (United States)

    Luo, Ming; Ding, Ling-Wen; Ge, Zhi-Juan; Wang, Zhen-Yu; Hu, Bo-Lun; Yang, Xiao-Bei; Sun, Qiao-Yang; Xu, Zeng-Fu


    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. A practical total synthesis of the microbial alkaline proteinase inhibitor (MAPI). (United States)

    Haebich, Dieter; Hillisch, Alexander; El Sheikh, Sherif


    Diverse serine and cysteine proteases as well as alkaline proteinases and elastases play a crucial role in numerous biological processes. Natural peptide aldehydes such as the "microbial alkaline proteinase inhibitor" (MAPI, 1) are valuable tools to characterize novel enzymes and to study their function in nature. Within a drug discovery program we wanted to design and explore non-natural MAPI congeners with novel biological profiles. To that end we devised a simple, practical, and scalable synthesis of MAPI 1 from readily available amino acid building blocks. The modular nature of our approach allows convenient structural modification of the MAPI backbone.

  13. Alkaline proteinase inhibitor of Pseudomonas aeruginosa: a mutational and molecular dynamics study of the role of N-terminal residues in the inhibition of Pseudomonas alkaline proteinase. (United States)

    Feltzer, Rhona E; Trent, John O; Gray, Robert D


    Alkaline proteinase inhibitor of Pseudomonas aeruginosa is a 11.5-kDa, high affinity inhibitor of the serralysin class of zinc-dependent proteinases secreted by several Gram-negative bacteria. X-ray crystallography of the proteinase-inhibitor complex reveals that five N-terminal inhibitor residues occupy the extended substrate binding site of the enzyme and that the catalytic zinc is chelated by the alpha-amino and carbonyl groups of the N-terminal residue of the inhibitor. In this study, we assessed the effect of alteration of inhibitor residues 2-5 on its affinity for Pseudomonas alkaline proteinase (APR) as derived from the ratio of the dissociation and associate rate constants for formation of the enzyme-inhibitor complex. The largest effect was observed at position Ser-2, which occupies the S1' pocket of the enzyme and donates a hydrogen bond to the carboxyl group of the catalytic Glu-177 of the proteinase. Substitution of Asp, Arg, or Trp at this position increased the dissociation constant KD by 35-, 180-, and 13-fold, respectively. Mutation at positions 3-5 of the trunk also resulted in a reduction in enzyme-inhibitor affinity, with the exception of an I4W mutant, which exhibited a 3-fold increase in affinity. Molecular dynamics simulation of the complex formation between the catalytic domain of APR and the S2D mutant showed that the carboxyl of Asp-2 interacts with the catalytic zinc, thereby partially neutralizing the negative charge that otherwise would clash with the carboxyl group of Glu-177 of APR. Simulation of the interaction between the alkaline proteinase and the I4W mutant revealed a major shift in the loop comprised of residues 189-200 of the enzyme that allowed formation of a stacking interaction between the aromatic rings of Ile-4 of the inhibitor and Tyr-158 of the proteinase. This new interaction could account for the observed increase in enzyme-inhibitor affinity.

  14. The potency and specificity of the interaction between the IA3 inhibitor and its target aspartic proteinase from Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Phylip, L H; Lees, W E; Brownsey, B G;


    The yeast IA3 polypeptide consists of only 68 residues, and the free inhibitor has little intrinsic secondary structure. IA3 showed subnanomolar potency toward its target, proteinase A from Saccharomyces cerevisiae, and did not inhibit any of a large number of aspartic proteinases with similar...... sequences/structures from a wide variety of other species. Systematic truncation and mutagenesis of the IA3 polypeptide revealed that the inhibitory activity is located in the N-terminal half of the sequence. Crystal structures of different forms of IA3 complexed with proteinase A showed that residues...... by the nontarget aspartic proteinases, it was not cleaved by proteinase A. The random coil IA3 polypeptide escapes cleavage by being stabilized in a helical conformation upon interaction with the active site of proteinase A. This results, paradoxically, in potent selective inhibition of the target enzyme....

  15. Isolation and characterization of a proteinase inhibitor from marama beans. (United States)

    Elfant, M; Bryant, L; Starcher, B


    A protease inhibitor was purified from the African marama bean (Tylosema esculenturm). The inhibitor is present in large amounts, representing about 10.5% of the total protein. The molecular weight is slightly larger than soybean trypsin inhibitor and was estimated at 23,000 by SDS-gel electrophoresis or 24,500 by amino acid analysis. The amino acid composition was atypical of most other plant inhibitors with a cysteine content of only one or possibly two residues/mole and a blocked amino terminus. Inhibition studies indicated virtually no inhibition of chymotrypsin activity. Elastase, however, was inhibited to the same extent as trypsin, requiring about 2 moles of inhibitor for complete inhibition of the enzyme.

  16. Structurally unique recombinant Kazal-type proteinase inhibitor retains activity when terminally extended and glycosylated. (United States)

    Kludkiewicz, Barbara; Kodrík, Dalibor; Grzelak, Krystyna; Nirmala, Xavier; Sehnal, Frantisek


    Recombinant derivatives of the Kazal-type serine proteinase inhibitor GmSPI2 (36 amino acid residues), which is a component of insect silk, were prepared in the expression vector Pichia pastoris. The rhSPI2 had a C-terminal hexahistidine tag attached to the GmSPI2 sequence, rtSPI2 was extended with GluAlaAla at the N-terminus, and rfSPI2 included this N-terminal extension and a C-terminal tail of 22 residues (myc epitope and hexahistidine). A portion of the secreted rfSI2 was O-glycosylated with a trimannosyl or hexamannosyl. The native inhibitor was active slightly on trypsin and highly on subtilisin and proteinase K. The extended C-terminus in rhSPI2 and rfSPI2 enhanced activity on the two latter enzymes and rendered rfSPI2 active on elastase and pronase, but abolished the inhibition of trypsin. The glycosylation of rfSPI2 reduced its inhibitory activity to a level comparable with the native inhibitor. The rtSPI2 with tripeptide extension at the N-terminus and no C-terminal modification was clearly less active than the native inhibitor. None of the tested compounds inhibited alpha-chymotrypsin and the non-serine proteinases.

  17. A Kunitz proteinase inhibitor from corms of Xanthosoma blandum with bactericidal activity. (United States)

    Lima, Thaís B; Silva, Osmar N; Migliolo, Ludovico; Souza-Filho, Carlos R; Gonçalves, Eduardo G; Vasconcelos, Ilka M; Oliveira, José T A; Amaral, André C; Franco, Octávio L


    Bacterial infections directly affect the world's population, and this situation has been aggravated by indiscriminate use of antimicrobial agents, which can generate resistant microorganisms. In this report, an initial screening of proteins with antibacterial activity from corms of 15 species of the Xanthosoma genus was conducted. Since Xanthosoma blandum corms showed enhanced activity toward bacteria, a novel protein with bactericidal activity was isolated from this particular species. Edman degradation was used for protein N-termini determination; the primary structure showed similarities with Kunitz inhibitors, and this protein was named Xb-KTI. This protein was further challenged against serine proteinases from different sources, showing clear inhibitory activities. Otherwise, no hemolytic activity was observed for Xb-KTI. The results demonstrate the biotechnological potential of Xb-KTI, the first proteinase inhibitor with antimicrobial activity described in the Xanthosoma genus.

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

    Institute of Scientific and Technical Information of China (English)


    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.

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

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


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

  20. Characterization of the Proteinase that Initiates the Degradation of the Trypsin Inhibitor in Germinating Mung Beans (Vigna radiata). (United States)

    Wilson, K A; Tan-Wilson, A L


    The proteinase (proteinase F) responsible for the initial proteolysis of the mung bean (Vigna radiata) trypsin inhibitor (MBTI) during germination has been purified 1400-fold from dry beans. The enzyme acts as an endopeptidase, cleaving the native inhibitor, MBTI-F, to produce the first modified inhibitor form, MBTI-E. The cleavage of the Asp76-Lys77 peptide bond of MBTI-F occurs at a pH optimum of 4.5, with the tetrapeptide Lys-Asp-Asp-Asp being released. Proteinase F exhibited no activity against the modified inhibitor forms MBTI-E and MBTI-C. Vicilin, the major storage protein of the mung bean, does not serve as a substrate for proteinase F between pH 4 and 7. Proteinase F is inhibited by phenylmethylsulfonyl fluoride, chymostatin, p-hydroxymercuribenzoate, and p-chlorophenylsulfonate, but not by iodoacetate and CuCl(2). It is not activated by dithiothreitol, and is stable for extended periods of time (10 months, 4 degrees C, pH 4.0) in the absence of reducing agents. An apparent molecular weight of 65,000 was found for proteinase F by gel filtration. Subcellular fractionation in glycerol suggests that greater than 85% of the proteinase F activity is found in the protein bodies of the ungerminated mung bean. The same studies indicate that at least 56% of the MBTI of the seed is also localized in the protein bodies.

  1. Alpha-1 antitrypsin reduces ovariectomy-induced bone loss in mice (United States)

    Alpha-1antitrypsin (AAT) is a multifunctional protein with proteinase inhibitor and anti-inflammatory activities. Recent studies showed that AAT has therapeutic effect for diseases associated with inflammation, such as type 1 diabetes and arthritis. Proinflammatory cytokines are primary mediators of...

  2. 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


    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.

  3. 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. (United States)

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


    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.

  4. Determination of human serum alpha1-acid glycoprotein and albumin binding of various marketed and preclinical kinase inhibitors. (United States)

    Zsila, Ferenc; Fitos, Ilona; Bencze, Gyula; Kéri, György; Orfi, László


    There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been marketed already for the treatment of cancer. This time 139 validated kinase targets are in the focus of drug research of pharmaceutical companies and big efforts are made for the development of new, druglike kinase inhibitors. Plasma protein binding is an important factor of the ADME profiling of a drug compound. Human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma. Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach. HSA binding was also evaluated by affinity chromatography. Protein binding interactions of twenty-six kinase inhibitors are characterized. The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed. Structural, biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed.

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

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


    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.

  6. Suppression of collagen-induced arthritis with a serine proteinase inhibitor (serpin) derived from myxoma virus. (United States)

    Brahn, Ernest; Lee, Sarah; Lucas, Alexandra; McFadden, Grant; Macaulay, Colin


    Many viruses encode virulence factors to facilitate their own survival by modulating a host's inflammatory response. One of these factors, secreted from cells infected with myxoma virus, is the serine proteinase inhibitor (serpin) Serp-1. Because Serp-1 had demonstrated anti-inflammatory properties in arterial injury models and viral infections, it was cloned and evaluated for therapeutic efficacy in collagen-induced arthritis (CIA). Clinical severity was significantly lower in the Serp-1 protocols (pproteinase inhibitors in inflammatory joint diseases, such as rheumatoid arthritis, should be investigated further.

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

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


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

  8. Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph. (United States)

    Bania, Jacek; Samborski, Jaroslaw; Bogus, Mieczyslawa; Polanowski, Antoni


    The relatively little-investigated entomopathogen Conidiobolus coronatus secretes several proteinases into culture broth. Using a combination of ion-exchange and size-exclusion chromatography, we purified to homogeneity a serine proteinase of Mr 30,000-32,000, as ascertained by SDS-PAGE. The purified enzyme showed subtilisin-like activity. It very effectively hydrolyzed N-Suc-Ala(2)-Pro-Phe-pNa with a Km-1.36 x 10(-4) M and Kcat-24 s(-1), and N-Suc-Ala(2)-Pro-Leu-pNa with Km-6.65 x 10(-4) M and Kcat-11 s(-1). The specificity index k(cat)/K(m) for the tested substrates was calculated to be 176,340 s(-1) M(-1) and 17,030 s(-1) M(-1), respectively. Using oxidized insulin B chain as a substrate, the purified proteinase exhibited specificity to aromatic and hydrophobic amino-acid residues, such as Phe, Leu, and Gly at the P1 position, splitting primarily the peptide bonds: Phe(1)-Val(2), Leu(15)-Tyr(16), and Gly(23)-Phe(24). The proteinase appeared to be sensitive to the specific synthetic inhibitors of the serine proteinases DFP (diisopropyl flourophosphate) and PMSF (phenyl-methylsulfonyl fluoride) as well as to some naturally occurring protein inhibitors of chymotrypsin. It is worth noting that the enzyme exhibited the highest sensitivity to inhibition by AMCI-1 (with an association constant of 3 x 10(10) M(-1)), an inhibitor of cathepsin G/chymotrypsin from the larval hemolymph of Apis mellifera, reinforcing the possibility of involvement of inhibitors from hemolymph in insect innate immunity. The substrate specificity and proteinase inhibitor effects indicate that the purified proteinase from the fermentation broth of Conidiobolus coronatus is a subtilisin-like serine proteinase.

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

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


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

  10. Ethylene-regulated expression of a tomato fruit ripening gene encoding a proteinase inhibitor I with a glutamic residue at the reactive site.


    Margossian, L J; Federman, A D; Giovannoni, J.J.; Fischer, R L


    We report the isolation from tomato (Lycopersicon esculentum) of an ethylene-responsive member of the proteinase inhibitor gene family. DNA sequence analysis of a full-length cDNA clone indicates that the ethylene-responsive gene is distantly related to the tomato proteinase inhibitor I gene, having 53% sequence identity. The predicted amino acid sequence reveals 47% and 45% sequence identity with the tomato and potato proteinase inhibitor I polypeptides, respectively. Additionally, the ethyl...

  11. Purification and characterization of proteinase inhibitors from wild soja (Glycine soja) seeds. (United States)

    Deshimaru, Masanobu; Hanamoto, Ryuji; Kusano, Chiho; Yoshimi, Shingo; Terada, Shigeyuki


    Nine proteinase inhibitors, I-VIIa, VIIb, and VIII, were isolated from wild soja seeds by ammonium sulfate fractionation and successive chromatographies on SP-Toyopearl 650M, Sephacryl S-200SF, and DEAE-Toyopearl 650S columns. Reverse-phase HPLC finally gave pure inhibitors. All of the inhibitors inhibited trypsin with dissociation constants of 3.2-6.2 x 10(-9) M. Some of the inhibitors inhibited chymotrypsin and elastase as well. Two inhibitors (VIIb and VIII) with a molecular weight of 20,000 were classified as a soybean Kunitz inhibitor family. Others (I-VIla) had a molecular weight of about 8,000, and were stable to heat and extreme pH, suggesting that these belonged to the Bowman-Birk inhibitor family. Partial amino acid sequences of four inhibitors were also analyzed. The complete sequence of inhibitor IV was ascertained from the nucleotide sequences of cDNA clones encoding isoinhibitors homologous to soybean C-II.

  12. 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


    of antigens by guinea pig accessory cells. The proteinase inhibitor benzyloxycarbonyl-phenylalanylalanine-diazomethyl-ketone, which selectively inhibits cysteine proteinases, was used to block this set of enzymes in cultured cells. We demonstrate that the selective inhibition of the cysteine proteinases...... 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....

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

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


    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.

  14. The reaction of serpins with proteinases involves important enthalpy changes. (United States)

    Boudier, C; Bieth, J G


    When active serpins are proteolytically inactivated in a substrate-like reaction, they undergo an important structural transition with a resultant increase in their conformational stability. We have used microcalorimetry to show that this conformational alteration is accompanied by an important enthalpy change. For instance, the cleavage of alpha(1)-proteinase inhibitor by Pseudomonas aeruginosa elastase, Staphylococcus aureus V8 proteinase, or papain and that of antithrombin by leukocyte elastase are characterized by large enthalpy changes (DeltaH = -53 to -63 kcal mol(-1)). The former reaction also has a large and negative heat capacity (DeltaC(p)() = -566 cal K(-1) mol(-1)). In contrast, serpins release significantly less heat when they act as proteinase inhibitors. For example, the inhibition of pancreatic elastase, leukocyte elastase, and pancreatic chymotrypsin by alpha(1)-proteinase inhibitor and that of pancreatic trypsin and coagulation factor Xa by antithrombin are accompanied by a DeltaH of -20 to -31 kcal mol(-1). We observe no heat release upon proteolytic cleavage of inactive serpins or following inhibition of serine proteinases by canonical inhibitors or upon acylation of chymotrypsin by N-trans-cinnamoylimidazole. We suggest that part of the large enthalpy change that occurs during the structural transition of serpins is used to stabilize the proteinase in its inactive state.

  15. Proteinases involved in the degradation of trypsin inhibitor in germinating mung beans. (United States)

    Wilson, K A; Tan-Wilson, A L


    The mung bean (Vigna radiata (L.) Wilczek) trypsin inhibitor (MBTI) is rapidly modified by limited proteolysis during the early stages of seedling growth. Using an electrophoretic assay that separates the unmodified inhibitor (MBTI-F) and the first two modified species (MBTI-E and -C), a pH optimum of approximately 4 was found for the modification reaction. The inhibitor modifying activity is initially low in ungerminated seeds, with the reaction F leads to E being the primary reaction catalyzed. Activity catalyzing the production of MBTI-C appears on the first day of germination. This activity (F leads to E leads to C) increases up to 6 days after inhibition, at which time the cotyledons begin to abscise. The activity converting MBTI-F and -E to MBTI-C was strongly inhibited by phenylmethylsulfonyl fluoride (3.3 mM) but only weakly by iodoacetate (9 mM) and not at all by pepstatin A (9 microM), leupeptin (18 microM), or EDTA (5 mM). These results suggest the involvement of proteinases other than the major endopeptidase of the germinating seed, vicilin peptidohydrolase. This conclusion is further supported by gel filtration of the extracts of cotyledons on Sephacryl S-200. At least three proteinases are present in germinated cotyledons capable of modifying MBTI-F to MBTI-C and/or -E. All are distinguishable from vicilin peptidohydrolase on the basis of their molecular weight and inhibition by low molecular weight organic reagents.

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

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


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

  17. Bio-physical evaluation and in vivo delivery of plant proteinase inhibitor immobilized on silica nanospheres. (United States)

    Khandelwal, Neha; Doke, Dhananjay S; Khandare, Jayant J; Jawale, Priyanka V; Biradar, Ankush V; Giri, Ashok P


    Recombinant expression of Capsicum annuum proteinase inhibitors (CanPI-13) and its application via synthetic carrier for the crop protection is the prime objective of our study. Herein, we explored proteinase inhibitor peptide immobilization on silica based nanospheres and rods followed by its pH mediated release in vitro and in vivo. Initial studies suggested silica nanospheres to be a suitable candidate for peptide immobilization. Furthermore, the interactions were characterized biophysically to ascertain their conformational stability and biological activity. Interestingly, bioactive peptide loading at acidic pH on nanospheres was found to be 62% and showed 56% of peptide release at pH 10, simulating gut milieu of the target pest Helicoverpa armigera. Additionally, in vivo study demonstrated significant reduction in insect body mass (158 mg) as compared to the control insects (265 mg) on 8th day after feeding with CanPI-13 based silica nanospheres. The study confirms that peptide immobilized silica nanosphere is capable of affecting overall growth and development of the feeding insects, which is known to hamper fecundity and fertility of the insects. Our study illustrates the utility and development of peptide-nanocarrier based platform in delivering diverse biologically active complexes specific to gut pH of H. armigera.

  18. Cloning of Proteinase Inhibitor Gene StPI in Diploid Potato and Its Expression Analysis

    Institute of Scientific and Technical Information of China (English)


    A full-length cDNA of proteinase inhibitor gene with completed open reading frame of 116 amino acids was cloned from Ralstonia solanacearum (Rs) resistant potato leaves using the rapid amplification of cDNA ends (RACE) method and designated as StPI. BLAST search against NCBI showed that the StPI gene shared 89% identity with potato proteinase inhibitor Ⅰ precursor in nucleotide and 74% in amino acid. Analysis of semi-quantitative RT-PCR indicated that this gene was induced by Rs as well as up-regulated by jasmonic acid (JA). The StPI gene expression reached the highest level during 6-12 h post Rs-inoculation or JA-treatment, and then leveled off. Moreover, this gene was strongly induced by JA and its mRNA accumulation increased more quickly than that of Rs-inoculation. The StPI gene may play a role in potato resistance against Rs. The induction of StPI by Rs invasion may have a similar signal transduction pathway with JA treatment.

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

  20. Design of dimerization inhibitors of HIV-1 aspartic proteinase: A computer-based combinatorial approach (United States)

    Caflisch, Amedeo; Schramm, Hans J.; Karplus, Martin


    Inhibition of dimerization to the active form of the HIV-1 aspartic proteinase (HIV-1 PR) may be a way to decrease the probability of escape mutations for this viral protein. The Multiple Copy Simultaneous Search (MCSS) methodology was used to generate functionality maps for the dimerization interface of HIV-1 PR. The positions of the MCSS minima of 19 organic fragments, once postprocessed to take into account solvation effects, are in good agreement with experimental data on peptides that bind to the interface. The MCSS minima combined with an approach for computational combinatorial ligand design yielded a set of modified HIV-1 PR C-terminal peptides that are similar to known nanomolar inhibitors of HIV-1 PR dimerization. A number of N-substituted 2,5-diketopiperazines are predicted to be potential dimerization inhibitors of HIV-1 PR.

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

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


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

  2. Effects of pH on the association between the inhibitor cystatin and the proteinase chymopapain. (United States)

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


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

  3. Effects of cysteine proteinase inhibitors scN and E-64 on southern corn rootworm larval development (United States)

    The southern corn rootworm (SCRW) can be a serious pest of peanut pods. A laboratory bioassay was developed to test feeding cysteine proteinase inhibitors soyacystatin N (scN) and E-64 against southern corn rootworm reared on artificial diet to determine the effects on larvae development and mortal...

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

    DEFF Research Database (Denmark)

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


    The present study on the rat shows that i.v. administration of the proteinase inhibitor aprotinin reduces the urinary output of immunoreactive epidermal growth factor (EGF) while the amount of immunoreactive EGF in the kidneys is increased. This indicates that the EGF-precursor in the rat kidney ...

  5. Isolation of tomato proteinase inhibitor Ⅱ gene and the function of its intron

    Institute of Scientific and Technical Information of China (English)


    The genomic DNA sequence of tomato proteinase inhibitor Ⅱ gene (named tin2i, whose accession number in GenBank is AF007240) was isolated by PCR techniques. The intron sequence (TPI), with a length of 109 bp, owns typical structures of GT/AG dinucleotides at both ends and high content of AT base pairs which accounts for 80.7% of the total nucleotides. As shown by recombination experiment, the TPI sequence could efficiently promote the expression of the reporter gene gusA and this effect was independent of the position and orientation of the intron, thus showing its role as an enhancer. Such experiments as gel retardation assays, GUS histochemical staining and GUS fluorometric assays further demonstrated that TPI sequence maybe has promoter-like activity.

  6. Cystein proteinase inhibitor stefin A as an indicator of efficiency of tumor treatment in mice. (United States)

    Korolenko, T A; Poteryaeva, O N; Falameeva, O V; Levina, O A


    The concentration of stefin A (cystatin A in mice) was measured in animals with experimental tumors (LS lymphosarcoma, HA-1-hepatoma, and Lewis lung carcinoma) during effective antitumor therapy. In mice with these tumors serum concentrations of stefin A increased, while the concentration of cystatin C (extracellular cystein proteinase inhibitor) decreased. The concentration of stefin A in tumor tissue in Lewis lung carcinoma was higher than in LS lymphosarcoma and HA-1-hepatoma ascitic cells, which can be explained by the degree of their malignancy. The content of stefin A in tumor tissue was similar to that in the liver and spleen of tumor-bearing animals, while its concentration in the liver and spleen of tumor-bearing animals was lower than in intact mice. The level of stefin A is an important marker of malignancy and an indicator of the efficiency of antitumor therapy.

  7. High sequence variability among hemocyte-specific Kazal-type proteinase inhibitors in decapod crustaceans. (United States)

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


    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.

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

    Institute of Scientific and Technical Information of China (English)


    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.

  9. A serine proteinase inhibitor isolated from Tamarindus indica seeds and its effects on the release of human neutrophil elastase. (United States)

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


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

  10. Solution structure of PMP-C: a new fold in the group of small serine proteinase inhibitors. (United States)

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


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

  11. Synthesis of the proteinase inhibitor LEKTI domain 6 by the fragment condensation method and regioselective disulfide bond formation. (United States)

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


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

  12. Free-energy analysis of enzyme-inhibitor binding: aspartic proteinase-pepstatin complexes. (United States)

    Kalra, P; Das, A; Jayaram, B


    Expeditious in silico determinations of the free energies of binding of a series of inhibitors to an enzyme are of immense practical value in structure-based drug design efforts. Some recent advances in the field of computational chemistry have rendered a rigorous thermodynamic treatment of biologic molecules feasible, starting from a molecular description of the biomolecule, solvent, and salt. Pursuing the goal of developing and making available a software for assessing binding affinities, we present here a computationally rapid, albeit elaborate, methodology to estimate and analyze the molecular thermodynamics of enzyme-inhibitor binding with crystal structures as the point of departure. The complexes of aspartic proteinases with seven inhibitors have been adopted for this study. The standard free energy of complexation is considered in terms of a thermodynamic cycle of six distinct steps decomposed into a total of 18 well-defined components. The model we employed involves explicit all-atom accounts of the energetics of electrostatic interactions, solvent screening effects, van der Waals components, and cavitation effects of solvation combined with a Debye-Huckel treatment of salt effects. The magnitudes and signs of the various components are estimated using the AMBER parm94 force field, generalized Born theory, and solvent accessibility measures. Estimates of translational and rotational entropy losses on complexation as well as corresponding changes in the vibrational and configurational entropy are also included. The calculated standard free energies of binding at this stage are within an order of magnitude of the observed inhibition constants and necessitate further improvements in the computational protocols to enable quantitative predictions. Some areas such as inclusion of structural adaptation effects, incorporation of site-dependent amino acid pKa shifts, consideration of the dynamics of the active site for fine-tuning the methodology are easily

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

    Directory of Open Access Journals (Sweden)

    Sandra Macedo-Ribeiro

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

  14. Neutrophil elastase reduces secretion of secretory leukoproteinase inhibitor (SLPI by lung epithelial cells: role of charge of the proteinase-inhibitor complex

    Directory of Open Access Journals (Sweden)

    Hiemstra Pieter S


    Full Text Available Abstract Background Secretory leukoproteinase inhibitor (SLPI is an important inhibitor of neutrophil elastase (NE, a proteinase implicated in the pathogenesis of lung diseases such as COPD. SLPI also has antimicrobial and anti-inflammatory properties, but the concentration of SLPI in lung secretions in COPD varies inversely with infection and the concentration of NE. A fall in SLPI concentration is also seen in culture supernatants of respiratory cells exposed to NE, for unknown reasons. We investigated the hypothesis that SLPI complexed with NE associates with cell membranes in vitro. Methods Respiratory epithelial cells were cultured in the presence of SLPI, varying doses of proteinases over time, and in different experimental conditions. The likely predicted charge of the complex between SLPI and proteinases was assessed by theoretical molecular modelling. Results We observed a rapid, linear decrease in SLPI concentration in culture supernatants with increasing concentration of NE and cathepsin G, but not with other serine proteinases. The effect of NE was inhibited fully by a synthetic NE inhibitor only when added at the same time as NE. Direct contact between NE and SLPI was required for a fall in SLPI concentration. Passive binding to cell culture plate materials was able to remove a substantial amount of SLPI both with and without NE. Theoretical molecular modelling of the structure of SLPI in complex with various proteinases showed a greater positive charge for the complex with NE and cathepsin G than for other proteinases, such as trypsin and mast cell tryptase, that also bind SLPI but without reducing its concentration. Conclusion These data suggest that NE-mediated decrease in SLPI is a passive, charge-dependent phenomenon in vitro, which may correlate with changes observed in vivo.

  15. Potato type I and II proteinase inhibitors: modulating plant physiology and host resistance. (United States)

    Turra, David; Lorito, Matteo


    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.

  16. Single-Step Purification and Characterization of A Recombinant Serine Proteinase Inhibitor from Transgenic Plants. (United States)

    Jha, Shweta; Agarwal, Saurabh; Sanyal, Indraneel; Amla, D V


    Expression of recombinant therapeutic proteins in transgenic plants has a tremendous impact on safe and economical production of biomolecules for biopharmaceutical industry. The major limitation in their production is downstream processing of recombinant protein to obtain higher yield and purity of the final product. In this study, a simple and rapid process has been developed for purification of therapeutic recombinant α1-proteinase inhibitor (rα1-PI) from transgenic tomato plants, which is an abundant serine protease inhibitor in human serum and chiefly inhibits the activity of neutrophil elastase in lungs. We have expressed rα1-PI with modified synthetic gene in transgenic tomato plants at a very high level (≃3.2 % of total soluble protein). The heterologous protein was extracted with (NH4)2SO4 precipitation, followed by chromatographic separation on different matrices. However, only immunoaffinity chromatography resulted into homogenous preparation of rα1-PI with 54 % recovery. The plant-purified rα1-PI showed molecular mass and structural conformation comparable to native serum α1-PI, as shown by mass spectrometry and optical spectroscopy. The results of elastase inhibition assay revealed biological activity of the purified rα1-PI protein. This work demonstrates a simple and efficient one-step purification of rα1-PI from transgenic plants, which is an essential prerequisite for further therapeutic development.

  17. 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


    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.

  18. The N-terminal octapeptide acts as a dimerization inhibitor of SARS coronavirus 3C-like proteinase. (United States)

    Wei, Ping; Fan, Keqiang; Chen, Hao; Ma, Liang; Huang, Changkang; Tan, Lei; Xi, Dong; Li, Chunmei; Liu, Ying; Cao, Aoneng; Lai, Luhua


    The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS. Accurate determination of the dimer dissociation constant and the role of the N-finger (residues 1-7) will provide more insights into the enzyme catalytic mechanism of SARS 3CL proteinase. The dimer dissociation constant of the wild-type protein was determined to be 14.0microM by analytical ultracentrifugation method. The N-finger fragment of the enzyme plays an important role in enzyme dimerization as shown in the crystal structure. Key residues in the N-finger have been studied by site-directed mutagenesis, enzyme assay, and analytical ultracentrifugation. A single mutation of M6A was found to be critical to maintain the dimer structure of the enzyme. The N-terminal octapeptide N8 and its mutants were also synthesized and tested for their potency as dimerization inhibitors. Peptide cleavage assay confirms that peptide N8 is a dimerization inhibitor with a K(i) of 2.20mM. The comparison of the inhibitory activities of N8 and its mutants indicates that the hydrophobic interaction of Met-6 and the electrostatic interaction of Arg-4 contribute most for inhibitor binding. This study describes the first example of inhibitors targeting the dimeric interface of SARS 3CL proteinase, providing a novel strategy for drug design against SARS and other coronaviruses.

  19. [Molecular cloning and analysis of cDNA sequences encoding serine proteinase and Kunitz type inhibitor in venom gland of Vipera nikolskii viper]. (United States)

    Ramazanova, A S; Fil'kin, S Iu; Starkov, V G; Utkin, Iu N


    Serine proteinases and Kunitz type inhibitors are widely represented in venoms of snakes from different genera. During the study of the venoms from snakes inhabiting Russia we have cloned cDNAs encoding new proteins belonging to these protein families. Thus, a new serine proteinase called nikobin was identified in the venom gland of Vipera nikolskii viper. By amino acid sequence deduced from the cDNA sequence, nikobin differs from serine proteinases identified in other snake species. Nikobin amino acid sequence contains 15 unique substitutions. This is the first serine proteinase of viper from Vipera genus for which a complete amino acid sequence established. The cDNA encoding Kunitz type inhibitor was also cloned. The deduced amino acid sequence of inhibitor is homologous to those of other proteins from that snakes of Vipera genus. However there are several unusual amino acid substitutions that might result in the change of biological activity of inhibitor.

  20. Conformational changes of ovine α-1-proteinase inhibitor: The influence of heparin binding (United States)

    Gupta, Vivek Kumar; Gowda, Lalitha R.


    α-1-Proteinase inhibitor (α-1-PI), the archetypal serpin causes rapid, irreversible stoichiometric inhibition of redundant circulating serine proteases and is associated with emphysema, inflammatory response and maintenance of protease-inhibitor equilibrium in vascular and peri-vascular spaces. A homogenous preparation of heparin octasaccharide binds to ovine and human α-1-PI and enhances their protease inhibitory activity phenomenally. Size-exclusion chromatography and dynamic light scattering experiments reveal that ovine α-1-PI undergoes a decrease in the Stokes' radius upon heparin binding. A strong binding; characterizes this α-1-PI-heparin interaction as revealed by the binding constant ( Kα) 1.98 ± 0.2 × 10 -6 M and 2.1 ± 0.2 × 10 -6 M determined by fluorescence spectroscopy and equilibrium dialysis, respectively. The stoichiometry of heparin binding to ovine α-1-PI was 1.1 ± 0.2:1. The Stern-Volmer constants ( Ksv) for heparin activated ovine and human α-1-PI were found to be 5.13 × 10 -6 M and 5.67 × 10 -6 M, respectively, significantly higher than the native inhibitors. FTIR and CD spectroscopy project the systematic structural reorientations that α-1-PI undergoes upon heparin binding characterized by a decrease in α-helical content and a concomitant increase in β-turn and random coil elements. It is likely that these conformational changes result in the movement of the α-1-PI reactive site loop into an extended structure that is better poised to combat the cognate protease and accelerate the inhibition.

  1. Bowman-Birk proteinase inhibitor from Clitoria fairchildiana seeds: Isolation, biochemical properties and insecticidal potential. (United States)

    Dantzger, Miriam; Vasconcelos, Ilka Maria; Scorsato, Valéria; Aparicio, Ricardo; Marangoni, Sergio; Macedo, Maria Lígia Rodrigues


    Herein described is the biochemical characterisation, including in vitro and in vivo assays, for a proteinase inhibitor purified from Clitoria fairchildiana seeds (CFPI). Purification was performed by hydrophobic interaction and gel filtration chromatography. Kinetic studies of the purified inhibitor showed a competitive-type inhibitory activity against bovine trypsin and chymotrypsin, with an inhibition stoichiometry of 1:1 for both enzymes. The inhibition constants against trypsin and chymotrypsin were 3.3 × 10(-10) and 1.5 × 10(-10)M, respectively, displaying a tight binding property. SDS-PAGE showed that CFPI has a single polypeptide chain with an apparent molecular mass of 15 kDa under non-reducing conditions. However, MALDI-TOF analysis demonstrated a molecular mass of 7.973 kDa, suggesting that CFPI is dimeric in solution. The N-terminal sequence of CFPI showed homology with members of the Bowman-Birk inhibitor family. CFPI remained stable to progressive heating for 30 min to each temperature range of 37 up to 100 °C and CD analysis exhibited no changes in spectra at 207 nm after heating at 90 °C and subsequent cooling. Moreover, CFPI was active over a wide pH range (2-10). In contrast, reduction with DTT resulted in a loss of inhibitory activity against trypsin and chymotrypsin. CFPI also exhibited significant inhibitory activity against larval midgut trypsin enzymes from Anagasta kuehniella (76%), Diatraea saccharalis (59%) and Heliothis virescens (49%). Its insecticidal properties were further analysed by bioassays and confirmed by negative impact on A. kuehniella development.

  2. Purification and characterization of native and recombinant SaPIN2a, a plant sieve element-localized proteinase inhibitor. (United States)

    Wang, Zhen-Yu; Ding, Ling-Wen; Ge, Zhi-Juan; Wang, Zhaoyu; Wang, Fanghai; Li, Ning; Xu, Zeng-Fu


    SaPIN2a encodes a proteinase inhibitor in nightshade (Solanum americanum), which is specifically localized to the enucleate sieve elements. It has been proposed to play an important role in phloem development by regulating proteolysis in sieve elements. In this study, we purified and characterized native SaPIN2a from nightshade stems and recombinant SaPIN2a expressed in Escherichia coli. Purified native SaPIN2a was found as a charge isomer family of homodimers, and was weakly glycosylated. Native SaPIN2a significantly inhibited serine proteinases such as trypsin, chymotrypsin, and subtilisin, with the most potent inhibitory activity on subtilisin. It did not inhibit cysteine proteinase papain and aspartic proteinase cathepsin D. Recombinant SaPIN2a had a strong inhibitory effect on chymotrypsin, but its inhibitory activities toward trypsin and especially toward subtilisin were greatly reduced. In addition, native SaPIN2a can effectively inhibit midgut trypsin-like activities from Trichoplusia ni and Spodoptera litura larvae, suggesting a potential for the production of insect-resistant transgenic plants.

  3. Structural and functional characteristics of plant proteinase inhibitor-II (PI-II) family. (United States)

    Rehman, Shazia; Aziz, Ejaz; Akhtar, Wasim; Ilyas, Muhammad; Mahmood, Tariq


    Plant proteinase inhibitor-II (PI-II) proteins are one of the promising defensive proteins that helped the plants to resist against different kinds of unfavorable conditions. Different roles for PI-II have been suggested such as regulation of endogenous proteases, modulation of plant growth and developmental processes and mediating stress responses. The basic knowledge on genetic and molecular diversity of these proteins has provided significant insight into their gene structure and evolutionary relationships in various members of this family. Phylogenetic comparisons of these family genes in different plants suggested that the high rate of retention of gene duplication and inhibitory domain multiplication may have resulted in the expansion and functional diversification of these proteins. Currently, a large number of transgenic plants expressing PI-II genes are being developed for enhancing the defensive capabilities against insects, bacteria and pathogenic fungi. Much emphasis is yet to be given to exploit this ever expanding repertoire of genes for improving abiotic stress resistance in transgenic crops. This review presents an overview about the current knowledge on PI-II family genes, their multifunctional role in plant defense and physiology with their potential applications in biotechnology.

  4. Cystatin like thiol proteinase inhibitor from pancreas of Capra hircus: purification and detailed biochemical characterization. (United States)

    Priyadarshini, Medha; Bano, Bilqees


    A thiol proteinase inhibitor from Capra hircus (goat) pancreas (PTPI) isolated by ammonium sulphate precipitation (20-80%) and gel filtration chromatography on Sephacryl S-100HR, with 20.4% yield and 500-fold purification, gave molecular mass of 44 kDa determined by its electrophoretic and gel filtration behavior, respectively. The stokes radius, diffusion and sedimentation coefficients of PTPI were 27.3 A, 7.87 x 10(-7) cm(2) s(-1) and 3.83 s, respectively. It was stable in pH range 3-10 and up to 70 degrees C (critical temperature, E (a) = 21 kJ mol(-1)). Kinetic analysis revealed reversible and competitive mode of inhibition with PTPI showing the highest inhibitory efficiency against papain (K ( i ) = 5.88 nM). The partial amino acid sequence analysis showed that it shared good homology with bovine parotid and skin cystatin C. PTPI possessed 17.18% alpha helical content assessed by CD spectroscopy. The hydropathy plot of first 24 residues suggested that most amino acids of this stretch might be in the hydrophobic core of the protein.

  5. Use of recombinant Entamoeba histolytica cysteine proteinase 1 to identify a potent inhibitor of amebic invasion in a human colonic model. (United States)

    Meléndez-López, Samuel G; Herdman, Scott; Hirata, Ken; Choi, Min-Ho; Choe, Youngchool; Craik, Charles; Caffrey, Conor R; Hansell, Elisabeth; Chávez-Munguía, Bibiana; Chen, Yen Ting; Roush, William R; McKerrow, James; Eckmann, Lars; Guo, Jianhua; Stanley, Samuel L; Reed, Sharon L


    Cysteine proteinases are key virulence factors of the protozoan parasite Entamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of the E. histolytica genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on E. histolytica cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive E. histolytica and is highly expressed and released. Recombinant EhCP1 was expressed in Escherichia coli and refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.

  6. N-terminal extension of the yeast IA3 aspartic proteinase inhibitor relaxes the strict intrinsic selectivity. (United States)

    Winterburn, Tim J; Phylip, Lowri H; Bur, Daniel; Wyatt, David M; Berry, Colin; Kay, John


    Yeast IA(3) aspartic proteinase inhibitor operates through an unprecedented mechanism and exhibits a remarkable specificity for one target enzyme, saccharopepsin. Even aspartic proteinases that are very closely similar to saccharopepsin (e.g. the vacuolar enzyme from Pichia pastoris) are not susceptible to significant inhibition. The Pichia proteinase was selected as the target for initial attempts to engineer IA(3) to re-design the specificity. The IA(3) polypeptides from Saccharomyces cerevisiae and Saccharomyces castellii differ considerably in sequence. Alterations made by deletion or exchange of the residues in the C-terminal segment of these polypeptides had only minor effects. By contrast, extension of each of these wild-type and chimaeric polypeptides at its N-terminus by an MK(H)(7)MQ sequence generated inhibitors that displayed subnanomolar potency towards the Pichia enzyme. This gain-in-function was completely reversed upon removal of the extension sequence by exopeptidase trimming. Capture of the potentially positively charged aromatic histidine residues of the extension by remote, negatively charged side-chains, which were identified in the Pichia enzyme by modelling, may increase the local IA(3) concentration and create an anchor that enables the N-terminal segment residues to be harboured in closer proximity to the enzyme active site, thus promoting their interaction. In saccharopepsin, some of the counterpart residues are different and, consistent with this, the N-terminal extension of each IA(3) polypeptide was without major effect on the potency of interaction with saccharopepsin. In this way, it is possible to convert IA(3) polypeptides that display little affinity for the Pichia enzyme into potent inhibitors of this proteinase and thus broaden the target selectivity of this remarkable small protein.

  7. A Plant Proteinase Inhibitor from Enterolobium contortisiliquum Attenuates Pulmonary Mechanics, Inflammation and Remodeling Induced by Elastase in Mice. (United States)

    Theodoro-Júnior, Osmar Aparecido; Righetti, Renato Fraga; Almeida-Reis, Rafael; Martins-Oliveira, Bruno Tadeu; Oliva, Leandro Vilela; Prado, Carla Máximo; Saraiva-Romanholo, Beatriz Mangueira; Leick, Edna Aparecida; Pinheiro, Nathalia Montouro; Lobo, Yara Aparecida; Martins, Mílton de Arruda; Oliva, Maria Luiza Vilela; Tibério, Iolanda de Fátima Lopes Calvo


    Proteinase inhibitors have been associated with anti-inflammatory and antioxidant activities and may represent a potential therapeutic treatment for emphysema. Our aim was to evaluate the effects of a plant Kunitz proteinase inhibitor, Enterolobium contortisiliquum trypsin inhibitor (EcTI), on several aspects of experimental elastase-induced pulmonary inflammation in mice. C57/Bl6 mice were intratracheally administered elastase (ELA) or saline (SAL) and were treated intraperitoneally with EcTI (ELA-EcTI, SAL-EcTI) on days 1, 14 and 21. On day 28, pulmonary mechanics, exhaled nitric oxide (ENO) and number leucocytes in the bronchoalveolar lavage fluid (BALF) were evaluated. Subsequently, lung immunohistochemical staining was submitted to morphometry. EcTI treatment reduced responses of the mechanical respiratory system, number of cells in the BALF, and reduced tumor necrosis factor-α (TNF-α), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-12 (MMP-12), tissue inhibitor of matrix metalloproteinase (TIMP-1), endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS)-positive cells and volume proportion of isoprostane, collagen and elastic fibers in the airways and alveolar walls compared with the ELA group. EcTI treatment reduced elastase induced pulmonary inflammation, remodeling, oxidative stress and mechanical alterations, suggesting that this inhibitor may be a potential therapeutic tool for chronic obstructive pulmonary disease (COPD) management.

  8. A Plant Proteinase Inhibitor from Enterolobium contortisiliquum Attenuates Pulmonary Mechanics, Inflammation and Remodeling Induced by Elastase in Mice (United States)

    Theodoro-Júnior, Osmar Aparecido; Righetti, Renato Fraga; Almeida-Reis, Rafael; Martins-Oliveira, Bruno Tadeu; Oliva, Leandro Vilela; Prado, Carla Máximo; Saraiva-Romanholo, Beatriz Mangueira; Leick, Edna Aparecida; Pinheiro, Nathalia Montouro; Lobo, Yara Aparecida; Martins, Mílton de Arruda; Oliva, Maria Luiza Vilela; Tibério, Iolanda de Fátima Lopes Calvo


    Proteinase inhibitors have been associated with anti-inflammatory and antioxidant activities and may represent a potential therapeutic treatment for emphysema. Our aim was to evaluate the effects of a plant Kunitz proteinase inhibitor, Enterolobium contortisiliquum trypsin inhibitor (EcTI), on several aspects of experimental elastase-induced pulmonary inflammation in mice. C57/Bl6 mice were intratracheally administered elastase (ELA) or saline (SAL) and were treated intraperitoneally with EcTI (ELA-EcTI, SAL-EcTI) on days 1, 14 and 21. On day 28, pulmonary mechanics, exhaled nitric oxide (ENO) and number leucocytes in the bronchoalveolar lavage fluid (BALF) were evaluated. Subsequently, lung immunohistochemical staining was submitted to morphometry. EcTI treatment reduced responses of the mechanical respiratory system, number of cells in the BALF, and reduced tumor necrosis factor-α (TNF-α), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-12 (MMP-12), tissue inhibitor of matrix metalloproteinase (TIMP-1), endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS)-positive cells and volume proportion of isoprostane, collagen and elastic fibers in the airways and alveolar walls compared with the ELA group. EcTI treatment reduced elastase induced pulmonary inflammation, remodeling, oxidative stress and mechanical alterations, suggesting that this inhibitor may be a potential therapeutic tool for chronic obstructive pulmonary disease (COPD) management. PMID:28216579

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

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


    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.

  10. Distortion of the catalytic domain of tissue-type plasminogen activator by plasminogen activator inhibitor-1 coincides with the formation of stable serpin-proteinase complexes. (United States)

    Perron, Michel J; Blouse, Grant E; Shore, Joseph D


    Plasminogen activator inhibitor-1 (PAI-1) is a typical member of the serpin family that kinetically traps its target proteinase as a covalent complex by distortion of the proteinase domain. Incorporation of the fluorescently silent 4-fluorotryptophan analog into PAI-1 permitted us to observe changes in the intrinsic tryptophan fluorescence of two-chain tissue-type plasminogen activator (tPA) and the proteinase domain of tPA during the inhibition reaction. We demonstrated three distinct conformational changes of the proteinase that occur during complex formation and distortion. A conformational change occurred during the initial formation of the non-covalent Michaelis complex followed by a large conformational change associated with the distortion of the proteinase catalytic domain that occurs concurrently with the formation of stable proteinase-inhibitor complexes. Following distortion, a very slow structural change occurs that may be involved in the stabilization or regulation of the trapped complex. Furthermore, by comparing the inhibition rates of two-chain tPA and the proteinase domain of tPA by PAI-1, we demonstrate that the accessory domains of tPA play a prominent role in the initial formation of the non-covalent Michaelis complex.

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

    Sheffield, William P; Bhakta, Varsha


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

  12. 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.

  13. Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice. (United States)

    Quilis, Jordi; López-García, Belén; Meynard, Donaldo; Guiderdoni, Emmanuel; San Segundo, Blanca


    Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot-and-mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound- and pathogen-inducible mpi promoter. The mpi-pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi-pci rice, compared with larvae fed on wild-type plants, was observed. Expression of the mpi-pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi-pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi-pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi-pci fusion gene for dual resistance against insects and pathogens in rice plants.

  14. Effects of proteinase inhibitor from Adenanthera pavonina seeds on short- and long term larval development of Aedes aegypti. (United States)

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


    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

  15. The squash aspartic proteinase inhibitor SQAPI is widely present in the cucurbitales, comprises a small multigene family, and is a member of the phytocystatin family. (United States)

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


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

  16. A Novel Trypsin Inhibitor-Like Cysteine-Rich Peptide from the Frog Lepidobatrachus laevis Containing Proteinase-Inhibiting Activity. (United States)

    Wang, Yu-Wei; Tan, Ji-Min; Du, Can-Wei; Luan, Ning; Yan, Xiu-Wen; Lai, Ren; Lu, Qiu-Min


    Various bio-active substances in amphibian skins play important roles in survival of the amphibians. Many protease inhibitor peptides have been identified from amphibian skins, which are supposed to negatively modulate the activity of proteases to avoid premature degradation or release of skin peptides, or to inhibit extracellular proteases produced by invading bacteria. However, there is no information on the proteinase inhibitors from the frog Lepidobatrachus laevis which is unique in South America. In this work, a cDNA encoding a novel trypsin inhibitor-like (TIL) cysteine-rich peptide was identified from the skin cDNA library of L. laevis. The 240-bp coding region encodes an 80-amino acid residue precursor protein containing 10 half-cysteines. By sequence comparison and signal peptide prediction, the precursor was predicted to release a 55-amino acid mature peptide with amino acid sequence, IRCPKDKIYKFCGSPCPPSCKDLTPNCIAVCKKGCFCRDGTVDNNHGKCVKKENC. The mature peptide was named LL-TIL. LL-TIL shares significant domain similarity with the peptides from the TIL supper family. Antimicrobial and trypsin-inhibitory abilities of recombinant LL-TIL were tested. Recombinant LL-TIL showed no antimicrobial activity, while it had trypsin-inhibiting activity with a Ki of 16.5178 μM. These results suggested there was TIL peptide with proteinase-inhibiting activity in the skin of frog L. laevis. To the best of our knowledge, this is the first report of TIL peptide from frog skin.

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

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


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

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

    Directory of Open Access Journals (Sweden)

    Chalaire Katelyn C


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

  19. Activation of proteinase 3 contributes to Non-alcoholic Fatty Liver Disease (NAFLD) and insulin resistance. (United States)

    Toonen, Erik J M; Mirea, Andreea-Manuela; Tack, Cees J; Stienstra, Rinke; Ballak, Dov B; van Diepen, Janna A; Hijmans, Anneke; Chavakis, Triantafyllos; Dokter, Wim H; Pham, Christine T N; Netea, Mihai G; Dinarello, Charles A; Joosten, Leo A B


    Activation of inflammatory pathways is known to accompany development of obesity-induced non-alcoholic fatty liver disease (NAFLD), insulin resistance and type 2 diabetes. In addition to caspase-1, the neutrophil serine proteases proteinase 3, neutrophil elastase and cathepsin G are able to process the inactive pro-inflammatory mediators IL-1β and IL-18 to their bioactive forms, thereby regulating inflammatory responses. In the present study, we investigated whether proteinase 3 is involved in obesity-induced development of insulin resistance and NAFLD. We investigated the development of NAFLD and insulin resistance in mice deficient for neutrophil elastase/proteinase 3 and neutrophil elastase/cathepsin G and in wild-type mice treated with the neutrophil serine proteinase inhibitor human alpha-1 antitrypsin. Expression profiling of metabolically relevant tissues obtained from insulin resistant mice showed that expression of proteinase 3 was specifically upregulated in the liver, whereas neutrophil elastase, cathepsin G and caspase-1 were not. Neutrophil elastase/proteinase 3 deficient mice showed strongly reduced levels of lipids in the liver after fed a high fat diet. Moreover, these mice were resistant to high fat diet-induced weight gain, inflammation and insulin resistance. Injection of proteinase 3 exacerbated insulin resistance in caspase-1(-/-) mice, indicating that proteinase 3 acts independently of caspase-1. Treatment with alpha-1 antitrypsin during the last 10 days of a 16 week high fat diet reduced hepatic lipid content and decreased fasting glucose levels. We conclude that proteinase 3 is involved in NAFLD and insulin resistance and that inhibition of proteinase 3 may have therapeutic potential.

  20. Enzyme specificity of proteinase inhibitor region in amyloid precursor protein of Alzheimer's disease: different properties compared with protease nexin I. (United States)

    Kitaguchi, N; Takahashi, Y; Oishi, K; Shiojiri, S; Tokushima, Y; Utsunomiya, T; Ito, H


    Senile plaques, often surrounded by abnormally grown neurites, are characteristic of Alzheimer's diseased brain. The core of the plaque is mainly composed of amyloid beta protein (beta-AP), two of whose three precursors (APP) have serine proteinase inhibitor regions (APPI). APPI derivatives containing 60, 72 or 88 amino-acid fragments (APPI-60, APPI-72 and APPI-88, respectively) of the longest APP were produced in COS-1 cell culture medium, with the APPI cDNA ligated to the signal sequence of tissue plasminogen activator. The secreted APPIs were purified by sequential acetone precipitation followed by affinity chromatography using immobilized trypsin. These three APPIs and O-glycosylation-site-mutated APPI showed similar inhibitory activity against trypsin, chymotrypsin and plasmin. The purified APPI-72 was found to inhibit trypsin (Ki = 1.1 x 10(-10) M) and chymotrypsin (Ki = 5.8 x 10(-9) M) most strongly, and to inhibit leukocyte elastase (Ki = 7.9 x 10(-7) M) and several blood coagulation proteinases (Ki = 0.46-12 x 10(-7) M), but not urokinase or thrombin. The observed inhibition pattern was quite different from that of protease nexin I, one of serine proteinase inhibitors possessing neurite outgrowth activity. This suggests that the physiological roles of APPI are different from those of protease nexin I, and that APPI could not cause aberrant growth of neurite into the plaque. The presence of APPI having strong inhibitory activity in the brain might lead to the formation of amyloid deposits by preventing complete degradation of APPs.

  1. 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


    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.

  2. Glucose-6-phosphate isomerase is an endogenous inhibitor to myofibril-bound serine proteinase of crucian carp (Carassius auratus). (United States)

    Sun, Le-Chang; Zhou, Li-Gen; Du, Cui-Hong; Cai, Qiu-Feng; Hara, Kenji; Su, Wen-Jin; Cao, Min-Jie


    Glucose-6-phosphate isomerase (GPI) was purified to homogeneity from the skeletal muscle of crucian carp ( Carassius auratus ) by ammonium sulfate fractionation, column chromatographies of Q-Sepharose, SP-Sepharose, and Superdex 200 with a yield of 8.0%, and purification folds of 468. The molecular mass of GPI was 120 kDa as estimated by gel filtration, while on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two subunits (55 and 65 kDa) were identified, suggesting that it is a heterodimer. Interestingly, GPI revealed specific inhibitory activity toward a myofibril-bound serine proteinase (MBSP) from crucian carp, while no inhibitory activity was identified toward other serine proteinases, such as white croaker MBSP and crucian carp trypsin. Kinetic analysis showed that GPI is a competitive inhibitor toward MBSP, and the K(i) was 0.32 microM. Our present results indicated that the multifunctional protein GPI is an endogenous inhibitor to MBSP and may play a significant role in the regulation of muscular protein metabolism in vivo.

  3. A four-domain Kunitz-type proteinase inhibitor from Solen grandis is implicated in immune response. (United States)

    Wei, Xiumei; Yang, Jialong; Yang, Jianmin; Liu, Xiangquan; Liu, Meijun; Yang, Dinglong; Xu, Jie; Hu, Xiaoke


    Serine proteinase inhibitor (SPI) serves as a negative regulator in immune signal pathway by restraining the activities of serine proteinase (SP) and plays an essential role in the innate immunity. In the present study, a Kunitz-type SPI was identified from the mollusk razor clam Solen grandis (designated as SgKunitz). The full-length cDNA of SgKunitz was of 1284 bp, containing an open reading frame (ORF) of 768 bp. The ORF encoded four Kunitz domains, and their amino acids were well conserved when compared with those in other Kunitz-type SPIs, especially the six cysteines involved in forming of three disulfide bridges in each domain. In addition, the tertiary structure of all the four domains adopted a typical model of Kunitz-type SPI family, indicating SgKunitz was a new member of Kunitz-type SPI superfamily. The mRNA transcripts of SgKunitz were detected in all tested tissues of razor clam, including muscle, mantle, gonad, gill, hepatopancreas and hemocytes, and with the highest expression level in gill. When the razor clams were stimulated by LPS, PGN or β-1, 3-glucan, the expression level of SgKunitz mRNA in hemocytes was significantly up-regulated (P inhibitor of SP involving in the immune response of S. grandis, and provided helpful evidences to understand the regulation mechanism of immune signal pathway in mollusk.

  4. A five-domain Kazal-type serine proteinase inhibitor from black tiger shrimp Penaeus monodon and its inhibitory activities. (United States)

    Somprasong, Nawarat; Rimphanitchayakit, Vichien; Tassanakajon, Anchalee


    A novel five-domain Kazal-type serine proteinase inhibitor, SPIPm2, identified from the hemocyte cDNA library of black tiger shrimp Penaeus monodon was successfully expressed in the Escherichia coli expression system. The expressed recombinant SPIPm2 (rSPIPm2) as inclusion bodies was solubilized with a sodium carbonate buffer, pH10, and purified by gel filtration chromatography. The molecular mass of rSPIPm2 was determined using MALDI-TOF mass spectrometry to be 29.065 kDa. The inhibitory activities of rSPIPm2 were tested against trypsin, alpha-chymotrypsin, subtilisin and elastase. The inhibitor exhibited potent inhibitory activities against subtilisin and elastase, weak inhibitory activity against trypsin, and did not inhibit chymotrypsin. Tight-binding inhibition assay suggested that the molar ratios of SPIPm2 to subtilisin and elastase were 1:2 and 1:1, respectively. The inhibition against subtilisin and elastase was a competitive type with inhibition constants (Ki) of 0.52 and 3.27 nM, respectively. The inhibitory activity of SPIPm2 against subtilisin implies that, in shrimp, it may function as a defense component against proteinases from pathogenic bacteria but the elastase inhibitory function is not known.

  5. Crystal quality and inhibitor binding by aspartic proteinases; preparation of high quality crystals of mouse renin (United States)

    Badasso, M.; Sibanda, B. L.; Cooper, J. B.; Dealwis, C. G.; Wood, S. P.


    Renin from mouse submandibular glands has been highly purified and co-crystallized with a synthetic nonapeptide fragment of rat angiotensionogen in which the scissile Leu-Leu bond has been modified as a hydroxyethylene mimic of the transition state. The strong diffraction from these crystals compared to the native form is discussed in relation to the behaviour of other members of the aspartic proteinase family in crystallisation.

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

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


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

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

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


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

  8. A selective reversible azapeptide inhibitor of human neutrophil proteinase 3 derived from a high affinity FRET substrate. (United States)

    Epinette, Christophe; Croix, Cécile; Jaquillard, Lucie; Marchand-Adam, Sylvain; Kellenberger, Christine; Lalmanach, Gilles; Cadene, Martine; Viaud-Massuard, Marie-Claude; Gauthier, Francis; Korkmaz, Brice


    The biological functions of human neutrophil proteinase 3 (PR3) remain unclear because of its close structural resemblance to neutrophil elastase and its apparent functional redundancy with the latter. Thus, all natural inhibitors of PR3 preferentially target neutrophil elastase. We have designed a selective PR3 inhibitor based on the sequence of one of its specific, sensitive FRET substrates. This azapeptide, azapro-3, inhibits free PR3 in solution, PR3 bound to neutrophil membranes, and the PR3 found in crude lung secretions from patients with chronic inflammatory pulmonary diseases. But it does not inhibit significantly neutrophil elastase or cathepsin G. Unlike most of azapeptides, this inhibitor does not form a stable acyl-enzyme complex; it is a reversible competitive inhibitor with a K(i) comparable to the K(m) of the parent substrate. Low concentrations (60 μM) of azapro-3 totally inhibited the PR3 secreted by triggered human neutrophils (200,000 cells/100 μL) and the PR3 in neutrophil homogenates and in lung secretions of patients with lung inflammation for hours. Azapro-3 also resisted proteolysis by all proteases contained in these samples for at least 2h.

  9. Domain 2 of a Kazal serine proteinase inhibitor SPIPm2 from Penaeus monodon possesses antiviral activity against WSSV. (United States)

    Visetnan, Suwattana; Donpudsa, Suchao; Supungul, Premruethai; Tassanakajon, Anchalee; Rimphanitchayakit, Vichien


    A 5-domain Kazal type serine proteinase inhibitor SPIPm2 from Penaeus monodon is involved in innate immune defense against white spot syndrome virus (WSSV). To test which domains were involved, the 5 domains of SPIPm2 were over-expressed and tested against WSSV infection. By using hemocyte primary cell culture treated with each recombinant SPIPm2 domain along with WSSV, the expression of WSSV early genes ie1, WSV477 and late gene VP28 were substantially reduced as compared to other domains when the recombinant domain 2, rSPIPm2D2, was used. Injecting the WSSV along with rSPIPm2D2 but not with other domains caused delay in mortality rate of the infected shrimp. The results indicate that the SPIPm2D2 possesses strong antiviral activity and, hence, contributes predominantly to the antiviral activity of SPIPm2.

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

    Institute of Scientific and Technical Information of China (English)

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


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

  11. Arabidopsis cysteine proteinase inhibitor AtCYSb interacts with a Ca(2+)-dependent nuclease, AtCaN2. (United States)

    Guo, Kunyuan; Bu, Yuanyuan; Takano, Tetsuo; Liu, Shenkui; Zhang, Xinxin


    Plant cysteine proteinase inhibitors (cystatins) play important roles in plant defense mechanisms. Some proteins that interact with cystatins may defend against abiotic stresses. Here, we showed that AtCaN2, a Ca(2+)-dependent nuclease in Arabidopsis, is transcribed in senescent leaves and stems and interacts with an Arabidopsis cystatin (AtCYSb) in a yeast two-hybrid screen. The interaction between AtCYSb and AtCaN2 was confirmed by in vitro pull-down assay and bimolecular fluorescence complementation. Agarose gel electrophoresis showed that the nuclease activity of AtCaN2 against λDNA was inhibited by AtCYSb, which suggests that AtCYSb regulates nucleic acid degradation in cells.

  12. Prevalence of the serpin peptidase inhibitor (alpha-1-antitrypsin PI*S and PI*Z alleles in Brazilian children with liver disease

    Directory of Open Access Journals (Sweden)

    Guilherme Baldo


    Full Text Available Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin, member 1 (SERPINA1 deficiency is one of the main genetic causes related to liver disease in children. In SERPINA1 deficiency the most frequent SERPINA1 alleles found are the PI*S and PI*Z alleles. We used the polymerase chain reaction and the amplification created restriction site (ACRS technique to investigate the prevalence of the PI*S and PI*Z alleles in a group of Brazilian children (n = 200 with liver disease and established the general frequency of the PI*S allele in our population. We found a significant association of the PI*Z allele and liver disease, but no such relationship was found for the PI*S allele. Our results show that SERPINA1 deficiency due to the PI*Z allele, even when heterozygous, is a frequent cause of liver disease in our group of Brazilian children but that the PI*S allele does not confer an increased risk of hepatic disorders in our group of Brazilian children.

  13. 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)


    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.

  14. Discovery of small molecule inhibitors of ubiquitin-like poxvirus proteinase I7L using homology modeling and covalent docking approaches (United States)

    Katritch, Vsevolod; Byrd, Chelsea M.; Tseitin, Vladimir; Dai, Dongcheng; Raush, Eugene; Totrov, Maxim; Abagyan, Ruben; Jordan, Robert; Hruby, Dennis E.


    Essential for viral replication and highly conserved among poxviridae, the vaccinia virus I7L ubiquitin-like proteinase (ULP) is an attractive target for development of smallpox antiviral drugs. At the same time, the I7L proteinase exemplifies several interesting challenges from the rational drug design perspective. In the absence of a published I7L X-ray structure, we have built a detailed 3D model of the I7L ligand binding site (S2-S2' pocket) based on exceptionally high structural conservation of this site in proteases of the ULP family. The accuracy and limitations of this model were assessed through comparative analysis of available X-ray structures of ULPs, as well as energy based conformational modeling. The 3D model of the I7L ligand binding site was used to perform covalent docking and VLS of a comprehensive library of about 230,000 available ketone and aldehyde compounds. Out of 456 predicted ligands, 97 inhibitors of I7L proteinase activity were confirmed in biochemical assays (˜20% overall hit rate). These experimental results both validate our I7L ligand binding model and provide initial leads for rational optimization of poxvirus I7L proteinase inhibitors. Thus, fragments predicted to bind in the prime portion of the active site can be combined with fragments on non-prime side to yield compounds with improved activity and specificity.

  15. 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


    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.

  16. 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


    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.

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

    Institute of Scientific and Technical Information of China (English)


    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.

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

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


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

  19. Cysteine proteinase inhibitor Act d 4 is a functional allergen contributing to the clinical symptoms of kiwifruit allergy. (United States)

    Popovic, Milica M; Milovanovic, Mina; Burazer, Lidija; Vuckovic, Olga; Hoffmann-Sommergruber, Karin; Knulst, Andre C; Lindner, Buko; Petersen, Arnd; Jankov, Ratko; Gavrovic-Jankulovic, Marija


    Kiwifruit has become a frequent cause of fruit allergy in the recent years. The molecular basis of type I hypersensitivity to kiwifruit is attributed to 11 IUIS allergens, with Act d 1, Act d 2 and Act d 5 characterized in extenso. Evaluation of the allergenic properties of Act d 4, a cysteine proteinase inhibitor from green kiwifruit (Actinidia deliciosa) was performed in this study. Identity of the purified glycoprotein was determined by Edman degradation and by mass fingerprint whereby more than 90% of the primary structure of the mature kiwifruit cystatin was confirmed. Using MALDI TOF analysis, molecular masses of 10902.5 and 11055.2 Da were detected for Act d 4, respectively. Positive skin prick reactivity with Act d 4 was induced in three kiwifruit allergic patients, as well as the upregulation of CD63 and CD203c molecules in the basophile activation assay. The IgE reactivity was detected in dot blot analysis while Western blot analysis was negative using sera from six kiwifruit patients, suggesting the presence of conformational IgE epitopes on the Act d 4 molecule. As activator of effector cells in type I hypersensitivity Act d 4 is a functional allergen contributing to the clinical symptoms of kiwifruit allergy.

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

    Directory of Open Access Journals (Sweden)

    Bruno Tadeu Martins-Olivera


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

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

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


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

  2. Alpha-1 Antitrypsin Deficiency (United States)

    ... by blood tests showing the low levels of alpha-1 antitrypsin and abnormal liver tests. Other tests such as ultrasound imaging or tests using specialized X-ray techniques may be necessary. A liver biopsy may ...

  3. Leucaena leucocephala serine proteinase inhibitor: primary structure and action on blood coagulation, kinin release and rat paw edema. (United States)

    Oliva, M L; Souza-Pinto, J C; Batista, I F; Araujo, M S; Silveira, V F; Auerswald, E A; Mentele, R; Eckerskorn, C; Sampaio, M U; Sampaio, C A


    A serine proteinase inhibitor isolated from Leucaena leucocephala seeds (LlTI) was purified to homogeneity by acetone fractionation, ion exchange chromatography, gel filtration and reverse phase chromatography (HPLC). SDS-PAGE indicated a protein with M(r) 20000 and two polypeptide chains (alpha-chain, M(r) 15000, and beta-chain, M(r) 5000), the sequence being determined by automatic Edman degradation and by mass spectroscopy. LlTI is a 174 amino acid residue protein which shows high homology to plant Kunitz inhibitors, especially those double chain proteins purified from the Mimosoideae subfamily. LlTI inhibits plasmin (K(i) 3.2 x 10(-10) M), human plasma kallikrein (K(i) 6.3 x 10(-9) M), trypsin (K(i) 2.5 x 10(-8) M) and chymotrypsin (K(i) 1.4 x 10(-8) M). Factor XIIa activity is inhibited but K(i) was not determined, and factor Xa, tissue kallikrein and thrombin are not inhibited by LlTI. The action of LlTI on enzymes that participate in the blood clotting extrinsic pathway is confirmed by the prolongation of activated partial thromboplastin time, used as clotting time assay. The inhibition of the fibrinolytic activity of plasmin was confirmed on the hydrolysis of fibrin plates. LlTI inhibits kinin release from high molecular weight kininogen by human plasma kallikrein in vitro and, administered intravenously, causes a decrease in paw edema induced by carrageenin or heat in male Wistar rats. In addition, lower concentrations of bradykinin were found in limb perfusion fluids of LlTI-treated rats.

  4. A three-domain Kazal-type serine proteinase inhibitor exhibiting domain inhibitory and bacteriostatic activities from freshwater crayfish Procambarus clarkii. (United States)

    Li, Xin-Cang; Wang, Xian-Wei; Wang, Zong-Heng; Zhao, Xiao-Fan; Wang, Jin-Xing


    In crustaceans, Kazal-type serine proteinase inhibitors in hemolymph are believed to function as regulators of the host-defense reactions or inhibitors against proteinases from microorganisms. In this study, we report a Kazal-type serine proteinase inhibitor, named hcPcSPI1, from freshwater crayfish (Procambarus clarkii). We found that hcPcSPI1 is composed of a putative signal peptide, an RGD motif, and three tandem Kazal-type domains with the domain P1 residues L, L and E, respectively. Mainly, hcPcSPI1 was detected in hemocytes as well as in the heart, gills, and intestine at both the mRNA and protein levels. Quantitative real-time PCR analysis showed that hcPcSPI1 in hemocytes was upregulated by the stimulation of Esherichia coli (8099) or became decreased after a white spot syndrome virus (WSSV) challenge. In addition, hcPcSPI1 and its three independent domains were overexpressed and purified to explore their potential functions. All four proteins inhibited subtilisin A and proteinase K, but not alpha-chymotypsin or trypsin. Recombinant hcPcSPI1 could firmly attach to Gram-negative bacteria E. coli and Klebsiella pneumoniae; Gram-positive bacteria Bacillus subtilis, Bacillus thuringiensis and Staphylococcus aureus; fungi Candida albicans and Saccharomyce cerevisiae, and only domain 1 was responsible for the binding to E. coli and S. aureus. In addition, recombinant hcPcSPI1 was also found to possess bacteriostatic activity against the B. subtilis and B. thuringiensis. Domains 2 and 3 contributed mainly to these bacteriostatic activities. All results suggested that hcPcSPI1 might play important roles in the innate immunity of crayfish.

  5. Interaction between Kazal serine proteinase inhibitor SPIPm2 and viral protein WSV477 reduces the replication of white spot syndrome virus. (United States)

    Ponprateep, Sirikwan; Phiwsaiya, Kornsunee; Tassanakajon, Anchalee; Rimphanitchayakit, Vichien


    White spot syndrome (WSS) is a viral disease caused by white spot syndrome virus (WSSV) which leads to severe mortality in cultured penaeid shrimp. In response to WSSV infection in Penaeus monodon, a Kazal serine proteinase inhibitor SPIPm2, normally stored in the granules of granular and semi-granular hemocytes is up-regulated and found to deter the viral replication. By using yeast two-hybrid screening, we have identified a viral target protein, namely WSV477. Instead of being a proteinase, the WSV477 was reported to be a Cys2/Cys2-type zinc finger regulatory protein having ATP/GTP-binding activity. In vitro pull down assay confirmed the protein-protein interaction between rSPIPm2 and rWSV477. Confocal laser scanning microscopy demonstrated that the SPIPm2 and WSV477 were co-localized in the cytoplasm of shrimp hemocytes. Using RNA interference, the silencing of WSV477 resulted in down-regulated of viral late gene VP28, the same result obtained with SPIPm2. In this instance, the SPIPm2 does not function as proteinase inhibitor but inhibit the regulatory function of WSV477.

  6. 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


    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.

  7. The response to epidermal growth factor of human maxillary tumor cells in terms of tumor growth, invasion and expression of proteinase inhibitors. (United States)

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


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

  8. 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


    levels of antithrombin III (AT III), alpha 2-macroglobulin (alpha 2M) alpha 1-antitrypsin (alpha 1at), complement factors (factor B, C3, C4), pregnancy zone protein (PZP), corticosteroid binding globulin (CBG), sex hormone binding globulin (SHBG) and albumin were measured before treatment and during...

  9. A unique downstream estrogen responsive unit mediates estrogen induction of proteinase inhibitor-9, a cellular inhibitor of IL-1beta- converting enzyme (caspase 1). (United States)

    Krieg, S A; Krieg, A J; Shapiro, D J


    Recently, proteinase inhibitor 9 (PI-9) was identified as the first endogenous inhibitor of caspase 1 (IL-1beta-converting enzyme). The regulation of PI-9 expression, therefore, has great importance in the control of inflammatory processes. We reported that PI-9 mRNA and protein are rapidly and directly induced by estrogen in human liver cells. Using transient transfections to assay PI-9 promoter truncations and mutations, we demonstrate that this strong estrogen induction is mediated by a unique downstream estrogen responsive unit (ERU) approximately 200 nucleotides downstream of the transcription start site. Using primers flanking the ERU in chromatin immunoprecipitation assays, we demonstrate estrogen-dependent binding of ER to the cellular PI-9 promoter. The ERU consists of an imperfect estrogen response element (ERE) palindrome immediately adjacent to a direct repeat containing two consensus ERE half-sites separated by 13 nucleotides (DR13). In transient transfections, all four of the ERE half-sites in the imperfect ERE and in the DR13 were important for estrogen inducibility. Transfected chicken ovalbumin upstream transcription factor I and II down-regulated estrogen-mediated expression from the ERU. EMSAs using purified recombinant human ERalpha demonstrate high-affinity binding of two ER complexes to the ERU. Further EMSAs showed that one ER dimer binds to an isolated DR13, supporting the view that one ER dimer binds to the imperfect ERE and one ER dimer binds to DR13. Deoxyribonuclease I footprinting showed that purified ER protected all four of the half-sites in the ERU. Our finding that a direct repeat can function with an imperfect ERE palindrome to confer estrogen inducibility on a native gene extends the repertoire of DNA sequences able to function as EREs.

  10. Identification of proteinaceous inhibitors of a cysteine proteinase (an Arg-specific gingipain) from Porphyromonas gingivalis in rice grain, using targeted-proteomics approaches. (United States)

    Taiyoji, Mayumi; Shitomi, Yasuyuki; Taniguchi, Masayuki; Saitoh, Eiichi; Ohtsubo, Sadami


    Porphyromonas gingivalis is known to be a major etiologic agent in the onset and progression of chronic periodontitis. Among various virulence factors that this bacterium produces, Arg- and Lys-specific cysteine proteinases (gingipains) are believed to be major determinants of the pathogenicity of P. gingivalis. Here, we report on our finding that there are inhibitors of these cysteine proteinases in a rice protein fraction. Comprehensive affinity chromatography and MS analyses resulted in the identification of 17 Arg-gingipain (Rgp)-interacting proteins in the rice endosperm. Of these, four proteins (i.e., a 26 kDa globulin, a plant lipid transfer/trypsin-alpha amylase inhibitor, the RA17 seed allergen, and an alpha amylase/trypsin inhibitor) were estimated to account for 90% of the Rgp inhibitory activity in the rice protein fraction, using a two-dimensional gel system of double-layer reverse zymography. In addition, a synthetic peptide derived from an Rgp-interacting protein, cyanate hydratase, could inhibit the growth of P. gingivalis and showed inhibitory activity against both the Arg- and Lys-gingipains. These results suggest that these rice proteins may be useful as nutraceutical ingredients for the prevention and management of periodontal diseases.

  11. Safety and efficacy of alpha-1-antitrypsin augmentation therapy in the treatment of patients with alpha-1-antitrypsin deficiency

    Directory of Open Access Journals (Sweden)

    Irina Petrache


    Full Text Available Irina Petrache1, Joud Hajjar1, Michael Campos21Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA; 2Department of Medicine, Division of Pulmonary, Critical Care and Sleep Medicine, Miller School of Medicine, University of Miami, Florida, USA Abstract: Alpha-1-antitrypsin deficiency (AATD, also known as alpha1-proteinase inhibitor deficiency, is an autosomal co-dominant condition. The genotypes associated with AATD include null, deficient, and dysfunctional alpha-1-antitrypsin (A1AT variants, which result in low levels of circulating functional A1AT, unbalanced protease activity, and an increased risk of developing lung emphysema, the leading cause of morbidity in these patients. Furthermore, the most common abnormal genotype, Pi*ZZ may also cause trapping of abnormally folded protein polymers in hepatocytes causing liver dysfunction. A major focus of therapy for patients with lung disease due to AATD is to correct the A1AT deficiency state by augmenting serum levels with intravenous infusions of human plasma-derived A1AT. This strategy has been associated with effective elevations of A1AT levels and function in serum and lung epithelial fluid and observational studies suggest that it may lead to attenuation in lung function decline, particularly in patients with moderate impairment of lung function. In addition, an observational study suggests that augmentation therapy is associated with a reduction of mortality in subjects with AATD and moderate to severe lung impairment. More recent randomized placebo-controlled studies utilizing computer scan densitometry suggest that this therapy attenuates lung tissue loss. Augmentation therapy has a relative paucity of side effects, but it is highly expensive. Therefore, this therapy is recommended for patients with AATD who have a high-risk A1AT genotype with plasma A1AT below protective levels (11 µM and evidence of obstructive lung disease. In this article, we

  12. 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


    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.

  13. Alpha-1-antitrypsin deficiency: An overview of recent advances

    Directory of Open Access Journals (Sweden)

    El Hazmi Mohsen


    Full Text Available Alpha 1-antitrypsin (αl AT, a serpine, is one of the most important proteinase inhibitor in the serum and plays an essential role in protection of the lung tissues against the proteolytic attach of elastase. The gene for a1AT is located on chromosome 14 q 32 and is highly susceptible to mutations. A large number of variants of α 1 AT are known and some including PiZ and PiS result in a1AT deficiency. In patients with PiZ, the most severe and common α1AT deficient variant, the α1AT protein accumulates in the liver and results in severe hepatic diseases. Other clinical consequences of α1AT deficiency include emphysema in majority of the patients. This state is further aggravated in patients who smoke. Several treatment strategies have been suggested, including replacement therapy by purified α1AT or recombinant α1AT given intravenously or as aerosol. Synthetic peptides. lung transplantation and volume reduction surgery are under investigation and evaluation. This paper updates the information on α1 AT and its deficiency state.

  14. Purification and Partial Characterization of Trypsin-Specific Proteinase Inhibitors from Pigeonpea Wild Relative Cajanus platycarpus L.(Fabaceae active against Gut Proteases of Lepidopteran pest Helicoverpa armigera

    Directory of Open Access Journals (Sweden)

    Marri Swathi


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

  15. 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


    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.

  16. Evaluation of in vitro and in vivo effects of semipurified proteinase inhibitors from Theobroma seeds on midgut protease activity of Lepidopteran pest insects. (United States)

    Paulillo, Luis Cesar Maffei Sartini; Sebbenn, Alexandre Magno; de Carvalho Derbyshire, Maria Tereza Vitral; Góes-Neto, Aristóteles; de Paula Brotto, Marco Aurélio; Figueira, Antonio


    We have characterized in vitro and in vivo effects of trypsin inhibitors from Theobroma seeds on the activity of trypsin- and chymotrypsin-like proteins from Lepidopteran pest insects. The action of semipurified trypsin inhibitors from Theobroma was evaluated by the inhibition of bovine trypsin and chymotrypsin activities determined by the hydrolysis of N-Benzoyl-DL-Arginine-p-Nitroanilide (BAPA) and N-Succinyl-Ala-Ala-Pho-Phe p-Nitroanilide (S-(Ala)2ProPhe-pNA). Proteinase inhibitor activities from Theobroma cacao and T. obovatum seeds were the most effective in inhibiting trypsin-like proteins, whereas those from T. obovatum and T. sylvestre were the most efficient against chymotrypsin-like proteins. All larvae midgut extracts showed trypsin-like proteolytic activities, and the putative trypsin inhibitors from Theobroma seeds significantly inhibited purified bovine trypsin. With respect to the influence of Theobroma trypsin inhibitors on intact insects, the inclusion of T. cacao extracts in artificial diets of velvet bean caterpillars (Anticarsia gemmatalis) and sugarcane borer (Diatraea saccharalis) produced a significant increase in the percentage of adult deformation, which is directly related to both the survival rate of the insects and oviposition.

  17. Specificity of binding of the low density lipoprotein receptor-related protein to different conformational states of the clade E serpins plasminogen activator inhibitor-1 and proteinase nexin-1. (United States)

    Jensen, Jan K; Dolmer, Klavs; Gettins, Peter G W


    The low density lipoprotein receptor-related protein (LRP) is the principal clearance receptor for serpins and serpin-proteinase complexes. The ligand binding regions of LRP consist of clusters of cysteine-rich approximately 40-residue complement-like repeats (CR), with cluster II being the principal ligand-binding region. To better understand the specificity of binding at different sites within the cluster and the ability of LRP to discriminate in vivo between uncomplexed and proteinase-complexed serpins, we have systematically examined the affinities of plasminogen activator inhibitor-1 (PAI-1) and proteinase nexin-1 (PN-1) in their native, cleaved, and proteinase-complexed states to (CR)(2) and (CR)(3) fragments of LRP cluster II. A consistent blue shift of the CR domain tryptophan fluorescence suggested a common mode of serpin binding, involving lysines on the serpin engaging the acidic region around the calcium binding site of the CR domain. High affinity binding of non-proteinase-complexed PAI-1 and PN-1 occurred to all fragments containing three CR domains (3-59 nm) and most that contain only two CR domains, although binding energies to different (CR)(3) fragments differed by up to 18% for PAI-1 and 9% for PN-1. No detectable difference in affinity was seen between native and cleaved serpin. However, the presence of proteinase in complex with the serpin enhanced affinity modestly and presumably nonspecifically. This may be sufficient to give preferential binding of such complexes in vivo at the relevant physiological concentrations.

  18. 丝氨酸蛋白酶抑制剂PI-9的研究进展%The present understanding of serine proteinase inhibitor-9

    Institute of Scientific and Technical Information of China (English)

    雷晓晔; 周业江


    丝氨酸蛋白酶抑制剂-9(serine proteinase inhibitor9,PI-9)是丝氨酸蛋白酶抑制剂的重要成员,也是目前发现的颗粒蛋白酶B(granzyme B,GrB)唯一的内源性丝氨酸蛋白酶抑制剂.近年研究发现,PI-9能抑制GrB所致的靶细胞DNA断裂而阻断其诱导的细胞凋亡.在生理状况下,PI-9能阻止细胞毒淋巴细胞之间的相互攻击,维持机体免疫豁免部位的功能.近年研究发现PI-9还涉及-多种病理过程,诸如调节炎性介质反应,移植免疫应答及介导肿瘤免疫耐受等多种功能.%Serine proteinase inhibitor9(P1-9),a charac-teristic member of serpins,has been identified as the only inhibitor of granzyme B(GrB).Accumulated evidence suggested that PI-9 inhibits GrB-induced apoptosis by blocking DNA fragmentation of target cell.Physiologically,PI-9 could protect cytotoxic lymphocytes from committing autolysis or fratricide,and play an important role in facilitating immunologic tolerance of immune-privileged sites.In addition,evidences in recent years suggest that PI-9 Was also involved in vailous pathologic processes,such as inflammation,trans plantation and immune tolerance of tumor.

  19. Molecular characterization, expression and function analysis of a five-domain Kazal-type serine proteinase inhibitor from pearl oyster Pinctada fucata. (United States)

    Zhang, Dianchang; Ma, Jianjun; Jiang, Shigui


    Serine proteinase inhibitors represent an expanding superfamily of endogenous inhibitors that are regulate proteolytic events and involved in a variety of physiological and immunological processes. A five-domain Kazal-type serine proteinase inhibitor (poKSPI) was identified and characterized from pearl oyster Pinctada fucata based on expressed sequence tag (EST) analysis. The full-length cDNA was 737 bp with an open reading frame (ORF) 660 bp encoding a 219 amino acid protein a theoretical molecular weight (Mw) of 23.3 kDa and an isoelectric point (pI) of 8.40. A putative signal peptide of 19 amino acid residues and five tandem Kazal domains were identified. Four of the Kazal domains had the highly conserved motif sequences with six cysteine residues responsible for the formation of disulfide bridges. The deduced amino acid sequence of the poKSPI shared high homology with KSPIs from Hirudo medicinalis. The poKSPI mRNA could be detected in all examined tissues, the expression level of the poKSPI mRNA was the highest in mantle and gonad, while the lowest in haemocyte and intestine. After LPS challenge, the expression level of the poKSPI mRNA in digestive gland was significantly up-regulated at 4 h post-challenge and reached the peak at 12 h post-challenge, which was 4.23-fold higher than control group; the expression level of the poKSPI mRNA in gill was also significantly up-regulated at 8 and 12 h post-challenge, which were 4.48 and 2.26-fold higher than control group. After Vibrio alginolyticus challenge, the expression levels of the poKSPI mRNA in digestive gland were significantly up-regulated at 8, 12, 48 and 72 h post-challenge, which were 1.70, 1.79, 3.89 and 5.69-fold higher than control group, respectively; the expression level of the poKSPI mRNA in gill was significantly up-regulated at 24 h post-challenge, which was 5.30-fold higher than control group. The recombinant poKSPI protein could inhibit chymotrypsin and trypsin activities in dose

  20. Cysteine proteinases and cystatins

    Directory of Open Access Journals (Sweden)

    Adeliana S. Oliveira


    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.

  1. Hereditary fructose intolerance and alpha(1) antitrypsin deficiency. (United States)

    Hillebrand, G; Schneppenheim, R; Oldigs, H D; Santer, R


    A patient with coexisting hereditary fructose intolerance (HFI) and alpha(1) antitrypsin deficiency (alpha(1)ATD) is described. Protease inhibitor typing was not conclusive, presumably because of impaired N-glycosylation secondary to HFI. The case underlines the diagnostic role of molecular genetic techniques in inborn errors of metabolism.

  2. The Kinetic Characteristics of Proteinase A Inhibitor GLPAI%蛋白酶A抑制剂GLPAI动力学性质的研究

    Institute of Scientific and Technical Information of China (English)

    李屹松; 田亚平


    对一种从灵芝发酵液中提取得到的蛋白酶A抑制剂GLPAI(Ganoderma Lucidum pro-teinase A inhibitor)的动力学性质进行了研究,分别以胃蛋白酶、胰蛋白酶和蛋白酶A为底物考察了GLPAI的动力学性质,实验结果:GLPAI对上述3种蛋白酶的抑制类型均属于混合型抑制模式,对胃蛋白酶的Ki=4.64(μmol/L);对胰蛋白酶的Ki=33.5(μmo1/L);对蛋白酶A的Ki=2.7(μmol/L).

  3. 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


    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....... is not clarified. Using a rat model of diet-induced obesity (DIO), we characterized the pharmacological mechanisms underlying the appetite suppressive effect of tesofensine. DIO rats treated with tesofensine (2.0 mg/kg, s.c.) for 16 days showed significantly lower body weights than vehicle-treated DIO rats, being...

  4. NaStEP: a proteinase inhibitor essential to self-incompatibility and a positive regulator of HT-B stability in Nicotiana alata pollen tubes. (United States)

    Jiménez-Durán, Karina; McClure, Bruce; García-Campusano, Florencia; Rodríguez-Sotres, Rogelio; Cisneros, Jesús; Busot, Grethel; Cruz-García, Felipe


    In Solanaceae, the self-incompatibility S-RNase and S-locus F-box interactions define self-pollen recognition and rejection in an S-specific manner. This interaction triggers a cascade of events involving other gene products unlinked to the S-locus that are crucial to the self-incompatibility response. To date, two essential pistil-modifier genes, 120K and High Top-Band (HT-B), have been identified in Nicotiana species. However, biochemistry and genetics indicate that additional modifier genes are required. We recently reported a Kunitz-type proteinase inhibitor, named NaStEP (for Nicotiana alata Stigma-Expressed Protein), that is highly expressed in the stigmas of self-incompatible Nicotiana species. Here, we report the proteinase inhibitor activity of NaStEP. NaStEP is taken up by both compatible and incompatible pollen tubes, but its suppression in Nicotiana spp. transgenic plants disrupts S-specific pollen rejection; therefore, NaStEP is a novel pistil-modifier gene. Furthermore, HT-B levels within the pollen tubes are reduced when NaStEP-suppressed pistils are pollinated with either compatible or incompatible pollen. In wild-type self-incompatible N. alata, in contrast, HT-B degradation occurs preferentially in compatible pollinations. Taken together, these data show that the presence of NaStEP is required for the stability of HT-B inside pollen tubes during the rejection response, but the underlying mechanism is currently unknown.

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

    Institute of Scientific and Technical Information of China (English)

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


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

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

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


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

  7. 无核荔枝半胱氨酸蛋白酶抑制剂基因克隆及序列分析%Cloning and Sequence Analysis of a Cysteine Proteinase Inhibitor Gene of Seedless Litchi

    Institute of Scientific and Technical Information of China (English)

    刘兴地; 刘娜; 李明芳; 郑学勤


    [ Objective] This study aimed to clone the cysteine proteinase inhibitor gene of seedless litchi and analyze the sequence. [ Method] According to the EST sequence of cysteine proteinase inhibitor in constructed SSH subtractive library of seedless litchi abortion, nucleotide sequence of the cysteine proteinase inhibitor gene was obtained by using RACE technology and analyzed by using bioinformatics software. [ Result] A cysteine protease inhibitor gene was obtained with the sequence of 635 bp containing a 321 bp open reading frame. It was predicted that the encoded protein contained 106 amino acids with conserved domain of cysteine proteinase inhibitor and had relatively high homology with the cysteine proteinase inhibitor gene of several species. [ Conclusion] This study had laid the foundation for further exploring the physiological functions of this cysteine proteinase inhibitor gene in plants.%[目的]对无核荔枝的半胱氨酸蛋白酶抑制剂基因进行克隆,并对其序列下进行分析.[方法]根据构建的无核荔枝胚败育SSH消减文库的半胱氨酸蛋白酶抑制剂EST序列,通过RACE技术获得半胱氨酸蛋白酶抑制基因的核苷酸序列并应用生物信息学软件进行分析.[结果]获得一个635 bp的半胱氨酸蛋白酶抑制基因序列,预测该序列含有321 bp的开放阅读框,推导其编码的蛋白质含106个氨基酸,具有半胱氨酸蛋白酶抑制剂保守区,与多个物种的半胱氨酸蛋白酶抑制剂基因具有较高的同源性.[结论]为进一步研究半胱氨酸蛋白酶抑制剂在植物中的生理功能奠定了基础.

  8. Cinanserin is an inhibitor of the 3C-like proteinase of severe acute respiratory syndrome coronavirus and strongly reduces virus replication in vitro. (United States)

    Chen, Lili; Gui, Chunshan; Luo, Xiaomin; Yang, Qingang; Günther, Stephan; Scandella, Elke; Drosten, Christian; Bai, Donglu; He, Xichang; Ludewig, Burkhard; Chen, Jing; Luo, Haibin; Yang, Yiming; Yang, Yifu; Zou, Jianping; Thiel, Volker; Chen, Kaixian; Shen, Jianhua; Shen, Xu; Jiang, Hualiang


    The 3C-like proteinase (3CLpro) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is one of the most promising targets for anti-SARS-CoV drugs due to its crucial role in the viral life cycle. In this study, a database containing structural information of more than 8,000 existing drugs was virtually screened by a docking approach to identify potential binding molecules of SARS-CoV 3CLpro. As a target for screening, both a homology model and the crystallographic structure of the binding pocket of the enzyme were used. Cinanserin (SQ 10,643), a well-characterized serotonin antagonist that has undergone preliminary clinical testing in humans in the 1960s, showed a high score in the screening and was chosen for further experimental evaluation. Binding of both cinanserin and its hydrochloride to bacterially expressed 3CLpro of SARS-CoV and the related human coronavirus 229E (HCoV-229E) was demonstrated by surface plasmon resonance technology. The catalytic activity of both enzymes was inhibited with 50% inhibitory concentration (IC50) values of 5 microM, as tested with a fluorogenic substrate. The antiviral activity of cinanserin was further evaluated in tissue culture assays, namely, a replicon system based on HCoV-229E and quantitative test assays with infectious SARS-CoV and HCoV-229E. All assays revealed a strong inhibition of coronavirus replication at nontoxic drug concentrations. The level of virus RNA and infectious particles was reduced by up to 4 log units, with IC50 values ranging from 19 to 34 microM. These findings demonstrate that the old drug cinanserin is an inhibitor of SARS-CoV replication, acting most likely via inhibition of the 3CL proteinase.

  9. Biochemical, immunological and kinetic characterization and partial sequence analysis of a thiol proteinase inhibitor from Bubalus bubalis kidney: An attempt targeting kidney disorders. (United States)

    Shamsi, Anas; Ahmed, Azaj; Bano, Bilqees


    In the present study a thiol proteinase inhibitor was isolated from buffalo kidney making use of ammonium sulphate precipitation and gel filtration chromatography on Sephacryl S-100HR column. Purified inhibitor is homogeneous as it displayed a single band in gel electrophoresis both under reducing and non-reducing environment and is of 65KDa as revealed by gel filtration and SDS PAGE. Kinetic studies revealed the presence of reversible accompanied with competitive mode of inhibition; showing maximum efficacy against papain (Ki=2.90×10(-4)). It was maximally active at pH 8.0 and was stable for a period of 30, 60 and 90 days at 37, 4 and -20°C respectively. Immunological studies confirmed its purity of epitopes as a single precipitin line is obtained in immunodiffusion. N-terminal analysis revealed that it shared a good homology with mouse kidney cystatin as well as with Human Cys C and Cys E thereby advocating its use as a model for various human oriented studies which targets how the kidney cystatin level varies in accordance with various drugs that are currently being used as a target for variety of diseases.

  10. Alpha 1-antitrypsin does not inhibit human monocyte caspase-1.

    Directory of Open Access Journals (Sweden)

    Mohd Akhlakur Rahman

    Full Text Available Alpha 1-antitrypsin (A1AT is a 52 kDa serine protease inhibitor produced largely by hepatocytes but also by mononuclear phagocytes. A1AT chiefly inhibits neutrophil elastase and proteinase-3 but has also been reported to have immune modulatory functions including the ability to inhibit caspases. Its clinical availability for infusion suggests that A1AT therapy might modulate caspase related inflammation. Here we tested the ability of A1AT to modulate caspase-1 function in human mononuclear phagocytes.Purified plasma derived A1AT was added to active caspase-1 in a cell-free system (THP-1 lysates as well as added exogenously to cell-culture models and human whole blood models of caspase-1 activation. Functional caspase-1 activity was quantified by the cleavage of the caspase-1 specific fluorogenic tetrapeptide substrate (WEHD-afc and the release of processed IL-18 and IL-1β.THP-1 cell lysates generated spontaneous activation of caspase-1 both by WEHD-afc cleavage and the generation of p20 caspase-1. A1AT added to this cell free system was unable to inhibit caspase-1 activity. Release of processed IL-18 by THP-1 cells was also unaffected by the addition of exogenous A1AT prior to stimulation with LPS/ATP, a standard caspase-1 activating signal. Importantly, the A1AT exhibited potent neutrophil elastase inhibitory capacity. Furthermore, A1AT complexed to NE (and hence conformationally modified also did not affect THP-1 cell caspase-1 activation. Finally, exogenous A1AT did not inhibit the ability of human whole blood samples to process and release IL-1β.A1AT does not inhibit human monocyte caspase-1.

  11. A Kazal-type serine proteinase inhibitor from chicken liver (clTI-1): purification, primary structure, and inhibitory properties. (United States)

    Kubiak, Agnieszka; Jakimowicz, Piotr; Polanowski, Antoni


    Low-molecular-mass trypsin inhibitor (clTI-1; chicken liver Trypsin Inhibitor-1) was purified from chicken liver by extraction with perchloric acid, ammonium sulfate precipitation, a combination of ethanol-acetone fractionation followed by gel filtration, ion-exchange chromatography and RP-HPLC on a C18 column. The inhibitor occurs in two isoforms with molecular masses of 5938.56 and 6026.29 Da (determined by MALDI TOFF mass spectrometry). The complete amino acid sequences of both isoforms were determined (UniProtKB/Swiss-Prot P85000; ISK1L_CHICK). The inhibitor shows a high homology to Kazal-type family inhibitors, especially to trypsin/acrosin inhibitors and pancreatic secretory trypsin inhibitors. clTI-1 inhibits both bovine and porcine trypsin (K(a)=1.1 x 10(9) M(-1) and 2.5 x 10(9) M(-1), respectively). Significant differences were shown in the inhibition of the anionic and cationic forms of chicken trypsin (K(a)=4.5 x 10(8) M(-1) and 1.2 x 10(10) M(-1)). Weak interaction with human plasmin (K(a)=1.2 x 10(7) M(-1)) was also revealed.

  12. DNA structures decorated with cathepsin G/secretory leukocyte proteinase inhibitor stimulate IFNI production by plasmacytoid dendritic cells

    DEFF Research Database (Denmark)

    Skrzeczynska-Moncznik, Joanna; Wlodarczyk, Agnieszka; Banas, Magdalena;


    psoriasis. Here, we demonstrate that IFNI production in pDCs is stimulated by DNA structures containing the neutrophil serine protease cathepsin G (CatG) and the secretory leukocyte protease inhibitor (SLPI), which is a controlling inhibitor of serine proteases. We also demonstrate the presence...... of neutrophil-derived DNA structures containing CatG and SLPI in lesional skin samples from psoriasis patients. These findings suggest a previously unappreciated role for CatG in psoriasis by linking CatG and its inhibitor SLPI to the IFNI-dependent regulation of immune responses by pDCs in psoriatic skin....

  13. Triangular gold nanoparticles conjugated with peptide ligands: a new class of inhibitor for Candida albicans secreted aspartyl proteinase. (United States)

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


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

  14. Elastase, α1-proteinase inhibitor, and interleukin-8 in children and young adults with end-stage kidney disease undergoing continuous ambulatory peritoneal dialysis. (United States)

    Polańska, Bożena; Augustyniak, Daria; Makulska, Irena; Niemczuk, Maria; Jankowski, Adam; Zwolińska, Danuta


    Peritoneal dialysis is one of the main modality of treatment in end-stage kidney diseases (ESKD) in children. In our previous work in chronic kidney disease patients, in pre-dialyzed period and on hemodialysis, the neutrophils were highly activated. The aim of this study was to assess an inflammatory condition and neutrophil activation in ESKD patients undergoing continuous ambulatory peritoneal dialysis (CAPD). Thirteen CAPD patients without infection, both sexes, aged 2.5-24 years, and group of healthy subjects (C) were studied. For comparative purposes the conservatively treated (CT) group of ESKD patients was included. Neutrophil elastase in complex with α1-proteinase inhibitor (NE-α1PI; ELISA), α1-proteinase inhibitor (α1PI; radial immunodiffusion) and interleukin-8 (IL-8; ELISA) were measured in the blood samples from CAPD, CT, and C group and in the peritoneal dialysate fluid (PDF) samples of patients on CAPD. A significantly increased plasma NE-α1PI levels (median 176.5 μg/L, range 85.2-373.2 μg/L; p < 0.00005), serum IL-8 (median 18.6 pg/mL, range 15.73-35.28 pg/mL; p < 0.05), and slightly decreased serum α1PI (median 1,540 mg/L, range 1,270-1,955; p ≤ 0.05) compared to the control groups were found. There were no significant differences of analyzed parameters between CAPD and CT patients. The concentration ratio of NE-α1PI, α1PI and IL-8 in blood/PDF was 29.97, 8.24, and 4.48, respectively. There were significantly positive correlations between serum and PDF concentration of α1PI and IL-8 (r = 0.613, p < 0.05; r = 0.59; p < 0.005, respectively). The results of our study demonstrate that neutrophils are highly activated in non-infected CAPD patients. The pivotal marker of this activation is NE-α1PI. It may contribute to chronic inflammation and tissues injury.

  15. Matrix metalloproteinase-2 and tissue inhibitor of metallo-proteinase-2 in colorectal carcinoma invasion and metastasis


    Li, Bing-hui; zhao,Peng; Liu, Shi-Zheng; Yu, Yue-Ming; Han, Mei; Wen, Jin-kun


    AIM: To explore the relationship between matrix metallopr-oteinase-2 (MMP-2) and tissue inhibitor of metallopr-oteinase-2 (TIMP-2) in the development of colorectal carcinoma and to provide a valuable marker for clinical diagnosis.

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

  17. 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;


    . Neutrophils from patients with A1AT-deficiency carrying the (PI)ZZ mutation in the A1AT gene appeared structurally and functionally normal, but A1AT produced in leukocytes of these patients lacked the ability to bind proteases efficiently. We conclude that A1AT generation and release from neutrophils add......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......1AT 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...

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

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


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

  19. Potential Use of Proteinase Inhibitors, Avidin, and Other Bio-reagents for Synergizing Bt Performance and Delaying Resistance Development to Bt (United States)

    After being ingested by target insects, the insecticidal proteins from Bacillus thuringiensis (Bt) need to go through a proteolytic process by insect midgut proteinases to become activated. At the same time, Bt can be hydrolyzed and degraded by midgut proteinases to become non-toxic to target insect...

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

    Institute of Scientific and Technical Information of China (English)

    康鲁平; 齐荔红; 张俊平; 周斌


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

  1. Entamoeba histolytica: correlation of assessment methods to measure erythrocyte digestion, and effect of cysteine proteinases inhibitors in HM-1:IMSS and HK-9:NIH strains. (United States)

    Mora-Galindo, Juan; Anaya-Velázquez, Fernando; Ramírez-Romo, Susana; González-Robles, Arturo


    Entamoeba histolytica trophozoites are able to degrade human erythrocytes; the loss of erythrocyte cellular matrix and degradation of plasma membrane were observed, along with the decrease in the average size of digestive vacuoles. Ninety-six percent of hemoglobin ingested was hydrolyzed by trophozoites within 3h, as evidenced by electrophoresis. Accordingly, X-ray spectroscopy revealed the presence of iron inside vacuoles after erythrophagocytosis, the concentration of which decreased to control levels in a similar period. Quantification of erythrocyte digestion at the early and late periods was determined by a spectrophotometric procedure, with t(1/2)=1.67 h and 35-min for HM-1:IMSS and HK-9:NIH trophozoites, respectively. In the latter, activity was due to the combined action of intracellular enzymatic activity and exocytosis. E-64c and leupeptin totally inhibited erythrocyte digestion within a 3-h period, thereafter hydrolysis took place at lower rate. Our results suggest that erythrocyte digestion in E. histolytica proceeds in different ways in these two amebic strains, and can be blocked by proteinase inhibitors.

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

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


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

  3. The recombinant prepro region of TvCP4 is an inhibitor of cathepsin L-like cysteine proteinases of Trichomonas vaginalis that inhibits trichomonal haemolysis. (United States)

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


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

  4. A Kazal-type serine proteinase inhibitor from Cyclina sinensis is involved in immune response and signal pathway initiation. (United States)

    Ren, Yipeng; Zhang, Hao; Pan, Baoping; Yan, Chuncai


    Serine protease inhibitors (SPIs) are an important group of protease inhibitors involved in a variety of biological processes. In the present study, a Kazal-type serine protease inhibitor homolog gene (designated as CsKPI) was identified from a Cyclina sinensis cDNA library. The open reading frame consists of 456 bp and encodes a protein of 151 amino acid residues with a theoretical molecular mass of 16.85 kDa and an isoelectric point of 5.74. Furthermore, using quantitative real-time PCR, we focused on the expression patterns of CsKPI found in tissues and on the stimulation of this gene's expression by bacteria. The results show that a higher-level mRNA expression of CsKPI was detected in hemocytes (P < 0.05) and was significantly upregulated at 3 h (P < 0.01) upon receiving bacterial challenges with Vibrio anguillarum. In addition, after the CsKPI gene was silenced by RNA interference, the expression of the CsTLR2 and CsMyD88 genes was extremely significantly decreased (P < 0.01) in C. sinensis. Finally, the recombinant CsKPI (rCsKPI) protein was purified and shown to exhibit less inhibitory activity than C-lyz against V. anguillarum in vitro. Hence, we propose that CsKPI plays an important role in the innate immunity and mediates TLR2 and MyD88-dependent pathway initiation in C. sinensis.

  5. 丝氨酸蛋白酶抑制剂B9与相关免疫细胞%Serine proteinase inhibitor B9 and related immune cells

    Institute of Scientific and Technical Information of China (English)

    邓常文; 张星星; 白冲


    Serine proteinase inhibitor B9 (Serpin B9),one member of the protease inhibitor superfamily including human serine proteinase inhibitor 9 (PI-9) and rat serine proteinase inhibitor homologous protein SPI-6,is an endogenous protease inhibitor mainly against GrB.Serpin B9 regulate apoptosis,immune reaction,DNA vaccines' abilities in T lymphocytes,dendritic cells,natural killer cells,neutrophils,mesenchymal stem cells and tumor cells.Study on the function of Serpin B9 could not only facilitate further exploration of the mechanism of immune related diseases,but also provide new theoretical basis for the treatment of tumor and the clinical applicution of stem cells.%丝氨酸蛋白酶抑制剂B9(Serpin B9)是蛋白酶抑制剂超家族成员,包括人丝氨酸蛋白酶抑制剂(PI-9)及鼠丝氨酸蛋白酶抑制剂同源蛋白(SPI-6),是主要针对颗粒蛋白酶B(GrB)的内源性蛋白酶抑制剂.Serpin B9的调节在T淋巴细胞、树突状细胞、自然杀伤细胞、中性粒细胞、间充质干细胞、肿瘤细胞有抑制凋亡、维持细胞平衡、调节免疫反应、增强DNA疫苗能力等功能.研究Serpin B9的作用机理,将有利于相关免疫疾病发生机制的探索,并为临床干细胞治疗、抗肿瘤治疗等提供新的理论依据.

  6. 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.


    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.

  7. Manduca sexta hemolymph proteinase 21 activates prophenoloxidase-activating proteinase 3 in an insect innate immune response proteinase cascade. (United States)

    Gorman, Maureen J; Wang, Yang; Jiang, Haobo; Kanost, Michael R


    Melanization, an insect immune response, requires a set of hemolymph proteins including pathogen recognition proteins that initiate the response, a cascade of mostly unknown serine proteinases, and phenoloxidase. Until now, only initial and final proteinases in the pathways have been conclusively identified. Four such proteinases have been purified from the larval hemolymph of Manduca sexta: hemolymph proteinase 14 (HP14), which autoactivates in the presence of microbial surface components, and three prophenoloxidase-activating proteinases (PAP1-3). In this study, we have used two complementary approaches to identify a serine proteinase that activates proPAP3. Partial purification from hemolymph of an activator of proPAP3 resulted in an active fraction with two abundant polypeptides of approximately 32 and approximately 37 kDa. Labeling of these polypeptides with a serine proteinase inhibitor, diisopropyl fluorophosphate, indicated that they were active serine proteinases. N-terminal sequencing revealed that both were cleaved forms of the previously identified hemolymph serine proteinase, HP21. Surprisingly, cleavage of proHP21 had occurred not at the predicted activation site but more N-terminal to it. In vitro reactions carried out with purified HP14 (which activates proHP21), proHP21, proPAP3, and site-directed mutant forms of the latter two proteinases confirmed that HP21 activates proPAP3 by limited proteolysis. Like the HP21 products purified from hemolymph, HP21 that was activated by HP14 in the in vitro reactions was not cleaved at its predicted activation site.

  8. Knock-down of transcript abundance of a family of Kunitz proteinase inhibitor genes in white clover (Trifolium repens) reveals a redundancy and diversity of gene function. (United States)

    Islam, Afsana; Leung, Susanna; Burgess, Elisabeth P J; Laing, William A; Richardson, Kim A; Hofmann, Rainer W; Dijkwel, Paul P; McManus, Michael T


    The transcriptional regulation of four phylogenetically distinct members of a family of Kunitz proteinase inhibitor (KPI) genes isolated from white clover (Trifolium repens; designated Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5) has been investigated to determine their wider functional role. The four genes displayed differential transcription during seed germination, and in different tissues of the mature plant, and transcription was also ontogenetically regulated. Heterologous over-expression of Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5 in Nicotiana tabacum retarded larval growth of the herbivore Spodoptera litura, and an increase in the transcription of the pathogenesis-related genes PR1 and PR4 was observed in the Tr-KPI1 and Tr-KPI4 over-expressing lines. RNA interference (RNAi) knock-down lines in white clover displayed significantly altered vegetative growth phenotypes with inhibition of shoot growth and a stimulation of root growth, while knock-down of Tr-KPI1, Tr-KPI2 and Tr-KPI5 transcript abundance also retarded larval growth of S. litura. Examination of these RNAi lines revealed constitutive stress-associated phenotypes as well as altered transcription of cellular signalling genes. These results reveal a functional redundancy across members of the KPI gene family. Further, the regulation of transcription of at least one member of the family, Tr-KPI2, may occupy a central role in the maintenance of a cellular homeostasis.

  9. 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


    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.

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


    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.

  11. The remarkable efficiency of a Pin-II proteinase inhibitor sans two conserved disulfide bonds is due to enhanced flexibility and hydrogen bond density in the reactive site loop. (United States)

    Joshi, Rakesh S; Mishra, Manasi; Tamhane, Vaijayanti A; Ghosh, Anirban; Sonavane, Uddhavesh; Suresh, C G; Joshi, Rajendra; Gupta, Vidya S; Giri, Ashok P


    Capsicum annuum (L.) expresses diverse potato type II family proteinase inhibitors comprising of inhibitory repeat domain (IRD) as basic functional unit. Most IRDs contain eight conserved cysteines forming four disulfide bonds, which are indispensible for their stability and activity. We investigated the functional significance of evolutionary variations in IRDs and their role in mediating interaction between the inhibitor and cognate proteinase. Among the 18 IRDs encoded by C. annuum, IRD-7, -9, and -12 were selected for further characterization on the basis of variation in their reactive site loop, number of conserved cysteine residues, and higher theoretical ΔGbind for interaction with Helicoverpa armigera trypsin. Moreover, inhibition kinetics showed that IRD-9, despite loss of some of the disulfide bonds, was a more potent proteinase inhibitor among the three selected IRDs. Molecular dynamic simulations revealed that serine residues in the place of cysteines at seventh and eighth positions of IRD-9 resulted in an increase in the density of intramolecular hydrogen bonds and reactive site loop flexibility. Results of the serine residues chemical modification also supported this observation and provided a possible explanation for the remarkable inhibitory potential of IRD-9. Furthermore, this natural variant among IRDs showed special attributes like stability to proteolysis and synergistic inhibitory effect on other IRDs. It is likely that IRDs have coevolved selective specialization of their structure and function as a response towards specific insect proteases they encountered. Understanding the molecular mechanism of pest protease-plant proteinaceous inhibitor interaction will help in developing effective pest control strategies. An animated interactive 3D complement (I3DC) is available in Proteopedia at

  12. Expression and regulation of metalloproteinases-2, -9 and tissue inhibitors of metallo- proteinases in rat corpus luteum

    Institute of Scientific and Technical Information of China (English)


    The expression and regulation of metalloproteinases-2, -9 (MMP-2, -9) and their tissue inhibitors TIMP-1, -2, -3 mRNA were studied in this experiment. In the PMSG- hCG primed pseudopregnant rat, MMP-2, -9 mRNA levels were the highest at Day 1, decreased from Day 4, and reached the minimal level at Day 8, then increased at Day 14; no significant changes were observed in TIMP-2 mRNA expression from Day 1 to Day 14; TIMP-3 mRNA expression was the lowest at Day 1, increased from Day 4, reached the maximal level at Day 8, and persisted to Day 14. TNF-αcould significantly increase the expression of MMP-2, -9 and TIMP-1 mRNA in the in vitro perfused pseudopregnant CL, and decrease the expression of TIMP-3 mRNA, but had no effect on TIMP-2 mRNA expression. The results indicate that MMP-2, -9 and TIMP-1, -2, -3 might be involved in the regulation of CL function and maintenance of CL structure via their coordinated gene expression. TNF-α could inhibit luteal regression via increasing MMP-2, -9 and TIMP-1 mRNA in the in vitro perfused pseudopregnant ovary.

  13. Expression of gelatinases and tissue inhibitors of metallo- proteinases in the rhesus monkey (Macaca mulatta) corpus luteum

    Institute of Scientific and Technical Information of China (English)


    Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are believed to play important roles in the formation and regression of corpus luteum (CL). This study is to investigate the expression of gelatinases (MMP-2, -9) and TIMPs in the rhesus monkey CL in both early and late luteal phases and during the early stages of pregnancy. Ovaries were collected from regularly cycling rhesus monkey at D5 and D15 following ovulation and at D12, D18 and D26 of pregnancy. In situ hybridization revealed that in the CL MMP-2 mRNA was expressed during both formation and regression, while MMP-9 mRNA was mainly localized in the late luteal phase. Reduction of MMP-2, -9 transcripts in the CL was observed during pregnancy. MMP-2 mRNA in the CL reduced to an undetectable level at D26 of pregnancy. TIMP-1 mRNA was highly expressed in the CL in both early and late luteal phases and persisted throughout the early stages of pregnancy. Strong signal for TIMP-2 mRNA was also detected in both luteal phases, and the level of TIMP-2 mRNA gradually increased with the progresses of pregnancy. No TIMP-3 mRNA was detected in the macaque CL in this study. In conclusion, these results suggest that MMP-2, -9 and TIMP-1, -2 may have functional roles in rhesus monkey CL. Coordinated expression of MMP-2, -9 and TIMP-2 may play a role in the maintaining of luteal function during early pregnancy. The unchanged expression pattern of TIMP-1 indicates that it may have other functions in the primate CL than inhibition of MMPs.

  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


    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. Chronic obstructive pulmonary disease in alpha1-antitrypsin PI MZ heterozygotes

    DEFF Research Database (Denmark)

    Hersh, C P; Dahl, Morten; Ly, N P;


    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....

  16. 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


    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

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

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


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

  18. 丝氨酸蛋白酶抑制剂9与支气管哮喘相关免疫细胞%Serine proteinase inhibitor 9 and bronchial asthma related immune cells

    Institute of Scientific and Technical Information of China (English)

    邓常文; 张星星; 屈玉兰; 白冲


    丝氨酸蛋白酶抑制剂9(serine proteinase inhibitor 9,PI-9)是一种主要针对颗粒蛋白酶B的内源性蛋白酶抑制剂.研究发现,PI-9对支气管哮喘相关免疫细胞有调节细胞凋亡、改变细胞功能等作用.研究PI-9的作用机制将有利于支气管哮喘发病机制的探索,并为临床治疗提供新的理论依据.%PI-9 is an endogenous protease inhibitor of GrB.When interacting with bronchial asthma related immune cells,PI-9 plays an important role in regulating cell apoptosis and function.Studying the mechanism of PI-9 is not only beneficial for the exploration of bronchial asthma nosogenesis,but also provide theoretical base for novel clinical therapy.

  19. 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


    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.

  20. 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


    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.

  1. 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


    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

  2. High-Resolution structure of the stable plasminogen activator inhibitor type-1 variant 14-1B in its proteinase-cleaved form: A new tool for detailed interaction studies and modeling

    Energy Technology Data Exchange (ETDEWEB)

    Jensen, J.; Gettins, P. (UIC)


    Wild-type plasminogen activator inhibitor type-1 (PAI-1) rapidly converts to the inactive latent state under conditions of physiological pH and temperature. For in vivo studies of active PAI-1 in cell culture and in vivo model systems, the 14-1B PAI-1 mutant (N150H-K154T-Q319L-M354I), with its stabilized active conformation, has thus become the PAI-1 of choice. As a consequence of the increased stability, the only two forms likely to be encountered are the active or the cleaved form, the latter either free or complexed with target proteinase. We hereby report the first structure of the stable 14-1B PAI-1 variant in its reactive center cleaved form, to a resolution of 2.0 {angstrom}. The >99% complete structure represents the highest resolved structure of free cleaved PAI-1. This high-resolution structure should be of great use for drug target development and for modeling protein-protein interactions such as those of PAI-1 with vitronectin.

  3. cDNA cloning of glucose-6-phosphate isomerase from crucian carp (Carassius carassius) and expression of the active region as myofibril-bound serine proteinase inhibitor in Escherichia coli. (United States)

    Han, Long; Cao, Min-Jie; Shi, Chao-lan; Wei, Xiao-Nan; Li, Huan; Du, Cui-Hong


    Glucose-6-phosphate isomerase (GPI) (EC can act as a myofibril-bound serine proteinase (MBSP) inhibitor (MBSPI) in fish. In order to better understand the biological information of the GPI and its functional domain for inhibiting MBSP, the cDNA of GPI was cloned from crucian carp (Carassius carassius) with RT-PCR, nested-PCR and 3'-RACE. The result of sequencing showed that the GPI cDNA had an open reading frame of 1662bp encoding 553 amino acid residues. After constructing and comparing the three-dimensional structures of GPI and MBSP, the middle fragment of crucian carp GPI (GPI-M) was predicted as a functional domain for inhibiting MBSP. Then the crucian carp GPI-M gene was cloned and expressed in Escherichia coli. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the recombinant GPI-M (rGPI-M) with molecular mass of approximately 21kDa in the form of inclusion bodies. The rGPI-M was obtained at an electrophoresis level purity of approximately 95% after denaturation and dialysis renaturation.

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

    Energy Technology Data Exchange (ETDEWEB)

    Tuan, Han-Fang [Spallation Neutron Source, Oak Ridge National Laboratory, 1 Bethel Valley Road, Oak Ridge, TN 37831 (United States); Erskine, Peter [Laboratory for Protein Crystallography, Centre for Amyloidosis and Acute Phase Proteins, UCL Department of Medicine (Hampstead Campus), Rowland Hill Street, London NW3 2PF (United Kingdom); Langan, Paul [Bioscience Division, Mailstop M888, Los Alamos National Laboratory, Los Alamos, NM 87545 (United States); Department of Chemistry, University of Toledo, Toledo, OH 53606 (United States); Cooper, Jon [Laboratory for Protein Crystallography, Centre for Amyloidosis and Acute Phase Proteins, UCL Department of Medicine (Hampstead Campus), Rowland Hill Street, London NW3 2PF (United Kingdom); Coates, Leighton, E-mail: [Spallation Neutron Source, Oak Ridge National Laboratory, 1 Bethel Valley Road, Oak Ridge, TN 37831 (United States); Department of Chemistry, University of Toledo, Toledo, OH 53606 (United States)


    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.

  5. Controlled intracellular proteolysis during postpartal involution of the uterus: characterization and regulation of an alkaline proteinase. (United States)

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


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

  6. Interactions outside the proteinase-binding loop contribute significantly to the inhibition of activated coagulation factor XII by its canonical inhibitor from corn. (United States)

    Korneeva, Vera A; Trubetskov, Mikhail M; Korshunova, Alena V; Lushchekina, Sofya V; Kolyadko, Vladimir N; Sergienko, Olga V; Lunin, Vladimir G; Panteleev, Mikhail A; Ataullakhanov, Fazoil I


    Activated factor XII (FXIIa) is selectively inhibited by corn Hageman factor inhibitor (CHFI) among other plasma proteases. CHFI is considered a canonical serine protease inhibitor that interacts with FXIIa through its protease-binding loop. Here we examined whether the protease-binding loop alone is sufficient for the selective inhibition of serine proteases or whether other regions of a canonical inhibitor are involved. Six CHFI mutants lacking different N- and C-terminal portions were generated. CHFI-234, which lacks the first and fifth disulfide bonds and 11 and 19 amino acid residues at the N and C termini, respectively, exhibited no significant changes in FXIIa inhibition (Ki = 3.2 ± 0.4 nm). CHFI-123, which lacks 34 amino acid residues at the C terminus and the fourth and fifth disulfide bridges, inhibited FXIIa with a Ki of 116 ± 16 nm. To exclude interactions outside the FXIIa active site, a synthetic cyclic peptide was tested. The peptide contained residues 20-45 (Protein Data Bank code 1BEA), and a C29D substitution was included to avoid unwanted disulfide bond formation between unpaired cysteines. Surprisingly, the isolated protease-binding loop failed to inhibit FXIIa but retained partial inhibition of trypsin (Ki = 11.7 ± 1.2 μm) and activated factor XI (Ki = 94 ± 11 μm). Full-length CHFI inhibited trypsin with a Ki of 1.3 ± 0.2 nm and activated factor XI with a Ki of 5.4 ± 0.2 μm. Our results suggest that the protease-binding loop is not sufficient for the interaction between FXIIa and CHFI; other regions of the inhibitor also contribute to specific inhibition.

  7. Proteinases of the cornea and preocular tear film. (United States)

    Ollivier, F J; Gilger, B C; Barrie, K P; Kallberg, M E; Plummer, C E; O'Reilly, S; Gelatt, K N; Brooks, D E


    Maintenance and repair of corneal stromal extracellular matrix (ECM) requires a tightly coordinated balance of ECM synthesis, degradation and remodeling in which proteolytic enzymes (proteinases) perform important functions. There are natural proteinase inhibitors present in preocular tear film (PTF) and cornea simultaneously with proteinases that prevent excessive degradation of normal healthy tissue. Disorders occur when there is an imbalance between proteinases and proteinase inhibitors in favor of the proteinases, causing pathologic degradation of stromal collagen and proteoglycans in the cornea. Two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, are of major importance in terms of remodeling and degradation of the corneal stromal collagen. Immunohistochemical studies have shown different origins of MMP-2 and -9. MMP-2 is synthesized by corneal keratocytes and performs a surveillance function in the normal cornea, becoming locally activated to degrade collagen molecules that occasionally become damaged. Alternatively, MMP-9 may be produced by epithelial cells and polymorphonuclear neutrophils following corneal wounding. Because the cornea is in close contact with the preocular tear film (PTF), proteinases have been evaluated in the PTF. In damaged corneas, total proteolytic activity in the tear fluid was found to be significantly increased compared to normal eyes and contralateral eyes. Studies analyzing the proteolytic activity in serial PTF samples during corneal healing led to the following conclusions: ulcerative keratitis in animals is associated with initially high levels of tear film proteolytic activity, which decrease as ulcers heal; proteinase levels in melting ulcers remain elevated leading to rapid progression of the ulcers. The success of medical and surgical treatment of the corneal ulcers is reflected by the proteolytic activity in tears. In animals, successful treatment leads to a rapid reduction in tear film proteolytic activity that

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

    Institute of Scientific and Technical Information of China (English)


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

  9. Clinical experience in Europe with uroselective alpha1-antagonists. (United States)

    Debruyne, F M; Van der Poel, H G


    alpha1-Adrenoreceptors are thought to be involved in prostate smooth muscle contractions and could hence play a role in the dynamic component of intravesical obstruction associated with symptomatic BPH. Consequently, since the mid-eighties alpha receptor blocking agents have been used for the treatment of BPH. Non-selective alpha blockers are usually associated with systemic side-effects which resulted in an exclusion or withdrawal of many patients from this form of treatment. With the availability of so-called uroselective alpha blockers the management picture has changed since it was anticipated that these compounds cause lesser side-effects with at least the same, or even better, efficacy. Comparative clinical studies are essential for determining the eventual advantages of the uroselective alpha1-antagonists and a large number of such studies have been performed worldwide studying the various available compounds. European studies with terazosin showed clear superiority of the drug over the placebo while causing only limited side-effects. Various other studies using alpha-blocking agents such as doxazosin, tamsulosin and alfuzosin yielded identical results. Especially with tamsulosin and alfuzosin, the side-effects were comparable with those encountered in the placebo group. About 7% of the patients using tamsulosin experienced retrograde ejaculation in one study which did not occur in the alfuzosin studies. Important studies in Europe have also investigated the value of a combination of an alpha blocker with a 5alpha-reductase inhibitor. Comparable studies in which both alfuzosin and doxazosin were combined with the 5alpha-reductase inhibitor Proscar have shown that a combination is not superior to a blocker monotherapy and especially in the ALFIN study the results show that alfuzosin monotherapy is superior to Proscar in the management of symptomatic BPH. European studies have evaluated Quality of Life, sexuality as well as socio-economical outcome of the

  10. Recent advances in the discovery of alpha1-adrenoceptor agonists. (United States)

    Bishop, Michael J


    The alpha(1) adrenoceptors are three of nine well-characterized receptors that are activated by epinephrine and norepinephrine. Agonists acting at the alpha(1) adrenoceptors produce numerous physiological effects, and are used therapeutically for several indications. Many known alpha(1) adrenoceptor agonists are alpha(1A) selective, but the discovery of highly selective alpha(1B) and alpha(1D) adrenoceptor agonists has proven to be an extremely difficult goal to achieve. This review will focus on recent advances in the discovery, development and clinical utility of subtype-specific alpha(1) agonists as well as contributions to our understanding of agonist-receptor interactions.

  11. Multigene family for Bowman-Birk type proteinase inhibitors of wild soja and soybean: the presence of two BBI-A genes and pseudogenes. (United States)

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


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

  12. Molecular characteristics and expression analysis of Kazal-type serines proteinase inhibitor (KSPI) gene from Hyriopsis cumingii%三角帆蚌 KSPI cDNA 的分子特征及表达分析

    Institute of Scientific and Technical Information of China (English)

    徐龙威; 王芹; 汪桂玲; 李家乐


    Kazal-type serines proteinase inhibitor ( KSPI) is very important in the immune reaction of biological body , but the study of KSPI in Hyriopsis cumingii has been rarely reported .In this study , the full length of cDNA sequence of Kazal-type serines proteinase inhibitor(KSPI) gene (accession number:KT901291) of H.cumingii was cloned.It was 1029 bp in length, containing 5′and 3′-UTRs parts of 61 bp and 206 bp, and an ORF of 762 bp encoding 253 amino acids, and the molecular weight was 27397.5 u.The amino acid sequence contains five Kazal domain structures , and the result of homology analysis showed that it has a few of similarity with other species, suggesting that this gene belongs to the typical KSPI gene.Real-time quantitative PCR showed that KSPI gene expressed in a wide range of tissues including the adductor , foot, liver, blood, mantle, gill and gonad, and the highest expression was in mantle , on the contrary, the lowest in liver.After being infected with Aeromonas hydrophila, the expression of KSPI gene had significant increas-ing in 7 tissues compared with the control group .The results suggested that KSPI gene is very important in the immune reaction of Hyri-opsis cumingii.%Kazal型丝氨酸蛋白酶抑制因子( KSPI)在生物体的免疫应答过程中发挥着重要作用,但在三角帆蚌中尚未进行其相关研究与报道,为研究KSPI对三角帆蚌免疫过程的影响,采用RACE法克隆了三角帆蚌KSPI cDNA全序列(登录号:KT901291),获得了1029 bp的全长,其中包含5′、3′端非翻译区分别为61 bp、206 bp,开放阅读框762 bp,共编码氨基酸253个,分子质量为27397.5u,氨基酸序列包含5个Kazal结构域,与一些已知物种的KSPI编码的氨基酸序列进行同源性分析后发现,与各种物种间具有相似的结构域,属于典型的KSPI。实时荧光定量PCR分析结果表明KSPI在外套膜、血液、肝、腮、闭壳肌、性腺、斧足7个组织中均

  13. Cysteine peptidases and their inhibitors in breast and genital cancer.

    Directory of Open Access Journals (Sweden)

    Magdalena Milan


    Full Text Available Cysteine proteinases and their inhibitors probably play the main role in carcinogenesis and metastasis. The metastasis process need external proteolytic activities that pass several barriers which are membranous structures of the connective tissue which includes, the basement membrane of blood vessels. Activities of the proteinases are regulated by endogenous inhibitors and activators. The imbalance between cysteine proteinases and cystatins seems to be associated with an increase in metastatic potential in some tumors. It has also been reported that proteinase inhibitors, specific antibodies for these enzymes and inhibition of the urokinase receptor may prevent cancer cell invasion. Some proteinase inhibitor could serve as agents for cancer treatment.

  14. Molecular characterization of two kazal-type serine proteinase inhibitor genes in the surf clam Mesodesma donacium exposed to Vibrio anguillarum. (United States)

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


    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.

  15. Metalloproteinase activity secreted by fibrogenic cells in the processing of prolysyl oxidase. Potential role of procollagen C-proteinase. (United States)

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


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


    NARCIS (Netherlands)



    Antithrombin is a member of the serine proteinase inhibitor (serpin) family which contain a flexible reactive site loop that interacts with, and is cleaved by the target proteinase. In cleaved and latent serpins, the reactive site loop is inserted into a large central beta-sheet in the same molecule

  17. Inhibitors of lysosomal cysteine proteases

    Directory of Open Access Journals (Sweden)

    Lyanna O. L.


    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.

  18. Anti-apoptotic effects of Z alpha1-antitrypsin in human bronchial epithelial cells.

    LENUS (Irish Health Repository)

    Greene, C M


    alpha(1)-antitrypsin (alpha(1)-AT) deficiency is a genetic disease which manifests as early-onset emphysema or liver disease. Although the majority of alpha(1)-AT is produced by the liver, it is also produced by bronchial epithelial cells, amongst others, in the lung. Herein, we investigate the effects of mutant Z alpha(1)-AT (ZAAT) expression on apoptosis in a human bronchial epithelial cell line (16HBE14o-) and delineate the mechanisms involved. Control, M variant alpha(1)-AT (MAAT)- or ZAAT-expressing cells were assessed for apoptosis, caspase-3 activity, cell viability, phosphorylation of Bad, nuclear factor (NF)-kappaB activation and induced expression of a selection of pro- and anti-apoptotic genes. Expression of ZAAT in 16HBE14o- cells, like MAAT, inhibited basal and agonist-induced apoptosis. ZAAT expression also inhibited caspase-3 activity by 57% compared with control cells (p = 0.05) and was a more potent inhibitor than MAAT. Whilst ZAAT had no effect on the activity of Bad, its expression activated NF-kappaB-dependent gene expression above control or MAAT-expressing cells. In 16HBE14o- cells but not HEK293 cells, ZAAT upregulated expression of cIAP-1, an upstream regulator of NF-kappaB. cIAP1 expression was increased in ZAAT versus MAAT bronchial biopsies. The data suggest a novel mechanism by which ZAAT may promote human bronchial epithelial cell survival.

  19. Functional analysis of alpha 1 beta 1 integrin in human natural killer cells. (United States)

    Pérez-Villar, J J; Melero, I; Gismondi, A; Santoni, A; López-Botet, M


    Upon activation with interleukin (IL)-2 human natural killer (NK) cells acquire on their surface the alpha 1 beta 1 and alpha 2 beta 1 integrins and down-regulate the expression of alpha 6 beta 1. By employing alpha 1 beta 1-specific monoclonal antibody (mAb) HP-2B6, characterized in our laboratory, we examined the functional role of the alpha 1 beta 1 integrin in NK cells. Treatment with HP-2B6 mAb partially interfered with attachment of cultured NK cells to type I collagen, and combined with an anti-alpha 2 beta 1 (TEA 1/41) mAb, it completely abrogated cell adhesion to this extracelular matrix protein. In contrast, NK cell attachment to laminin was completely blocked by the anti-beta 1 LIA 1/2 mAb, but was unaffected by alpha 1 and alpha 2-specific mAb; as alpha 3 beta 1 and alpha 6 beta 1 were undetectable, the data indicate that the alpha 1 beta 1 integrin binding sites for type I collagen and laminin are different. Incubation with anti-alpha 1 HP-2B6 or its F(ab')2 fragments specifically induced a rapid homotypic aggregation of NK cells that was dependent on active metabolism, an intact cytoskeleton and the presence of divalent cations (Ca2+ and Mg2+); homotypic cell adhesion was selectively blocked by anti-CD18, CD11a or CD54 mAb. In addition, stimulation of cultured NK cells with the anti-alpha 1 HP-2B6 enhanced TNF-alpha production and induced tyrosine phosphorylation of a 110-kDa protein. Pretreatment with specific inhibitors of protein tyrosine kinase (PTK) activity (tyrphostin 25 and herbimycin A) completely abrogated the functional effects induced by the anti-alpha 1 HP-2B6 mAb. Our data show that ligation of the alpha 1 beta 1 integrin positively modulates IL-2-activated NK cell function via a PTK-dependent pathway.

  20. alpha 1-Antitrypsin and coeliac disease in spain. (United States)

    Klasen, E C; Polanco, I; Biemond, I; Vazquez, C; Peña, A S


    Ninety-three Spanish children suffering from coeliac disease and 103 control subjects from the same area were screened for the amount of alpha 1-antitrypsin (alpha 1AT) and for any electrophoretic variations in it. In this case-control study no significant differences were detected either in phenotype distribution or amount. The present results indicate that no genetic association exists between alpha 1AT and coeliac disease. PMID:6969683

  1. [Involvement of proteinases produced by both neurons and microglia in neuronal lesion and death pathways]. (United States)

    Nakanishi, H; Yamamoto, K


    Much attention has been paid to proteinases derived from not only neurons but also microglia in relation to neuronal death. There is accumulating evidence that intra- and extracellular proteinases in these cells are part of the basic machinery of neuronal death pathways. Some members of the ced-3/interleukin-1 beta converting enzyme (ICE) (caspase) family of cysteine proteinases have been thought to play a major role in apoptosis of not only non-neuronal cells but also neurons. Calpain has also been demonstrated to be a mediator of the neurodegenerative response. Recent studies have shown that excitotoxic and ischemic neuronal injury could be attenuated by inhibitors of caspases and calpain. Several recent studies have suggested the involvement of endosomal/lysosomal proteinases, including cathepsins B, D and E, in neuronal death induced by excitotoxins and ischemia. Furthermore, it has been reported that the extracellular tissue-type plasminogen activator/plasmin proteolytic cascade is involved in excitotoxic injury of the hippocampal neurons. In addition to such neuronal proteinases, microglial proteinases are believed to be important for the modification of neuronal functions positively or negatively. Cathepsins E and S derived from microglia have been suggested to contribute to neuronal survival through degradation and removal of beta-amyloid, damaged neurons and cellular debris. On the other hand, 6-hydroxydopamine-induced microglial cell death was inhibited by inhibitors of aspartic proteinases and caspases, suggesting the involvement of cathepsins E and D and caspases in microglial cell death. Therefore, identification of which proteinases play a causative role in neuronal death execution and clarification of the regulators and substrates for such proteinases is very important for understanding the molecular basis of the neuronal death pathways and to develop novel neuroprotective agents.

  2. New potential uroselective NO-donor alpha1-antagonists. (United States)

    Boschi, Donatella; Tron, Gian Cesare; Di Stilo, Antonella; Fruttero, Roberta; Gasco, Alberto; Poggesi, Elena; Motta, Gianni; Leonardi, Amedeo


    A recent uroselective alpha(1)-adrenoceptor antagonist, REC15/2739, has been joined with nitrooxy and furoxan NO-donor moieties to give new NO-donor alpha(1)-antagonists. All the compounds studied proved to be potent and selective ligands of human cloned alpha(1a)-receptor subtype. Derivatives 6 and 7 were able to relax the prostatic portion of rat vas deferens contracted by (-)-noradrenaline because of both their alpha(1A)-antagonist and their NO-donor properties.

  3. Pharmacological profiles of a novel alpha 1-adrenoceptor agonist, PNO-49B, at alpha 1-adrenoceptor subtypes. (United States)

    Muramatsu, I; Ohmura, T; Kigoshi, S


    The effects of a newly synthesized compound, PNO-49B, (R)-(-)-3'-(2-amino-1-hydroxyethyl)-4'-fluoromethanesulfonanilide hydrochloride, on alpha 1-adrenoceptor subtypes were examined in various tissues in which the following distribution of alpha 1-adrenoceptor subtypes has been suggested: dog carotid artery (alpha 1B), dog mesenteric artery (alpha 1N), rabbit thoracic aorta (alpha 1B + alpha 1L), rat liver (alpha 1B), rat vas deferens (alpha 1A + alpha 1L), rat cerebral cortex (alpha 1A + alpha 1B) and rat thoracic aorta (controversial subtype). PNO-49B (0.1-100 microM) produced concentration-dependent contractions in dog mesenteric artery, rabbit thoracic aorta, rat thoracic aorta and rat vas deferens; and the maximal amplitudes of contraction were almost the same as or slightly less than those of noradrenaline. By contrast, the maximal response to PNO-49B in dog carotid artery was markedly smaller than the response to noradrenaline. In rabbit thoracic aorta, the contractile response to PNO-49B was not affected by inactivation of the alpha 1B subtype with chloroethylclonidine (CEC), although the response to noradrenaline was attenuated by that treatment. The dissociation constants (KA) of PNO-49B were not different among the rat thoracic aorta, dog carotid and mesenteric arteries and rabbit thoracic aorta (CEC-pretreated). The contractile responses to PNO-49B were inhibited competitively by prazosin, HV723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)-ethyl)- amino(propyl)benzeneacetonitrile fumarate) and by WB4101 (2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4- benzodioxane).(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Trypanosoma cruzi: insights into naphthoquinone effects on growth and proteinase activity. (United States)

    Bourguignon, Saulo C; Cavalcanti, Danielle F B; de Souza, Alessandra M T; Castro, Helena C; Rodrigues, Carlos R; Albuquerque, Magaly G; Santos, Dilvani O; da Silva, Gabriel Gomes; da Silva, Fernando C; Ferreira, Vitor F; de Pinho, Rosa T; Alves, Carlos R


    In this study we compared the effects of naphthoquinones (α-lapachone, β-lapachone, nor-β-lapachone and Epoxy-α-lap) on growth of Trypanosoma cruzi epimastigotes forms, and on viability of VERO cells. In addition we also experimentally analyzed the most active compounds inhibitory profile against T. cruzi serine- and cysteine-proteinases activity and theoretically evaluated them against cruzain, the major T. cruzi cysteine proteinase by using a molecular docking approach. Our results confirmed β-lapachone and Epoxy-α-lap with a high trypanocidal activity in contrast to α-lapachone and nor-β-lapachone whereas Epoxy-α-lap presented the safest toxicity profile against VERO cells. Interestingly the evaluation of the active compounds effects against T. cruzi cysteine- and serine-proteinases activities revealed different targets for these molecules. β-Lapachone is able to inhibit the cysteine-proteinase activity of T. cruzi proteic whole extract and of cruzain, similar to E-64, a classical cysteine-proteinase inhibitor. Differently, Epoxy-α-lap inhibited the T. cruzi serine-proteinase activity, similar to PMSF, a classical serine-proteinase inhibitor. In agreement to these biological profiles in the enzymatic assays, our theoretical analysis showed that E-64 and β-lapachone interact with the cruzain specific S2 pocket and active site whereas Epoxy-α-lap showed no important interactions. Overall, our results infer that β-lapachone and Epoxy-α-lap compounds may inhibit T. cruzi epimastigotes growth by affecting T. cruzi different proteinases. Thus the present data shows the potential of these compounds as prototype of protease inhibitors on drug design studies for developing new antichagasic compounds.

  5. Purification and characterization of major extracellular proteinases from Trichophyton rubrum. (United States)

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


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

  6. Molecular diagnosis of intermediate and severe alpha(1)-antitrypsin deficiency

    DEFF Research Database (Denmark)

    Dahl, Morten; Nordestgaard, B G; Lange, P;


    We tested whether intermediate (MZ, SZ) and severe (ZZ) alpha(1)-antitrypsin deficiency affects lung function in the population at large.......We tested whether intermediate (MZ, SZ) and severe (ZZ) alpha(1)-antitrypsin deficiency affects lung function in the population at large....

  7. Alpha1-antitrypsin deficiency: a clinical-genetic overview

    Directory of Open Access Journals (Sweden)

    Abboud RT


    in patients with chronic irreversible airflow obstruction, especially in those with early onset of disease or positive family history. Testing is also recommended for immediate family members of those with AATD, asthmatics with persistent airflow obstruction, and infants and older subjects with unexplained liver disease. There are over 100 different AAT gene variants; most are rare and only some are associated with clinical disease.Keywords: AAT, AATD, ZZ, early onset emphysema, panacinar emphysema, neonatal jaundice and hepatitis, childhood liver disease, genetics of alpha1-antitrypsin, alpha1-antitrypsin laboratory testing and phenotyping

  8. Hereditary alpha-1-antitrypsin deficiency and its clinical consequences

    Directory of Open Access Journals (Sweden)

    Stolk Jan


    Full Text Available Abstract Alpha-1-antitrypsin deficiency (AATD is a genetic disorder that manifests as pulmonary emphysema, liver cirrhosis and, rarely, as the skin disease panniculitis, and is characterized by low serum levels of AAT, the main protease inhibitor (PI in human serum. The prevalence in Western Europe and in the USA is estimated at approximately 1 in 2,500 and 1 : 5,000 newborns, and is highly dependent on the Scandinavian descent within the population. The most common deficiency alleles in North Europe are PI Z and PI S, and the majority of individuals with severe AATD are PI type ZZ. The clinical manifestations may widely vary between patients, ranging from asymptomatic in some to fatal liver or lung disease in others. Type ZZ and SZ AATD are risk factors for the development of respiratory symptoms (dyspnoea, coughing, early onset emphysema, and airflow obstruction early in adult life. Environmental factors such as cigarette smoking, and dust exposure are additional risk factors and have been linked to an accelerated progression of this condition. Type ZZ AATD may also lead to the development of acute or chronic liver disease in childhood or adulthood: prolonged jaundice after birth with conjugated hyperbilirubinemia and abnormal liver enzymes are characteristic clinical signs. Cirrhotic liver failure may occur around age 50. In very rare cases, necrotizing panniculitis and secondary vasculitis may occur. AATD is caused by mutations in the SERPINA1 gene encoding AAT, and is inherited as an autosomal recessive trait. The diagnosis can be established by detection of low serum levels of AAT and isoelectric focusing. Differential diagnoses should exclude bleeding disorders or jaundice, viral infection, hemochromatosis, Wilson's disease and autoimmune hepatitis. For treatment of lung disease, intravenous alpha-1-antitrypsin augmentation therapy, annual flu vaccination and a pneumococcal vaccine every 5 years are recommended. Relief of breathlessness

  9. Urinary alpha1-antichymotrypsin: a biomarker of prion infection.

    Directory of Open Access Journals (Sweden)

    Gino Miele

    Full Text Available The occurrence of blood-borne prion transmission incidents calls for identification of potential prion carriers. However, current methods for intravital diagnosis of prion disease rely on invasive tissue biopsies and are unsuitable for large-scale screening. Sensitive biomarkers may help meeting this need. Here we scanned the genome for transcripts elevated upon prion infection and encoding secreted proteins. We found that alpha(1-antichymotrypsin (alpha(1-ACT was highly upregulated in brains of scrapie-infected mice. Furthermore, alpha(1-ACT levels were dramatically increased in urine of patients suffering from sporadic Creutzfeldt-Jakob disease, and increased progressively throughout the disease. Increased alpha(1-ACT excretion was also found in cases of natural prion disease of animals. Therefore measurement of urinary alpha(1-ACT levels may be useful for monitoring the efficacy of therapeutic regimens for prion disease, and possibly also for deferring blood and organ donors that may be at risk of transmitting prion infections.

  10. Changes in vascular alpha 1- and alpha 2-adrenoceptor responsiveness by selegiline treatment. (United States)

    Pelat, M; Verwaerde, P; Tran, M A; Berlan, M; Senard, J M; Montastruc, J L


    Pharmacoepidemiological studies have reported an excess of mortality with selegiline, a MAO B inhibitor used in the treatment of Parkinson's disease. The mechanism of this putative adverse effect remains unknown but an interaction with the sympathetic nervous system was suggested. The aim of the present study was to investigate the influence of selegiline (10 mg/daily, orally during one week) on vascular alpha1- and alpha2-adrenoceptor responsiveness in conscious unrestrained dogs. Selegiline significantly increased resting values of both systolic and diastolic blood pressures and noradrenaline plasma levels (HPLC) without changing heart rate. Moreover, spectral analysis of systolic blood pressure (Fast Fourier Transformation) showed that selegiline increased the relative energy of a low frequency band without modifying the total spectrum. ED 50 calculated from dose-pressor response curves with phenylephrine (after beta-blockade by propranolol), an index of alpha1-adrenoceptor response or with noradrenaline (after alpha1- and beta blockade by prazosin plus propranolol), an index of alpha2-adrenoceptor response, were significantly higher after selegiline. Selegiline failed to modify the number of platelet alpha2-adrenoceptors measured by [(3)H] RX 821002 binding. Yohimbine-induced increase in noradrenaline release was significantly more marked after selegiline. These results support the evidence that selegiline induces a vascular alpha1- and alpha2-adrenoceptor-hyposensitivity that can be explained by the increase in noradrenaline release elicited by the drug.

  11. Plasma levels of alpha-1-antichymotrypsin are elevated in patients with chronic heart failure, but are of limited prognostic value

    NARCIS (Netherlands)

    Lok, S. I.; Lok, D. J.; van der Weide, P.; Winkens, B.; de la Porte, P. W. Bruggink-Andre; Doevendans, P. A.; de Weger, R. A.; van der Meer, P.; de Jonge, N.


    Background There is increasing interest in utilising novel markers of cardiovascular disease risk in patients with chronic heart failure (HF). Recently, it was shown that alpha-1-antichymotrypsin (ACT), an acute-phase protein and major inhibitor of cathpesin G, plays a role in the pathophysiology of

  12. Specificity of proteinase K at P2 to P3' sub-sites and its comparison to other serine proteases. (United States)

    Qasim, Mohammad A


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

  13. Effect of alpha1-blockers on stentless ureteroscopic lithotripsy

    Directory of Open Access Journals (Sweden)

    Jianguo Zhu


    Full Text Available ABSTRACT Objective To evaluate the clinical efficiency of alpha1-adrenergic antagonists on stentless ureteroscopic lithotripsy treating uncomplicated lower ureteral stones. Materials and Methods From January 2007 to January 2013, 84 patients who have uncomplicated lower ureteral stones treated by ureteroscopic intracorporeal lithotripsy with the holmium laser were analyzed. The patients were divided into two groups, group A (44 patients received indwelled double-J stents and group B (40 patients were treated by alpha1-adrenergic antagonists without stents. All cases of group B were treated with alpha1 blocker for 1 week. Results The mean operative time of group A was significantly longer than group B. The incidences of hematuria, flank/abdominal pain, frequency/urgency after surgery were statistically different between both groups. The stone-free rate of each group was 100%. Conclusions The effect of alpha1-adrenergic antagonists is more significant than indwelling stent after ureteroscopic lithotripsy in treating uncomplicated lower ureteral stones.

  14. Increase in net activity of serine proteinases but not gelatinases after local endotoxin exposure in the peripheral airways of healthy subjects.

    Directory of Open Access Journals (Sweden)

    Margaretha E Smith

    Full Text Available We tested the hypothesis that activation of the innate immune response induces an imbalance in the proteolytic homeostasis in the peripheral airways of healthy subjects, towards excess serine or gelatinase proteinase activity. During bronchoscopy, 18 healthy human subjects underwent intra-bronchial exposure to endotoxin and contra-lateral exposure to vehicle. Bronchoalveolar lavage (BAL samples were harvested 24 or 48 hours (h later. We quantified archetype proteinases, anti-proteinases, inflammatory BAL cells, and, importantly, total plus net proteinase activities using functional substrate assays. As expected, endotoxin exposure increased the concentrations of polymorphonuclear leukocytes (PMN's and macrophages, of proteinases and the anti-proteinases tissue inhibitor of metalloproteinase-1, α-1-antitrypsin and, to a lesser extent, secretory leukoproteinase inhibitor, at both time points. Notably, at these time points, endotoxin exposure substantially increased the quantitative NE/SLPI ratio and the net serine proteinase activity corresponding to neutrophil elastase (NE. Endotoxin exposure also increased the total gelatinase activity corresponding to matrix metalloproteinase (MMP-9; an activity dominating over that of MMP-2. However, endotoxin exposure had no impact on net gelatinolytic activity at 24 or 48 h after exposure. Thus, local activation of the innate immune response induces an imbalance towards increased net serine proteinase activity in the proteolytic homeostasis of the peripheral airways in healthy subjects. Hypothetically, this serine proteinase activity can contribute to tissue remodelling and hypersecretion via NE from PMN's, if it is triggered repeatedly, as might be the case in chronic inflammatory airway disorders.

  15. Fibrinogen and alpha(1)-antitrypsin in COPD exacerbations

    DEFF Research Database (Denmark)

    Sylvan Ingebrigtsen, Truls; Marott, J. L.; Rode, L.


    Background We tested the hypotheses that fibrinogen and alpha(1)-antitrypsin are observationally and genetically associated with exacerbations in COPD. Methods We studied 13 591 individuals with COPD from the Copenhagen General Population Study (2003-2013), of whom 6857 were genotyped for FGB -455...... and exacerbations in instrumental variable analyses. Results Elevated fibrinogen and alpha(1)-antitrypsin levels were associated with increased risk of exacerbations in COPD, HR=1.14 (1.07 to 1.22, p...

  16. Exploring the role of CT densitometry: a randomised study of augmentation therapy in alpha1-antitrypsin deficiency

    DEFF Research Database (Denmark)

    Dirksen, A; Piitulainen, E; Parr, D G;


    for the assessment of the therapeutic effect of augmentation therapy in subjects with alpha(1)-antitrypsin (alpha(1)-AT) deficiency. In total, 77 subjects (protease inhibitor type Z) were randomised to weekly infusions of 60 mg x kg(-1) human alpha(1)-AT (Prolastin) or placebo for 2-2.5 yrs. The primary end......-point was change in CT lung density, and an exploratory approach was adopted to identify optimal methodology, including two methods of adjustment for lung volume variability and two statistical approaches. Other end-points were exacerbations, health status and physiological indices. CT was more sensitive than...... other measures of emphysema progression, and the changes in CT and forced expiratory volume in 1 s were correlated. All methods of densitometric analysis concordantly showed a trend suggestive of treatment benefit (p-values for Prolastin versus placebo ranged 0.049-0.084). Exacerbation frequency...

  17. The cysteine proteinases of the pineapple plant. (United States)

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


    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.

  18. Proteinase activity regulation by glycosaminoglycans

    Directory of Open Access Journals (Sweden)

    Tersariol I.L.S.


    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.

  19. Functional analysis of {alpha}1,3/4-fucosyltransferase VI in human hepatocellular carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Qiya; Guo, Bin; Wang, Yingming; Wu, Jun; Jiang, Wenjun; Zhao, Shenan [State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433 (China); Qiao, Shouyi, E-mail: [State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433 (China); Wu, Yanhua, E-mail: [State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433 (China)


    Highlights: Black-Right-Pointing-Pointer Human FUT6 is up-regulated in HCC tissues. Black-Right-Pointing-Pointer Expression of FUT6 promotes G0/G1-S transition and cell growth. Black-Right-Pointing-Pointer FUT6 confers a growth advantage in vivo. Black-Right-Pointing-Pointer FUT6 suppresses p21 expression through modulating PI3K/Akt signaling. -- Abstract: The {alpha}1,3/4-fucosyltransferases (FUT) subfamily are key enzymes in cell surface antigen synthesis during various biological processes. A novel role of FUTs in tumorigenesis has been discovered recently, however, the underlying mechanism remains largely unknown. Here, we characterized FUT6, a member of {alpha}1,3/4-FUT subfamily, in human hepatocellular carcinoma (HCC). In HCC tissues, the expression levels of FUT6 and its catalytic product SLe{sup x} were significantly up-regulated. Overexpression of FUT6 in HCC cells enhanced S-phase cell population, promoted cell growth and colony formation ability. Moreover, subcutaneously injection of FUT6-overexpressing cells in nude mice promoted cell growth in vivo. In addition, elevating FUT6 expression markedly induced intracellular Akt phosphorylation, and suppressed the expression of the cyclin-dependent kinases inhibitor p21. Bath application of the PI3K inhibitor blocked FUT6-induced Akt phosphorylation, p21 suppression and cell proliferation. Our results suggest that FUT6 plays an important role in HCC growth by regulating the PI3K/Akt signaling pathway.

  20. Deficiency of a alpha-1-antitrypsin influences systemic iron homeostasis (United States)

    Abstract Background: There is evidence that proteases and anti-proteases participate in the iron homeostasis of cells and living systems. We tested the postulate that alpha-1 antitrypsin (A1AT) polymorphism and the consequent deficiency of this anti-protease in humans are asso...

  1. Molecular characterization of alpha 1- and alpha 2-adrenoceptors. (United States)

    Harrison, J K; Pearson, W R; Lynch, K R


    Three 'alpha 1-adrenoceptors' and three 'alpha 2-adrenoceptors' have now been cloned. How closely do these receptors match the native receptors that have been identified pharmacologically? What are the properties of these receptors, and how do they relate to other members of the cationic amine receptor family? Kevin Lynch and his colleagues discuss these questions in this review.

  2. Alpha-1 antitrypsin is markedly decreased following pulmonary F. tularensis challenge

    Directory of Open Access Journals (Sweden)

    James Patrick Chambers


    Full Text Available Alpha-1 antitrypsin, a small glycoprotein clade A serpine serine protease inhibitor of neutrophil elastase has been shown to increase in humans following bacterial and viral infection. However, we report here significant reduction of this major inhibitor of elastase in plasma of F. tularensis LVS and SCHU S4 (Type A strain following pulmonary challenge. Consistent with an imbalance of protease-antiprotease function at the alveolar level in lungs of infected animals, increased elastase activity was observed in lung lavage fluids accompanied by decrease lung function, i.e., loss of lung elastance with concomitant increase of pulmonary hysteresistivity. These data are suggestive of targeted tissue destruction via unchecked neutrophhil elastase activity in infected animals.

  3. Multiple pathways for vacuolar sorting of yeast proteinase A

    DEFF Research Database (Denmark)

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


    ) overproduction of Vps10p suppressed the missorting phenotype associated with overproduction of proteinase A, 2) overproduction of proteinase A induced missorting of carboxypeptidase Y, 3) vacuolar sorting of proteinase A in a deltavps10 strain was readily saturated by modest overproduction of proteinase A, and 4...

  4. Involvement of Human Estrogen Related Receptor Alpha 1 (hERR Alpha 1) in Breast Cancer and Hormonally Insensitive Disease (United States)


    breast tumor biopsies: relationship to steroid receptor status and regulation by progestins . Cancer Res, 59: 529-532, 1999. 16 17. Speirs, V., Parkes, A... aromatase expression in the breast tissue by ERR alpha-1 orphan receptor. Cancer Res, 58: 5695-5700, 1998. 42. Yang, C. and Chen, S. Two organochlorine

  5. Molecular and enzymatic properties of a cathepsin L-like proteinase with distinct substrate specificity from northern shrimp (Pandalus borealis). (United States)

    Aoki, H; Ahsan, M N; Watabe, S


    We purified a cathepsin L-like proteinase to homogeneity from the hepatopancreas of northern shrimp Pandalus borealis by several chromatographic procedures. The purified proteinase showed the highest specificity for leucine residue at P2, a specificity pattern similar to cathepsins S and K whereas proline and arginine residues were not suitable as P2 substrates. However, unlike these proteinases, it accepted valine almost equally to the phenylalanine residue at P2. The shrimp cathepsin was strongly inhibited by E-64, leupeptin and antipain, while benzyloxycarbonyl-Phe-Tyr(t-Bu)-CHN2, a specific inhibitor of cathepsin L, remained largely ineffective. Next, we determined the primary structure of the shrimp enzyme by molecular cloning and investigated the residues constituting the S2 subsite, which is possibly involved in its unusual substrate specificity. The deduced amino acid sequence of the shrimp proteinase shared the highest identity of 65% with a cathepsin L-like proteinase from lobster, but its identity to the well-characterized mammalian cathepsins S, L, and K fell within narrower ranges of 52-55%. However, the shrimp proteinase differed from these cathepsins in some key residues including, for example, the unique occurrence of cysteine and glutamine residues at the structurally important S2 subsite. Interestingly, transcripts of this proteinase were exclusively detected in the shrimp gut coinciding with its broad pH activity and stability profiles, which is also unusual as a cysteine proteinase. These results suggest that the shrimp enzyme is homologous to mammalian cathepsins S, L, and K, but is distinct from each of these proteinases in both enzymatic and structural properties.

  6. Taraxalisin -- a serine proteinase from dandelion Taraxacum officinale Webb s.l. (United States)

    Rudenskaya, G N; Bogacheva, A M; Preusser, A; Kuznetsova, A V; Dunaevsky YaE; Golovkin, B N; Stepanov, V M


    Latex of dandelion roots contains a serine proteinase that hydrolyzes a chromogenic peptide substrate Glp-Ala-Ala-Leu-pNA optimally at pH 8.0. Maximal activity of the proteinase in the roots is attained in April, at the beginning of plant development after the winter period. The protease was isolated by ammonium sulfate precipitation of the root extract followed by affinity chromatography on a Sepharose-Ala-Ala-Leu-mrp and gel filtration on Superose 6R performed in FPLC regime. Pure serine proteinase named taraxalisin was inactivated by specific inhibitors of serine proteinases, diisopropylfluorophosphate (DFP) and phenylmethylsulfonylfluoride (PMSF). Its molecular mass is 67 kDa and pI 4.5. pH stability range is 6-9 in the presence of 2 mM Ca2+, temperature optimum is at 40 degrees C; Km=0.37+/-0.06 mM. The substrate specificity of taraxalisin towards synthetic peptides and insulin B-chain is comparable with that of two other subtilisin-like serine proteinases, cucumisin and macluralisin. The taraxalisin N-terminal sequence traced for 15 residues revealed 40% coinciding residues when aligned with that of subtilisin Carlsberg.

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

    Directory of Open Access Journals (Sweden)

    Blom Nikolaj


    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:

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

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


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

  9. A triticale water-deficit-inducible phytocystatin inhibits endogenous cysteine proteinases in vitro. (United States)

    Chojnacka, Magdalena; Szewińska, Joanna; Mielecki, Marcin; Nykiel, Małgorzata; Imai, Ryozo; Bielawski, Wiesław; Orzechowski, Sławomir


    Water-deficit is accompanied by an increase in proteolysis. Phytocystatins are plant inhibitors of cysteine proteinases that belong to the papain and legumain family. A cDNA encoding the protein inhibitor TrcC-8 was identified in the vegetative organs of triticale. In response to water-deficit, increases in the mRNA levels of TrcC-8 were observed in leaf and root tissues. Immunoblot analysis indicated that accumulation of the TrcC-8 protein occurred after 72h of water-deficit in the seedlings. Using recombinant protein, inhibitory activity of TrcC-8 against cysteine proteases from triticale and wheat tissues was analyzed. Under water-deficit conditions, there are increases in cysteine proteinase activities in both plant tissues. The cysteine proteinase activities were inhibited by addition of the recombinant TrcC-8 protein. These results suggest a potential role for the triticale phytocystatin in modulating cysteine proteinase activities during water-deficit conditions.

  10. Inhibitory effects of human alpha 2-macroglobulin on Trypanosoma cruzi epimastigote proteinases. (United States)

    Ramos, A; Remedi, M S; Sánchez, C; Bonacci, G; Vides, M A; Chiabrando, G


    The inactivation of Trypanosoma cruzi proteinases by human alpha 2-macroglobulin (alpha 2-M), a major plasma proteinase inhibitor was studied. Evidences regarding the interaction between alpha 2-M and proteolytic enzymes contained in crude cell-free extracts of T. cruzi were derived from electrophoretic and enzymatic assays. The former showed conformational and structural changes occurring in alpha 2-M, as judged by the appearance of transformed 'fast' form on native PAGE; generation of bands of approximately 90 kDa on reduced SDS-PAGE and formation of covalent complexes enzyme-inhibitor on SDS-PAGE. On the other hand, the total proteolytic activity on azocasein dropped significantly in the presence of alpha 2-M, although partial activity was still maintained. The proteinases detected as a double band of 44 and 53 kDa on gelatin SDS-PAGE were also inhibited by alpha 2-M. Results suggest that the study of specific interactions between alpha 2-M and T. cruzi-proteinases, probably with cruzipain, could be biologically important in the fate of T. cruzi-infection and Chagas' disease.

  11. Crystal structure of the Bowman-Birk Inhibitor from Vigna unguiculata seeds in complex with beta-trypsin at 1.55 A resolution and its structural properties in association with proteinases. (United States)

    Barbosa, João Alexandre R G; Silva, Luciano P; Teles, Rozeni C L; Esteves, Gisele F; Azevedo, Ricardo B; Ventura, Manuel M; de Freitas, Sonia M


    The structure of the Bowman-Birk inhibitor from Vigna unguiculata seeds (BTCI) in complex with beta-trypsin was solved and refined at 1.55 A to a crystallographic R(factor) of 0.154 and R(free) of 0.169, and represents the highest resolution for a Bowman-Birk inhibitor structure to date. The BTCI-trypsin interface is stabilized by hydrophobic contacts and hydrogen bonds, involving two waters and a polyethylene glycol molecule. The conformational rigidity of the reactive loop is characteristic of the specificity against trypsin, while hydrophobicity and conformational mobility of the antichymotryptic subdomain confer the self-association tendency, indicated by atomic force microscopy, of BTCI in complex and free form. When BTCI is in binary complexes, no significant differences in inhibition constants for producing a ternary complex with trypsin and chymotrypsin were detected. These results indicate that binary complexes present no conformational change in their reactive site for both enzymes confirming that these sites are structurally independent. The free chymotrypsin observed in the atomic force microscopy assays, when the ternary complex is obtained from BTCI-trypsin binary complex and chymotrypsin, could be related more to the self-association tendency between chymotrypsin molecules and the flexibility of the reactive site for this enzyme than to binding-related conformational changes.

  12. 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.


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

  13. Collagen-induced expression of collagenase-3 by primary chondrocytes is mediated by integrin alpha 1 and discoidin domain receptor 2 : a protein kinase C-dependent pathway

    NARCIS (Netherlands)

    Vonk, Lucienne A.; Doulabi, Behrouz Z.; Huang, ChunLing; Helder, Marco N.; Everts, Vincent; Bank, Ruud A.


    Methods. Goat articular chondrocytes and chondrons were cultured on collagen coatings. Small interfering RNA (siRNA) oligonucleotides targeted against ITG alpha 1 and DDR2 were transfected into primary chondrocytes. Chemical inhibitors for mitogen-activated protein kinase kinase (MEK1) (PD98059), fo

  14. 血清视黄醇结合蛋白与半胱氨酸蛋白酶抑制剂C对肾脏疾病的诊断价值%Diagnosis value of serum retinal-binding protein and cysteine proteinase inhibitor C in renal diseases

    Institute of Scientific and Technical Information of China (English)

    陈晓婷; 张炳峰; 金菲


    Objective:To evaluate the value of serum retinal-binding protein(RBP) and cysteine proteinase inhibitor C(Cys C) in the diagnosis of renal diseases.Methods:A total of 165 patients with renal disease (patient group) and 177 healthy subjects (control group) were enrolled in the study.Serum RBP,Cys C,creatinine(Cr) and urine Cr were assayed and compared; creatinine clearance rate(Ccr)of the two groups were calculated and compared.Correlation between serum RBP with Cys C,creatinine (Cr) and Ccr were analyzed.ROC curve for the diagnosis of renal disease were drawn and the area under ROC curve was calculated.Results:Compared with the control group,the serum levels of RBP,Cys C and Cr in patient group were higher,but Ccr was lower; Serum levels of RBP in patient group was positively correlated to Cr,while Cys C levels negatively correlated to Ccr (r =0.726,0.705,-0.803,both P <0.01).The area under the ROC curve of RBP,Cys C and Cr were 0.856,0.917 and 0.810,respectively; the diagnosis sensitivity were 81.2%,91.5% and 63.3% ; and the diagnosis specificity were 73.2%,78.2% and 95.2%.Conclusion:The value of serum levels of RBP in the diagnosis of renal disease was lower than that of Cys C.%目的:探讨血清视黄醇结合蛋白(retinal-binding protein,RBP)与半胱氨酸蛋白酶抑制剂C(cysteine proteinase inhibitor C,Cys C)在肾脏疾病中的诊断价值.方法:选取165例肾脏疾病患者和177例健康对照者,分别检测并比较两者血清中RBP,Cys C,血肌酐和尿肌酐水平以及肌酐清除率(creatinine clearance rate,Ccr);对血清中RBP的含量与Cys C、肌酐、Ccr等指标的含量作相关性分析;作RBP、Cys C与肌酐对肾脏疾病诊断的ROC曲线,计算ROC曲线下面积.结果:与健康对照组相比,患者组血清RBP、Cys C和肌酐水平显著增高,而Ccr水平明显降低(P均<0.000 1);患者组血清中RBP与Cys C、肌酐呈明显正相关,与Ccr呈明显负相关(r分别为0.726,0.705,-0.803,P均<0.01).RBP

  15. 乳腺丝氨酸蛋白酶抑制剂在口腔鳞状细胞癌亚细胞中的表达及意义%Mammary serine proteinase inhibitor subcellular expression in oral squamous cell carcinoma and its clinical significance

    Institute of Scientific and Technical Information of China (English)

    罗军; 舒海荣; 应于康; 吴伟力; 季彤; 钟来平


    目的 检测乳腺丝氨酸蛋白酶抑制剂(mammary serine proteinase inhibitor,Maspin)在口腔鳞状细胞癌亚细胞中的表达,观察其与口腔鳞状细胞癌患者临床病理特征之间的关系,以期为临床提供参考.方法 应用免疫组织化学方法对45例口腔鳞状细胞癌的癌组织标本行Maspin蛋白含量的半定量测定,统计其亚细胞定位表达,并与各临床病理指标进行统计学分析.结果 Maspin蛋白在口腔鳞状细胞癌胞核中表达的强阳性率为24%(11/45),弱阳性率为11%(5/45),阴性率为64%(29/45),细胞核表达与肿瘤大小(P=0.019)、淋巴结转移(P=0.011)及术后转移(P=0.017)呈负相关,与患者术后的生存时间呈正相关(P=0.030);Maspin蛋白在口腔鳞状细胞癌胞质中表达的强阳性率为31%(14/45),弱阳性率为31%(14/45),阴性率为38%(17/45),细胞质表达与淋巴结转移(P=0.038)、术后转移(P=0.004)呈负相关,与患者术后的生存时间呈正相关(P=0.014).结论 Maspin蛋白在口腔鳞状细胞癌胞核或胞质中的表达对判断预后可能具有重要价值.%Objective To investigate the subcellular expression of mammary serine proteinase inhibitor(Maspin) in oral squamous cell carcinoma and its relationship to the clinicopathological features.Methods The Maspin protein subcellular expression was detected in 45 patients with oral squamous cell carcinoma by immunohistochemical staining.The relationship between the Maspin protein subcellular expression and the clinicopathological parameters was analyzed.Results The negative rate of nuclear maspin expression was 64%(29/45),and the weakly positive rate was 11%(5/45),and the strong positive rate was 24%(11/45).Nuclear maspin expression was negatively correlated with T stage(P=0.019),lymph node metastasis(P=0.038) and postoperative metastasis(P=0.004),but positively correlated with the patients′ survival rate (P=0.014).The negative rate of cytoplasmatic maspin expression was 38%(17/45),and the

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

    Institute of Scientific and Technical Information of China (English)

    黄日红; 吴泰华; 张中和


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

  17. Ethylene-regulated expression of a carnation cysteine proteinase during flower petal senescence. (United States)

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


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

  18. Suppression of human prostate cancer cell growth by alpha1-adrenoceptor antagonists doxazosin and terazosin via induction of apoptosis. (United States)

    Kyprianou, N; Benning, C M


    Recent evidence from our laboratory has demonstrated that alpha1-adrenoceptor antagonists doxazosin and terazosin induced apoptosis in prostate epithelial and smooth muscle cells in patients with benign prostatic hypertrophy (BPH; J. Urol., 159: 1810-1815, 1998; J. Urol., 161: 2002-2007, 1999). In this study, we investigated the biological action of three alpha1-adrenoceptor antagonists, doxazosin, terazosin, and tamsulosin, against prostate cancer cell growth. The antigrowth effect of the three alpha1-adrenoceptor antagonists was examined in two human prostate cancer cell lines, PC-3 and DU-145, and a prostate smooth muscle cell primary culture, SMC-1, on the basis of: (a) cell viability assay; (b) rate of DNA synthesis; and (c) induction of apoptosis. Our results indicate that treatment of prostate cancer cells with doxazosin or terazosin results in a significant loss of cell viability, via induction of apoptosis in a dose-dependent manner, whereas tamsulosin had no effect on prostate cell growth. Neither doxazosin nor terazosin exerted a significant effect on the rate of cell proliferation in prostate cancer cells. Exposure to phenoxybenzamine, an irreversible inhibitor of alpha1-adrenoceptors, does not abrogate the apoptotic effect of doxazosin or terazosin against human prostate cancer or smooth muscle cells. This suggests that the apoptotic activity of doxazosin and terazosin against prostate cells is independent of their capacity to antagonize alpha1-adrenoceptors. Furthermore, an in vivo efficacy trial demonstrated that doxazosin administration (at tolerated pharmacologically relevant doses) in SCID mice bearing PC-3 prostate cancer xenografts resulted in a significant inhibition of tumor growth. These findings demonstrate the ability of doxazosin and terazosin (but not tamsulosin) to suppress prostate cancer cell growth in vitro and in vivo by inducing apoptosis without affecting cell proliferation. This evidence provides the rationale for targeting both

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

    Institute of Scientific and Technical Information of China (English)

    黄明; 刘逸尘; 张亦陈; 孙妍; 孙金生


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

  20. 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.


    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

  1. Purification and some physico-chemical and enzymic properties of a calcium ion-activated neutral proteinase from rabbit skeletal muscle (United States)

    Azanza, Jean-Louis; Raymond, Jacques; Robin, Jean-Michel; Cottin, Patrick; Ducastaing, André


    Ca2+-activated neutral proteinase was purified from rabbit skeletal muscle by a method involving DEAE-Sephacel chromatography, affinity chromatography on organomercurial–Sepharose and gel filtration on Sephacryl S-200 and Sephadex G-150. The SDS (sodium dodecyl sulphate)/polyacrylamide-gel-electrophoresis data show that the purified enzyme contains only one polypeptide chain of mol.wt. 73000. The purification procedure used allowed us to eliminate a contaminant containing two components of mol.wt. about 30000 each. Whole casein or α1-casein were hydrolysed with a maximum rate at 30°C, pH7.5, and with 5mm-CaCl2, but myofibrils were found to be a very susceptible substrate for this proteinase. This activity is associated with the destruction of the Z-discs, which is caused by the solubilization of the Z-line proteins. The activity of the proteinase in vitro is not limited to the removal of Z-line. SDS/polyacrylamide-gel electrophoresis on larger plates showed the ability of the proteinase to degrade myofibrils more extensively than previously supposed. This proteolysis resulted in the production of a 30000-dalton component as well as in various other higher- and lower-molecular-weight peptide fragments. Troponin T, troponin I, α-tropomyosin, some high-molecular-weight proteins (M protein, heavy chain of myosin) and three unidentified proteins are degraded. Thus the number of proteinase-sensitive regions in the myofibrils is greater than as previously reported by Dayton, Goll, Zeece, Robson & Reville [(1976) Biochemistry 15, 2150–2158]. The Ca2+-activated neutral proteinase is not a chymotrypsin- or trypsin-like enzyme, but it reacted with all the classic thiol-proteinase inhibitors for cathepsin B, papain, bromelain and ficin. Thus the proteinase was proved to have an essential thiol group. Antipain and leupeptin are also inhibitors of the Ca2+-activated neutral proteinase. ImagesFig. 1.Fig. 2.Fig. 3. PMID:534501

  2. Proteinase K improves quantitative acylation studies. (United States)

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


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

  3. alpha 1-Antitrypsin-levels and phenotypes in Crohn's disease in the Netherlands. (United States)

    Klasen, E C; Biemond, I; Weterman, I T


    A group of 310 unrelated patients suffering from Crohn's disease has been screened for quantitative and electrophoretic variations of alpha 1-antitrypsin (alpha 1AT). A comparison was made betweeen patients and healthy controls. The distribution of electrophoretic alpha 1AT variants in the patients showed no significant deviation from the controls. The alpha 1AT quantities are significantly higher in the Crohn's disease population than in the controls. PMID:6969207

  4. Beta2-Microglobulin and Alpha1-Microglobulin as Markers of Balkan Endemic Nephropathy, a Worldwide Disease

    NARCIS (Netherlands)

    Stefanovic, Vladisav; Djukanovic, Ljubica; Cukuranovic, Rade; Bukvic, Danica; Lezaic, Visnja; Maric, Ivko; Ogrizovic, Sanja Simic; Jovanovic, Ivan; Vlahovic, Predrag; Pesic, Ivana; Djordjevic, Vidosava


    Background: aEuro,Urine beta2-microglobulin (beta2-MG) was mainly used as a tubular marker of Balkan endemic nephropathy (BEN) but recently alpha1-microglobulin (alpha1-MG) was proposed for the diagnosis of BEN. In this study, the potential of urine beta2-MG, alpha1-MG, albumin, and total protein in

  5. Increased outer arm and core fucose residues on the N-glycans of mutated alpha-1 antitrypsin protein from alpha-1 antitrypsin deficient individuals. (United States)

    McCarthy, Cormac; Saldova, Radka; O'Brien, M Emmet; Bergin, David A; Carroll, Tomás P; Keenan, Joanne; Meleady, Paula; Henry, Michael; Clynes, Martin; Rudd, Pauline M; Reeves, Emer P; McElvaney, Noel G


    Alpha-1 antitrypsin (AAT) is the major physiological inhibitor of a range of serine proteases, and in the lung, it maintains a protease-antiprotease balance. AAT deficiency (AATD) is an autosomal co-dominant condition with the Z mutation being the most common cause. Individuals homozygous for Z (PiZZ) have low levels of circulating mutant Z-AAT protein leading to premature emphysematous lung disease. Extensive glycoanalysis has been performed on normal AAT (M-AAT) from healthy individuals and the importance of glycosylation in affecting the immune modulatory roles of AAT is documented. However, no glycoanalysis has been carried out on Z-AAT from deficient individuals to date. In this study, we investigate whether the glycans present on Z-AAT differ to those found on M-AAT from healthy controls. Plasma AAT was purified from 10 individuals: 5 AATD donors with the PiZZ phenotype and 5 PiMM healthy controls. Glycoanalysis was performed employing N-glycan release, exoglycosidase digestion and UPLC analysis. No difference in branched glycans was identified between AATD and healthy controls. However, a significant increase in both outer arm (α1-3) (p = 0.04) and core (α1-6) fucosylated glycans (p < 0.0001) was found on Z-AAT compared to M-AAT. This study has identified increased fucosylation on N-glycans of Z-AAT indicative of ongoing inflammation in AATD individuals with implications for early therapeutic intervention.

  6. alpha-1-antitrypsin in breast milk of healthy Nigerian mothers. (United States)

    Omeme, J A; Lantos, J D; Ihongbe, J C


    Alpha-1-antitryspin (x-1-AT) may play a possible role as effector of immunological stasis. This study examines the levels of this glycoprotein in 73 breast milk samples from 60 healthy Nigerian mothers. Levels of x-1-AT were measured by single radial immunodiffusion according to the method of Mancini. Serum protein was measured by Lowry's method, albumin by Doumas' method. Highest mean levels of x-1-AT were found in colostrum (25 mg/dl). The level was significantly higher compared to transitional milk (14.2 mg/dl) or mature milk (165 mg/dl) (p0.001). Breast milk contains substantial amounts of x-1-AT which is not destroyed by pasturization at 56 degrees Centigrade. The immunological protective properties of breast milk are ideal for newborn babies, particularly those who are low birthweight and are thus most susceptible to neonatal necrotizing enterocolitis.

  7. An ECLIPSE View of Alpha-1 Antitrypsin Deficiency. (United States)

    Lomas, David A


    Chronic obstructive pulmonary disease (COPD) is a multicomponent condition that is estimated to become the third leading cause of death in 2020. The ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints) study, funded by GlaxoSmithKline, is an observational study designed to define outcomes that can be used as endpoints in clinical trials in individuals with COPD. It allowed us to describe the heterogeneity of COPD, the stability of the exacerbation phenotype, and the factors associated with a progressive decline in lung function and the progression of emphysema on computed tomography scans. The cohort was also used to define genetic factors and biomarkers associated with COPD and disease progression. This review considers how the results from ECLIPSE can inform our understanding of the lung disease associated with alpha-1 antitrypsin deficiency.

  8. Loss of Smyhc1 or Hsp90alpha1 function results in different effects on myofibril organization in skeletal muscles of zebrafish embryos.

    Directory of Open Access Journals (Sweden)

    Marta Codina

    Full Text Available BACKGROUND: Myofibrillogenesis requires the correct folding and assembly of sarcomeric proteins into highly organized sarcomeres. Heat shock protein 90alpha1 (Hsp90alpha1 has been implicated as a myosin chaperone that plays a key role in myofibrillogenesis. Knockdown or mutation of hsp90alpha1 resulted in complete disorganization of thick and thin filaments and M- and Z-line structures. It is not clear whether the disorganization of these sarcomeric structures is due to a direct effect from loss of Hsp90alpha1 function or indirectly through the disorganization of myosin thick filaments. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we carried out a loss-of-function analysis of myosin thick filaments via gene-specific knockdown or using a myosin ATPase inhibitor BTS (N-benzyl-p-toluene sulphonamide in zebrafish embryos. We demonstrated that knockdown of myosin heavy chain 1 (myhc1 resulted in sarcomeric defects in the thick and thin filaments and defective alignment of Z-lines. Similarly, treating zebrafish embryos with BTS disrupted thick and thin filament organization, with little effect on the M- and Z-lines. In contrast, loss of Hsp90alpha1 function completely disrupted all sarcomeric structures including both thick and thin filaments as well as the M- and Z-lines. CONCLUSION/SIGNIFICANCE: Together, these studies indicate that the hsp90alpha1 mutant phenotype is not simply due to disruption of myosin folding and assembly, suggesting that Hsp90alpha1 may play a role in the assembly and organization of other sarcomeric structures.

  9. Separation of basic drug enantiomers by capillary electrophoresis using chicken alpha1-acid glycoprotein: insight into chiral recognition mechanism. (United States)

    Matsunaga, Hisami; Sadakane, Yutaka; Haginaka, Jun


    Recombinant chicken alpha(1)-acid glycoprotein (alpha(1)-AGP) was prepared by the Escherichia coli expression system and completely deglycosylated alpha(1)-AGP (cd-alpha(1)-AGP) was obtained by treatments of native alpha(1)-AGP with a mixture of endoglycosidase and N-glycosidase. The average molecular masses of chicken alpha(1)-AGP, cd-alpha(1)-AGP and recombinant alpha(1)-AGP were estimated to be about 29 200, 21 700 and 20 700, respectively, by matrix-assisted laser desorption-time of flight-mass spectrometry. We compared the chiral recognition ability of chicken alpha(1)-AGP, cd-alpha(1)-AGP and recombinant alpha(1)-AGP using them as chiral selectors in capillary electrophoresis. The chicken alpha(1)-AGP showed higher resolution for eperisone, pindolol and tolperisone than cd-alpha(1)-AGP or recombinant alpha(1)-AGP. Recombinant alpha(1)-AGP still showed chiral recognition for three basic drugs tested. By addition of propranolol as a competitor in the separation solution in CE, no enantioseparations of three basic drugs were observed with chicken alpha(1)-AGP, cd-alpha(1)-AGP or recombinant alpha(1)-AGP. These results reveal that the protein domain of the chicken alpha(1)-AGP is responsible for the chiral recognition ability, and that the chiral recognition site(s) for basic drugs exists on the protein domain.

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

    DEFF Research Database (Denmark)

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


    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...... eight genera. We mapped these activity profiles on an independently obtained molecular phylogeny of the symbionts and show that total proteinase activity in lower attine symbionts peaks at ca. pH 6. The higher attine symbionts that have no known free-living relatives had much higher proteinase...... activities than the lower attine symbionts. Their total in vitro proteinase activity peaked at pH values around 5, which is close to the pH that the ants maintain in their fungus gardens, suggesting that the pH optimum of fungal proteinases may have changed after the irreversible domestication...

  11. Gene targeted therapeutics for liver disease in alpha-1 antitrypsin deficiency

    Directory of Open Access Journals (Sweden)

    Caitriona McLean


    Full Text Available Caitriona McLean*, Catherine M Greene*, Noel G McElvaneyRespiratory Research Division, Dept. Medicine, Royal College of Surgeons in Ireland, Education and Research Centre, Beaumont Hospital, Dublin 9, Ireland; *Each of these authors contributed equally to this workAbstract: Alpha-1 antitrypsin (A1AT is a 52 kDa serine protease inhibitor that is synthesized in and secreted from the liver. Although it is present in all tissues in the body the present consensus is that its main role is to inhibit neutrophil elastase in the lung. A1AT deficiency occurs due to mutations of the A1AT gene that reduce serum A1AT levels to <35% of normal. The most clinically significant form of A1AT deficiency is caused by the Z mutation (Glu342Lys. ZA1AT polymerizes in the endoplasmic reticulum of liver cells and the resulting accumulation of the mutant protein can lead to liver disease, while the reduction in circulating A1AT can result in lung disease including early onset emphysema. There is currently no available treatment for the liver disease other than transplantation and therapies for the lung manifestations of the disease remain limited. Gene therapy is an evolving field which may be of use as a treatment for A1AT deficiency. As the liver disease associated with A1AT deficiency may represent a gain of function possible gene therapies for this condition include the use of ribozymes, peptide nucleic acids (PNAs and RNA interference (RNAi, which by decreasing the amount of aberrant protein in cells may impact on the pathogenesis of the condition.Keywords: alpha-1 antitrypsin deficiency, siRNA, peptide nucleic acid, ribozymes

  12. Investigation of Serine-Proteinase-Catalyzed Peptide Splicing in Analogues of Sunflower Trypsin Inhibitor 1 (SFTI-1). (United States)

    Karna, Natalia; Łęgowska, Anna; Malicki, Stanisław; Dębowski, Dawid; Golik, Przemysław; Gitlin, Agata; Grudnik, Przemysław; Wladyka, Benedykt; Brzozowski, Krzysztof; Dubin, Grzegorz; Rolka, Krzysztof


    Serine-proteinase-catalyzed peptide splicing was demonstrated in analogues of the trypsin inhibitor SFTI-1: both single peptides and two-peptide chains (C- and N-terminal peptide chains linked by a disulfide bridge). In the second series, peptide splicing with catalytic amount of proteinase was observed only when formation of acyl-enzyme intermediate was preceded by hydrolysis of the substrate Lys-Ser peptide bond. Here we demonstrate that with an equimolar amount of the proteinase, splicing occurs in all the two-peptide-chain analogues. This conclusion was supported by high resolution crystal structures of selected analogues in complex with trypsin. We showed that the process followed a direct transpeptidation mechanism. Thus, the acyl-enzyme intermediate was formed and was immediately used for a new peptide bond formation; products associated with the hydrolysis of the acyl-enzyme were not observed. The peptide splicing was sequence- not structure-specific.

  13. Purification and Characterization of Two High-Molecular-Weight Cystine Proteinase Inhibitors (CPIs) from Silver Carp Eggs%鲢鱼卵中两种高分子半胱氨酸蛋白酶抑制因子的纯化与鉴定

    Institute of Scientific and Technical Information of China (English)

    李树红; 蒋然然; 杨娟; 刘玲; 钟海霞; 陈志光; 李美良; 李冉


    以鲢鱼卵为材料,通过匀浆、酸处理和超滤制备半胱氨酸蛋白酶抑制因子(cystine proteinase inhibitors,CPIs)粗提液,进而经Sephacryl S-100分子筛层析、Blue Sepharose 6 Fast Flow染料亲和层析、SP-Sepharose Fast Flow阳离子交换层析、ConA Sepharose 4B亲和层析,获得两种纯化的高分子CPIs,即ConA不吸附部分a-1和吸附部分的糖蛋白a-2.二者分别被纯化了102.62倍和274.28倍,酶活回收率分别为2.02%和1.42%.通过TSK G2000 SWXL凝胶过滤高效液相色谱结合十二烷基硫酸钠-聚丙烯酰胺凝胶电泳及其反相酶谱法分析,表明a-2及再次经高效液相色谱法回收的a-1在电泳图上均呈单一带,a-2为单一峰,且a-1的分子质量为139 ku,a-2的分子质量为92 ku.二者均能抑制半胱氨酸蛋白酶(木瓜蛋白酶和鲢鱼组织蛋白酶L)但不抑制丝氨酸蛋白酶(胰蛋白酶和胰凝乳蛋白酶).根据a-1和a-2的分子质量及抑制活性特征和糖蛋白特性,推测二者可能为鲢鱼卵Kininogens的不同形式.

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

    Mat Amin, Nakisah


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

  15. A chestnut seed cystatin differentially effective against cysteine proteinases from closely related pests. (United States)

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


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

  16. Roles of the Picornaviral 3C Proteinase in the Viral Life Cycle and Host Cells. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Di Sun


    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.

  18. Alpha macroglobulins and the low-density-lipoprotein-related protein alpha-2-macroglobulin receptor in experimental renal fibrosis

    NARCIS (Netherlands)

    van Goor, H; Diamond, [No Value; Ding, GH; Kaysen, GA


    In this study, we evaluated the location of non-specific proteinase inhibitors and their receptor in experimental glomerular and interstitial fibrosis. The alpha macroglobulins alpha-2-macroglobulin (alpha 2M) and alpha-1-inhibitor 3 (alpha 1I3) are proteinase inhibitors, including metalloproteinase

  19. Role of alpha-1 antitrypsin in human health and disease. (United States)

    de Serres, F; Blanco, I


    Alpha-1 antitrypsin (AAT) deficiency is an under-recognized hereditary disorder associated with the premature onset of chronic obstructive pulmonary disease, liver cirrhosis in children and adults, and less frequently, relapsing panniculitis, systemic vasculitis and other inflammatory, autoimmune and neoplastic diseases. Severe AAT deficiency mainly affects Caucasian individuals and has its highest prevalence (1 : 2000-1 : 5000 individuals) in Northern, Western and Central Europe. In the USA and Canada, the prevalence is 1: 5000-10 000. Prevalence is five times lower in Latin American countries and is rare or nonexistent in African and Asian individuals. The key to successful diagnosis is by measuring serum AAT, followed by the determination of the phenotype or genotype if low concentrations are found. Case detection allows implementation of genetic counselling and, in selected cases, the application of augmentation therapy. Over the past decade, it has been demonstrated that AAT is a broad-spectrum anti-inflammatory, immunomodulatory, anti-infective and tissue-repair molecule. These new capacities are promoting an increasing number of clinical studies, new pharmacological formulations, new patent applications and the search for alternative sources of AAT (including transgenic and recombinant AAT) to meet the expected demand for treating a large number of diseases, inside and outside the context of AAT deficiency.

  20. Environmental arsenic exposure, selenium and sputum alpha-1 antitrypsin

    DEFF Research Database (Denmark)

    Burgess, Jefferey L; Kurzius-Spencer, Margaret; Poplin, Gerald S;


    Exposure to arsenic in drinking water is associated with increased respiratory disease. Alpha-1 antitrypsin (AAT) protects the lung against tissue destruction. The objective of this study was to determine whether arsenic exposure is associated with changes in airway AAT concentration and whether...... this relationship is modified by selenium. A total of 55 subjects were evaluated in Ajo and Tucson, Arizona. Tap water and first morning void urine were analyzed for arsenic species, induced sputum for AAT and toenails for selenium and arsenic. Household tap-water arsenic, toenail arsenic and urinary inorganic...... arsenic and metabolites were significantly higher in Ajo (20.6±3.5 μg/l, 0.54±0.77 μg/g and 27.7±21.2 μg/l, respectively) than in Tucson (3.9±2.5 μg/l, 0.16±0.20 μg/g and 13.0±13.8 μg/l, respectively). In multivariable models, urinary monomethylarsonic acid (MMA) was negatively, and toenail selenium...

  1. Purification and characterization of a keratinolytic serine proteinase from Streptomyces albidoflavus. (United States)

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


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

  2. Purification and characterization of an elastinolytic proteinase secreted by cercariae of Schistosoma mansoni. (United States)

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


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

  3. Pharmacological tolerance to alpha 1-adrenergic receptor antagonism mediated by terazosin in humans.


    Vincent, J; Dachman, W; Blaschke, T F; Hoffman, B. B.


    Chronic administration of alpha 1-receptor antagonists is associated with loss of clinical efficacy, especially in congestive heart failure, although the mechanism is uncertain. To evaluate changes in venous alpha 1-adrenoceptor responsiveness during chronic alpha 1-adrenoceptor blockade, dose-response curves to phenylephrine and angiotensin II were constructed in 10 healthy subjects before, during, and after administration of terazosin 1 mg orally for 28 d. Terazosin initially shifted the do...

  4. Aspects of preanalytical variation of lactoferrin and elastase/alpha 1-protease inhibitor complexes

    DEFF Research Database (Denmark)

    Antonsen, S; Qvist, N; Wanscher, M


    , if possible. Therefore, we have evaluated the influence of various aspects of sampling and handling conditions on the results obtained when measuring ELA-PI and LAC. Blood samples from both healthy persons as well as patients, who had undergone laparotomy the day before, were investigated. We confirmed...... the previous observations of higher concentrations of ELA-PI and LAC in serum compared to plasma. This was more pronounced in patients than in healthy adults. In EDTA-blood the most important change was seen in samples from patients when stored at room temperature. In this situation increases of LAC...... specimens drawn from healthy persons. EDTA-plasma obtained by venous puncture following minimal stasis contained 10% higher concentrations of LAC compared to samples drawn from intravenous catheters, while no difference was observed in the case of ELA-PI. However, in one individual prolonged venous stasis...

  5. Recent advances in the molecular pharmacology of the alpha 1-adrenergic receptors. (United States)

    Guarino, R D; Perez, D M; Piascik, M T


    This review is intended to discuss recent developments in the molecular pharmacology of the alpha 1-adrenergic receptor (alpha 1-AR) subtypes. After a brief historical development, we will focus on the more contemporary issues having to do with this receptor family. Emphasis will be put on recent data regarding the cloning, nomenclature, signalling mechanisms, and genomic organization of the alpha 1-AR subtypes. We will also highlight recent mutational studies that identify key amino acid residues involved in ligand binding, as well as the role of the alpha 1-AR subtypes in regulating physiologic processes.

  6. The prevalence of alpha-1 antitrypsin deficiency in Ireland.

    LENUS (Irish Health Repository)

    Carroll, Tomas P


    BACKGROUND: Alpha-1 antitrypsin deficiency (AATD) results from mutations in the SERPINA1 gene and classically presents with early-onset emphysema and liver disease. The most common mutation presenting with clinical evidence is the Z mutation, while the S mutation is associated with a milder plasma deficiency. AATD is an under-diagnosed condition and the World Health Organisation recommends targeted detection programmes for AATD in patients with chronic obstructive pulmonary disease (COPD), non-responsive asthma, cryptogenic liver disease and first degree relatives of known AATD patients. METHODS: We present data from the first 3,000 individuals screened following ATS\\/ERS guidelines as part of the Irish National Targeted Detection Programme (INTDP). We also investigated a DNA collection of 1,100 individuals randomly sampled from the general population. Serum and DNA was collected from both groups and mutations in the SERPINA1 gene detected by phenotyping or genotyping. RESULTS: The Irish National Targeted Detection Programme identified 42 ZZ, 44 SZ, 14 SS, 430 MZ, 263 MS, 20 IX and 2 rare mutations. Analysis of 1,100 randomly selected individuals identified 113 MS, 46 MZ, 2 SS and 2 SZ genotypes. CONCLUSION: Our findings demonstrate that AATD in Ireland is more prevalent than previously estimated with Z and S allele frequencies among the highest in the world. Furthermore, our targeted detection programme enriched the population of those carrying the Z but not the S allele, suggesting the Z allele is more important in the pathogenesis of those conditions targeted by the detection programme.

  7. The prevalence of alpha-1 antitrypsin deficiency in Ireland

    Directory of Open Access Journals (Sweden)

    Morris Valerie B


    Full Text Available Abstract Background Alpha-1 antitrypsin deficiency (AATD results from mutations in the SERPINA1 gene and classically presents with early-onset emphysema and liver disease. The most common mutation presenting with clinical evidence is the Z mutation, while the S mutation is associated with a milder plasma deficiency. AATD is an under-diagnosed condition and the World Health Organisation recommends targeted detection programmes for AATD in patients with chronic obstructive pulmonary disease (COPD, non-responsive asthma, cryptogenic liver disease and first degree relatives of known AATD patients. Methods We present data from the first 3,000 individuals screened following ATS/ERS guidelines as part of the Irish National Targeted Detection Programme (INTDP. We also investigated a DNA collection of 1,100 individuals randomly sampled from the general population. Serum and DNA was collected from both groups and mutations in the SERPINA1 gene detected by phenotyping or genotyping. Results The Irish National Targeted Detection Programme identified 42 ZZ, 44 SZ, 14 SS, 430 MZ, 263 MS, 20 IX and 2 rare mutations. Analysis of 1,100 randomly selected individuals identified 113 MS, 46 MZ, 2 SS and 2 SZ genotypes. Conclusion Our findings demonstrate that AATD in Ireland is more prevalent than previously estimated with Z and S allele frequencies among the highest in the world. Furthermore, our targeted detection programme enriched the population of those carrying the Z but not the S allele, suggesting the Z allele is more important in the pathogenesis of those conditions targeted by the detection programme.

  8. The prevalence of alpha-1 antitrypsin deficiency in Ireland.

    LENUS (Irish Health Repository)

    Carroll, Tomas P


    Abstract Background Alpha-1 antitrypsin deficiency (AATD) results from mutations in the SERPINA1 gene and classically presents with early-onset emphysema and liver disease. The most common mutation presenting with clinical evidence is the Z mutation, while the S mutation is associated with a milder plasma deficiency. AATD is an under-diagnosed condition and the World Health Organisation recommends targeted detection programmes for AATD in patients with chronic obstructive pulmonary disease (COPD), non-responsive asthma, cryptogenic liver disease and first degree relatives of known AATD patients. Methods We present data from the first 3,000 individuals screened following ATS\\/ERS guidelines as part of the Irish National Targeted Detection Programme (INTDP). We also investigated a DNA collection of 1,100 individuals randomly sampled from the general population. Serum and DNA was collected from both groups and mutations in the SERPINA1 gene detected by phenotyping or genotyping. Results The Irish National Targeted Detection Programme identified 42 ZZ, 44 SZ, 14 SS, 430 MZ, 263 MS, 20 IX and 2 rare mutations. Analysis of 1,100 randomly selected individuals identified 113 MS, 46 MZ, 2 SS and 2 SZ genotypes. Conclusion Our findings demonstrate that AATD in Ireland is more prevalent than previously estimated with Z and S allele frequencies among the highest in the world. Furthermore, our targeted detection programme enriched the population of those carrying the Z but not the S allele, suggesting the Z allele is more important in the pathogenesis of those conditions targeted by the detection programme.

  9. Multiple proteinases from two Microsporum species. (United States)

    Simpanya, M F; Baxter, M


    Enzyme expression of 67 isolates of two Microsporum species, M. canis and M. cookei, were compared in both shake and stationary cultures using substrate copolymerized SDS-PAGE. Most M. canis isolates expressed more proteolytic bands in shake culture, while M. cookei isolates expressed more in stationary culture. M. canis isolates expressed up to six proteinases of different relative mobilities (122, 64, 62, 45, 31 and 25 kDa). M. cookei expressed up to seven proteinases in stationary culture (67, 66, 64, 62, 45, 42 and 39 kDa). Those of 67 and 66 kDa were not expressed in shake culture. The proteinases expressed by M. cookei were similar to those expressed by M. canis except for 122 and 25 kDa. With the exception of isolates from non-infected cats, 25 kDa was also commonly expressed by isolates from infected hosts in the shake culture treatment. The differences in enzyme expression obtained may reflect differences in the contrasting ecological roles of the two species.

  10. Gene targeted therapeutics for liver disease in alpha-1 antitrypsin deficiency.

    LENUS (Irish Health Repository)

    McLean, Caitriona


    Alpha-1 antitrypsin (A1AT) is a 52 kDa serine protease inhibitor that is synthesized in and secreted from the liver. Although it is present in all tissues in the body the present consensus is that its main role is to inhibit neutrophil elastase in the lung. A1AT deficiency occurs due to mutations of the A1AT gene that reduce serum A1AT levels to <35% of normal. The most clinically significant form of A1AT deficiency is caused by the Z mutation (Glu342Lys). ZA1AT polymerizes in the endoplasmic reticulum of liver cells and the resulting accumulation of the mutant protein can lead to liver disease, while the reduction in circulating A1AT can result in lung disease including early onset emphysema. There is currently no available treatment for the liver disease other than transplantation and therapies for the lung manifestations of the disease remain limited. Gene therapy is an evolving field which may be of use as a treatment for A1AT deficiency. As the liver disease associated with A1AT deficiency may represent a gain of function possible gene therapies for this condition include the use of ribozymes, peptide nucleic acids (PNAs) and RNA interference (RNAi), which by decreasing the amount of aberrant protein in cells may impact on the pathogenesis of the condition.

  11. Thyroid hormone receptor alpha1 follows a cooperative CRM1/calreticulin-mediated nuclear export pathway. (United States)

    Grespin, Matthew E; Bonamy, Ghislain M C; Roggero, Vincent R; Cameron, Nicole G; Adam, Lindsay E; Atchison, Andrew P; Fratto, Victoria M; Allison, Lizabeth A


    The thyroid hormone receptor alpha1 (TRalpha) exhibits a dual role as an activator or repressor of its target genes in response to thyroid hormone (T(3)). Previously, we have shown that TRalpha, formerly thought to reside solely in the nucleus bound to DNA, actually shuttles rapidly between the nucleus and cytoplasm. An important aspect of the shuttling activity of TRalpha is its ability to exit the nucleus through the nuclear pore complex. TRalpha export is not sensitive to treatment with the CRM1-specific inhibitor leptomycin B (LMB) in heterokaryon assays, suggesting a role for an export receptor other than CRM1. Here, we have used a combined approach of in vivo fluorescence recovery after photobleaching experiments, in vitro permeabilized cell nuclear export assays, and glutathione S-transferase pull-down assays to investigate the export pathway used by TRalpha. We show that, in addition to shuttling in heterokaryons, TRalpha shuttles rapidly in an unfused monokaryon system as well. Furthermore, our data show that TRalpha directly interacts with calreticulin, and point to the intriguing possibility that TRalpha follows a cooperative export pathway in which both calreticulin and CRM1 play a role in facilitating efficient translocation of TRalpha from the nucleus to cytoplasm.

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

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


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

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

    Institute of Scientific and Technical Information of China (English)

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


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

  14. Properties of a subtilisin-like proteinase from a psychrotrophic Vibrio species comparison with proteinase K and aqualysin I. (United States)

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


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

  15. GFR alpha-1 is expressed in parvalbumin GABAergic neurons in the hippocampus. (United States)

    Sarabi, A; Hoffer, B J; Olson, L; Morales, M


    Glial cell line derived neurotrophic factor (GDNF) is a potent survival factor for several types of neurons. GDNF binds with high affinity to GDNF-family receptor alpha-1 (GFR alpha-1). This receptor is expressed in different areas of the brain, including the hippocampus and dentate gyrus. By using in situ hybridization and immunohistochemistry, we found that 19% to 37% of glutamic acid decarboxylase (GAD) expressing neurons co-expressed GFR alpha-1 in the hippocampus. GFR alpha-1/GAD co-expression was found mainly in the stratum (s) pyramidale (29-37%) and s. oriens (20-25%). Further characterization of GFR alpha-1 expressing interneurons, based on their calcium-binding protein immunoreactivity, demonstrated that many parvalbumin (PV) immunoreactive neurons express GFR alpha-1 in the s. pyramidale of CA1 (72%), CA2 (70%) and CA3 (70%) subfields of the hippocampus. GFR alpha-1/PV double labeled neurons were also detected in the s. oriens of CA1 (52%), CA2 (27%) and CA3 (36%) subfields. The expression of GFR alpha-1 in principal neurons and in a specific sub-population of GABAergic neurons (PV-containing neurons) suggest that GDNF might modulate, in a selective manner, functions of the entire adult hippocampus.

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

    Directory of Open Access Journals (Sweden)

    Meng Guoliang


    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.

  17. Immobility from administration of the alpha1-adrenergic antagonist, terazosin, in the IVth ventricle in rats. (United States)

    Stone, Eric A; Lin, Yan; Quartermain, David


    Brain alpha1-adrenoceptors have been shown to be essential for motor activity and movement in mice using intraventricular injection of alpha1-antagonists. To facilitate subsequent neuroanatomical mapping of these receptors, the present study was undertaken to replicate these effects in the rat. Rats were administered the alpha1-antagonist, terazosin, in the absence and presence of the alpha1-agonist, phenylephrine, in the IVth ventricle and were tested for their motor activity responses to an environmental change. Terazosin was found to produce a dose-dependent, virtually complete cessation of behavioral activity that was reversed by coinfusion of phenylephrine. The results could not be explained by sedation. It is concluded that central alpha1-adrenoceptors are essential for behavioral activation in rats as in mice.

  18. Picornaviral 3C cysteine proteinases have a fold similar to the chymotrypsin-like serine proteinases

    Energy Technology Data Exchange (ETDEWEB)

    Allaire,M.; Chernaia, M.; Malcolm, B.; James, M.


    The picornavirus family includes several pathogens such as poliovirus, rhinovirus (the major cause of the common cold), hepatitis A virus and the foot-and-mouth disease virus. Picornaviral proteins are expressed by direct translation of the genomic RNA into a single, large polyprotein precursor. Proteolysis of the viral polyprotein into the mature proteins is assured by the viral 3C enzymes, which are cysteine proteinases. Here we report the X-ray crystal structure at 2.3 {angstrom} resolution of the 3C proteinase from hepatitis A virus (HAV-3C). The overall architecture of HAV-3C reveals a fold resembling that of the chymotrypsin family of serine proteinases, which is consistent with earlier predictions. Catalytic residues include Cys 172 as nucleophile and His 44 as general base. The 3C cleavage specificity for glutamine residues is defined primarily by His 191. The overall structure suggests that an inter-molecular (trans) cleavage releases 3C and that there is an active proteinase in the polyprotein.

  19. Diagnosis of alpha-1-antitrypsin deficiency by DNA analysis of children with liver disease

    Directory of Open Access Journals (Sweden)

    De TOMMASO Adriana Maria Alves


    Full Text Available Background - Alpha-1-antitrypsin deficiency is a genetic disorder which is transmitted in a co-dominant, autosomal form. Alpha-1-antitrypsin deficiency affects mainly the lungs and the liver leading, in the latter case, to neonatal cholestasis, chronic hepatitis or cirrhosis. A precise diagnosis of Alpha-1-antitrypsin deficiency may be obtained by biochemical or molecular analysis. Objective - The purpose of this study was to use DNA analysis to examine the presence of an alpha-1-antitrypsin deficiency in 12 children suspected of having this deficiency and who showed laboratory and clinical characteristics of the disease. Patients and Methods - Twelve patients, aged 3 months to 19 years, who had serum alpha-1-antitrypsin levels lower than normal and/or had hepatic disease of undefined etiology were studied. The mutant alleles S and Z of the alpha-1-antitrypsin gene were investigated in the 12 children. Alpha-1-antitrypsin gene organization was analyzed by amplification of genoma through the polymerase chain reaction and digestion with the restriction enzymes Xmnl (S allele and Taq 1 (Z allele. Results - Seven of the 12 patients had chronic liver disease of undefined etiology and the other five patients had low serum levels of alpha-1-antitrypsin as well as a diagnosis of neonatal cholestasis and/or chronic liver disease of undefined etiology. Five of the 12 patients were homozygous for the Z allele (ZZ and two had the S allele with another allele (*S different from Z. Conclusion - These results show that alpha-1-antitrypsin deficiency is relatively frequent in children with chronic hepatic disease of undefined etiology and/or low alpha-1-antitrypsin levels (41.6%. A correct diagnosis is important for effective clinical follow-up and for genetic counseling.

  20. The embryo's cystatin C and F expression functions as a protective mechanism against the maternal proteinase cathepsin S in mice. (United States)

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


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

  1. Crystal structure of 2A proteinase from hand, foot and mouth disease virus. (United States)

    Mu, Zhixia; Wang, Bei; Zhang, Xiaoyu; Gao, Xiaopan; Qin, Bo; Zhao, Zhendong; Cui, Sheng


    EV71 is responsible for several epidemics worldwide; however, the effective antiviral drug is unavailable to date. The 2A proteinase (2A(pro)) of EV71 presents a promising drug target due to its multiple roles in virus replication, inhibition of host protein synthesis and evasion of innate immunity. We determined the crystal structure of EV71 2A(pro) at 1.85Å resolution, revealing that the proteinase maintains a chymotrypsin-like fold. The active site is composed of the catalytic triads C110A, H21 and D39 with the geometry similar to that in other picornaviral 2A(pro), 3C(pro) and serine proteinases. The cI-to-eI2 loop at the N-terminal domain of EV71 2A(pro) adopts a highly stable conformation and contributes to the hydrophilic surface property, which are strikingly different in HRV2 2A(pro) but are similar in CVB4 2A(pro). We identified a hydrophobic motif "LLWL" followed by an acidic motif "DEE" at the C-terminus of EV71 2A(pro). The "LLWL" motif is folded into the β-turn structure that is essential for the positioning of the acidic motif. Our structural and mutagenesis study demonstrated that both the negative charging and the correct positioning of the C-terminus are essential for EV71 replication. Deletion of the "LLWL" motif abrogated the proteolytic activity, indicating that the motif is critical for maintaining the active proteinase conformation. Our findings provide the structural and functional insights into EV71 2A(pro) and establish a framework for structure-based inhibitor design.

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

    DEFF Research Database (Denmark)

    Zoega, Morten; Ravnsborg, Tina; Højrup, Peter;


    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...

  3. Design, synthesis and inhibitory effect of pentapeptidyl chloromethyl ketones on proteinase K. (United States)

    Kore, Anilkumar R; Shanmugasundaram, Muthian


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


    NARCIS (Netherlands)

    Haandrikman, Alfred J.; Kok, Jan; Venema, Gerard


    The production of enzymatically active proteinase by lactococci requires the joint presence of a proteinase gene, prtP, and a gene encoding a maturation protein, prtM. A 32-kDa protein produced by Escherichia coli upon expression of the prtM gene under the direction of the T7 RNA polymerase promoter

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

    DEFF Research Database (Denmark)

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


    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...... activities than the lower attine symbionts. Their total in vitro proteinase activity peaked at pH values around 5, which is close to the pH that the ants maintain in their fungus gardens, suggesting that the pH optimum of fungal proteinases may have changed after the irreversible domestication...... 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...

  6. The tricyclic antidepressants amitriptyline, nortriptyline and imipramine are weak antagonists of human and rat alpha1B-adrenoceptors. (United States)

    Nojimoto, F D; Mueller, A; Hebeler-Barbosa, F; Akinaga, J; Lima, V; Kiguti, L R de A; Pupo, A S


    Although it is long known that the tricyclic antidepressants amitriptyline, nortriptyline and imipramine inhibit the noradrenaline transporter and alpha(1)-adrenoceptors with similar affinities, which may lead to self-cancelling actions, the selectivity of these drugs for alpha(1)-adrenoceptor subtypes is unknown. The present study investigates the selectivity of amitriptyline, nortriptyline and imipramine for human recombinant and rat native alpha(1)-adrenoceptor subtypes. The selectivity of amitriptyline, nortriptyline and imipramine was investigated in HEK-293 cells expressing each of the human alpha(1)-subtypes and in rat native receptors from the vas deferens (alpha(1A)), spleen (alpha(1B)) and aorta (alpha(1D)) through [(3)H]prazosin binding, and noradrenaline-induced intracellular Ca(2+) increases and contraction assays. Amitriptyline, nortriptyline and imipramine showed considerably higher affinities for alpha(1A)- (approximately 25- to 80-fold) and alpha(1D)-adrenoceptors (approximately 10- to 25-fold) than for alpha(1B)-adrenoceptors in both contraction and [(3)H]prazosin binding assays with rat native and human receptors, respectively. In addition, amitriptyline, nortriptyline and imipramine were substantially more potent in the inhibition of noradrenaline-induced intracellular Ca(2+) increases in HEK-293 cells expressing alpha(1A)- or a truncated version of alpha(1D)-adrenoceptors which traffics more efficiently towards the cell membrane than in cells expressing alpha(1B)-adrenoceptors. Amitriptyline, nortriptyline and imipramine are much weaker antagonists of rat and human alpha(1B)-adrenoceptors than of alpha(1A)- and alpha(1D)-adrenoceptors. The differential affinities for these receptors indicate that the alpha(1)-adrenoceptor subtype which activation is most increased by the augmented noradrenaline availability resultant from the blockade of neuronal reuptake is the alpha(1B)-adrenoceptor. This may be important for the behavioural effects of these

  7. Polymorphism of caprine milk alphas1-casein in relation to performance of four Polish goat breeds. (United States)

    Barłowska, J; Litwińczuk, Z; Kedzierska-Matysek, M; Litwińczuk, A


    Polymorphism of goat milk alphas1-casein was determined and potential relations between genetic variants of this protein fraction and goat performance were evaluated. The investigations were performed on 598 goats assigned to of 4 breed groups (White improved 254 units, Coloured improved--124, White non-improved--146 and Coloured non-improved--74). For each goat, alphas1-casein polymorphism was determined in polyacrylamide gel by the PAGE-SDS method and percentage of milk alphas1-casein and gene frequency established. There was evaluated goat performance at successive lactations. In the goat population investigated, AA, AB, BB, AE, BE and EE alphas1-casein genotypes were identified. In all four breeds, alphas1-casein genotype EE clearly predominated (27.2-39.2%), recognized as "medium" and its share was higher in the groups of non-improved goats. It was conditioned by high frequency of gene E alphas1-casein (0.419-0.622). Generally, EE genotype percentage was higher in the non-improved goat groups. The improved goats, though, obtained higher productivity in each of the lactation studied. Analysis of relationships between alphas1-casein genetic variants and goats performance confirmed a significant influence on milk, protein and fat yields only in the Coloured improved goat group. There was revealed a more general tendency indicating a significant impact of "strong" alphas1-casein genotypes on a concentration of basic milk components, i.e. fat and protein, especially casein. In a group of goats producing milk of the highest casein content (over 2.4%) and protein (over 3.0%), the animals showing "strong" alphas1-casein variants dominated (85 and 70 %).

  8. Activation of the FAK-src molecular scaffolds and p130Cas-JNK signaling cascades by alpha1-integrins during colon cancer cell invasion. (United States)

    Van Slambrouck, Severine; Grijelmo, Clara; De Wever, Olivier; Bruyneel, Erik; Emami, Shahin; Gespach, Christian; Steelant, Wim F A


    Increased src tyrosine kinase expression and activity has been associated with colon cancer cell invasion and survival. Several signaling pathways are involved in the oncogenic activation of src during the adenoma to carcinoma progression and cellular invasion. In the present study, the synthetic ether lipid analog ET-18-OMe was shown to promote invasion of HCT-8/S11 colon cancer cells into collagen type I through the concomitant activation of src by phosphorylation at Tyr416 (5-30 min) in alpha1-integrin immunoprecipitates containing the integrin binding proteins talin and paxillin, as well as the phoshorylated and activated forms of focal adhesion kinase (FAK) at Tyr397 (a FAK kinase activation signal), Tyr576 and Tyr861. This was associated with the lateral redistribution of alpha1-integrins in focal aggregates and persistent activation of the p130Cas/JNK pathways at 5-30 min, with the subsequent induction and activation of the matrix metalloproteinases MMP-2 and MMP-9 (2-12 h). These activated molecular scaffolds and signaling cascades were not observed in immunoprecipitates of alpha2- and beta1-integrins, and tetraspanin CD9, an invasion and metastasis suppressor linked to integrins and FAK signaling. Our data demonstrate that the lateral redistribution and clustering of alpha1-integrins results in the recruitment of the FAK/src motility-promoting signaling complex involved in cancer cell invasion. Disruption of this proinvasive pathway was accomplished by the dominant negative mutant of src (K295R, kinase dead), src pharmacological inhibitor (PP1) and alpha1-integrin function blocking antibodies. These findings support the notion that the alpha1-integrin- and src-dependent signalosome is a relevant therapeutic target against tumor progression in colon cancer patients.

  9. Protein C Inhibitor-A Novel Antimicrobial Agent

    NARCIS (Netherlands)

    Malmström, E.; Mörgelin, M.; Malmsten, M.; Johansson, L.; Norrby-Teglund, A.; Shannon, O.; Schmidtchen, A.; Meijers, J.C.M.; Herwald, H.


    Protein C inhibitor (PCI) is a heparin-binding serine proteinase inhibitor belonging to the family of serpin proteins. Here we describe that PCI exerts broad antimicrobial activity against bacterial pathogens. This ability is mediated by the interaction of PCI with lipid membranes, which subsequentl

  10. 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: [Division of Animal Infectious Diseases, State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China)


    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.

  11. Disposition of Alpha-1-Antitrypsin in the Isolate Perfused Rabbit Lung

    Directory of Open Access Journals (Sweden)



    Full Text Available The potential for delivering large molecular weight proteins into the lungs to reach local or systemic sites of action was investigated by examining the disposition of alpha-1-antitrypsin in the isolated rabbit lung. Alpha-1-antitrypsin, a model protein, was measured in the periusion medium following intravascular administration and was found to remain constant, indicating limited uptake or metabolism by lung tissue. Intrabronchial instillation of 10 mg of alpha-1-antitrypsin in water resulted in no measurable concentration in the recirculating perfusate during the two hours experiment. These data suggest that transport of large proteins may be limited across lung-blood membrane barriers in either direction. Though this would limit the ability of inhaled drugs with large molecular weights to reach the general circulation, proteins which are used to treat respiratory diseases, such as alpha-1-antitrypsin, might be delivered locally by inhalation with only negligible systemic exposure.

  12. Alpha-1-antitrypsin phenotypes in Saudi Arabia: A study in the central province. (United States)

    Warsy, A S; El-Hazmi, M A; Sedrani, S H; Kinhal, M


    This study was conducted on 204 plasma samples obtained from Saudis living in the central province of Saudi Arabia, to determine the prevalence of alpha-1-antitrypsin (alpha1AT) phenotypes. The alpha1AT phenotypes were separated by isoelectric focusing on ampholine gels (pH 4-5). The prevalences of PiMM, MS, MZ, SZ, and ZZ were 0.8676, 0.0931, 0.0245, 0.0098, and 0.0049, respectively. The gene frequencies of the alpha1AT variants, i.e.., PiM, PiS, and PiZ, were 0.9265, 0.0515, 0.022, respectively. We describe and compare our results in a Saudi population with those reported for other populations.

  13. [Characterization of thermal denaturation process of proteinase K by spectrometry]. (United States)

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


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

  14. Proteinases as virulence factors in Leishmania spp. infection in mammals

    Directory of Open Access Journals (Sweden)

    Silva-Almeida Mariana


    Full Text Available Abstract Leishmania parasites cause human tegumentary and visceral infections that are commonly referred to as leishmaniasis. Despite the high incidence and prevalence of cases, leishmaniasis has been a neglected disease because it mainly affects developing countries. The data obtained from the analysis of patients’ biological samples and from assays with animal models confirm the involvement of an array of the parasite’s components in its survival inside the mammalian host. These components are classified as virulence factors. In this review, we focus on studies that have explored the role of proteinases as virulence factors that promote parasite survival and immune modulation in the mammalian host. Additionally, the direct involvement of proteinases from the host in lesion evolution is analyzed. The gathered data shows that both parasite and host proteinases are involved in the clinical manifestation of leishmaniasis. It is interesting to note that although the majority of the classes of proteinases are present in Leishmania spp., only cysteine-proteinases, metalloproteinases and, to a lesser scale, serine-proteinases have been adequately studied. Members from these classes have been implicated in tissue invasion, survival in macrophages and immune modulation by parasites. This review reinforces the importance of the parasite proteinases, which are interesting candidates for new chemo or immunotherapies, in the clinical manifestations of leishmaniasis.

  15. Effect of cyproterone acetate on alpha1-adrenoceptor subtypes in rat vas deferens

    Directory of Open Access Journals (Sweden)

    M. Campos


    Full Text Available Gonadal hormones regulate the expression of alpha1-adrenoceptor subtypes in several tissues. The present study was carried out to determine whether or not cyproterone acetate, an anti-androgenic agent, regulates the alpha1-adrenoceptor subtypes that mediate contractions of the rat vas deferens in response to noradrenaline. The actions of subtype selective alpha1-antagonists were investigated in vas deferens from control and cyproterone acetate-treated rats (10 mg/day, sc, for 7 days. Prazosin (pA2 ~9.5, phentolamine (pA2 ~8.3 and yohimbine (pA2 ~6.7 presented competitive antagonism consistent with activation of alpha1-adrenoceptors in vas deferens from both control and treated rats. The pA2 values estimated for WB 4101 (~9.5, benoxathian (~9.7, 5-methylurapidil (~8.5, indoramin (~8.7 and BMY 7378 (~6.8 indicate that alpha1A-adrenoceptors are involved in the contractions of the vas deferens from control and cyproterone acetate-treated rats. Treatment of the vas deferens from control rats with the alpha1B/alpha1D-adrenoceptor alkylating agent chloroethylclonidine had no effect on noradrenaline contractions, supporting the involvement of the alpha1A-subtype. However, this agent partially inhibited the contractions of vas deferens from cyproterone acetate-treated rats, suggesting involvement of multiple receptor subtypes. To further investigate this, the actions of WB 4101 and chloroethylclonidine were reevaluated in the vas deferens from rats treated with cyproterone acetate for 14 days. In these organs WB 4101 presented complex antagonism characterized by a Schild plot with a slope different from unity (0.65 ± 0.05. After treatment with chloroethylclonidine, the complex antagonism presented by WB 4101 was converted into classical competitive antagonism, consistent with participation of alpha1A-adrenoceptors as well as alpha1B-adrenoceptors. These results suggest that cyproterone acetate induces plasticity in the alpha1-adrenoceptor subtypes

  16. An Alpha-1A Adrenergic Receptor Agonist Prevents Acute Doxorubicin Cardiomyopathy in Male Mice (United States)

    Montgomery, Megan D.; Chan, Trevor; Swigart, Philip M.; Myagmar, Bat-erdene; Dash, Rajesh; Simpson, Paul C.


    Alpha-1 adrenergic receptors mediate adaptive effects in the heart and cardiac myocytes, and a myocyte survival pathway involving the alpha-1A receptor subtype and ERK activation exists in vitro. However, data in vivo are limited. Here we tested A61603 (N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide), a selective imidazoline agonist for the alpha-1A. A61603 was the most potent alpha-1-agonist in activating ERK in neonatal rat ventricular myocytes. A61603 activated ERK in adult mouse ventricular myocytes and protected the cells from death caused by the anthracycline doxorubicin. A low dose of A61603 (10 ng/kg/d) activated ERK in the mouse heart in vivo, but did not change blood pressure. In male mice, concurrent subcutaneous A61603 infusion at 10 ng/kg/d for 7 days after a single intraperitoneal dose of doxorubicin (25 mg/kg) increased survival, improved cardiac function, heart rate, and cardiac output by echocardiography, and reduced cardiac cell necrosis and apoptosis and myocardial fibrosis. All protective effects were lost in alpha-1A-knockout mice. In female mice, doxorubicin at doses higher than in males (35–40 mg/kg) caused less cardiac toxicity than in males. We conclude that the alpha-1A-selective agonist A61603, via the alpha-1A adrenergic receptor, prevents doxorubicin cardiomyopathy in male mice, supporting the theory that alpha-1A adrenergic receptor agonists have potential as novel heart failure therapies. PMID:28081170

  17. Bioisosteric phentolamine analogs as selective human alpha(2)- versus alpha(1)-adrenoceptor ligands. (United States)

    Bavadekar, Supriya A; Hong, Seoung-Soo; Lee, Sang-Ii; Miller, Duane D; Feller, Dennis R


    Phentolamine is known to act as a competitive, non-subtype-selective alpha-adrenoceptor antagonist. In an attempt to improve alpha(2)- versus alpha(1)-adrenoceptor selectivity and alpha(2)-adrenoceptor subtype-selectivity, two new chemical series of bioisosteric phentolamine analogs were prepared and evaluated. These compounds were evaluated for binding affinities on alpha(1)- (alpha(1A)-, alpha(1B)-, alpha(1D)-) and alpha(2)- (alpha(2A)-, alpha(2B)-, alpha(2C)-) adrenoceptor subtypes that had been stably expressed in human embryonic kidney and Chinese hamster ovary cell lines, respectively. Methylation of the phenolic hydroxy group and replacement of the 4-methyl group of phentolamine with varying lipophilic substituents yielded bioisosteric analogs selective for the alpha(2)- versus alpha(1)-adrenoceptors. Within the alpha(2)-adrenoceptors, these analogs bound with higher affinity at the alpha(2A)- and alpha(2C)-subtypes as compared to the alpha(2B)-subtype. In particular, the t-butyl analog was found to be the most selective, its binding at the alpha(2C)-adrenoceptor (Ki=3.6 nM) being 37- to 173-fold higher than that at the alpha(1)-adrenoceptors, and around 2- and 19-fold higher than at the alpha(2A)- and alpha(2B)-adrenoceptors, respectively. Data from luciferase reporter gene assays confirmed the functional antagonist activities of selected compounds from the bioisosteric series on human alpha(1A)- and alpha(2C)-adrenoceptors. Thus, the results with these bioisosteric analogs of phentolamine provide a lead to the rational design of potent and selective alpha(2)-adrenoceptor ligands that may be useful in improving the therapeutic profile of this drug class for human disorders.

  18. Heme-Scavenging Role of alpha1-Microglobulin in Chronic Ulcers.


    Allhorn, Maria; Lundqvist, Katarina; Schmidtchen, Artur; Åkerström, Bo


    Chronic venous ulcers are characterized by chronic inflammation. Heme and iron, originating from blood cell hemolysis as well as extravascular necrosis, have been implicated as important pathogenic factors due to their promotion of oxidative stress. It was recently reported that the plasma and tissue protein alpha1-microglobulin is involved in heme metabolism. The protein binds heme, and a carboxy-terminally processed form, truncated alpha1-microglobulin, also degrades heme. Here, we show the...

  19. Effect of extracellular pH on recombinant alpha1beta2gamma2 and alpha1beta2 GABAA receptors. (United States)

    Mercik, Katarzyna; Pytel, Maria; Cherubini, Enrico; Mozrzymas, Jerzy W


    Recently, we have reported that extracellular protons allosterically modulated neuronal GABA(A) receptors [Mozrzymas, J.W., Zarnowska, E.D., Pytel, M., Mercik, K., 2003a. Modulation of GABA(A) receptors by hydrogen ions reveals synaptic GABA transient and a crucial role of desensitiztion process. Journal of Neuroscience 23, 7981-7992]. However, GABAARs in neurons are heterogeneous and the effect of hydrogen ions depends on the receptor subtype. In particular, gamma2 subunit sets the receptor sensibility to several modulators including protons. However, the mechanisms whereby protons modulate gamma2-containing and gamma2-free GABAARs have not been fully elucidated. To this end, current responses to ultrafast GABA applications were recorded for alpha1beta2gamma2 and alpha1beta2 receptors at different pH values. For both receptor types, increase in pH induced a decrease in amplitudes of currents elicited by saturating [GABA] but this effect was stronger for alpha1beta2 receptors. In the case of alpha1beta2gamma2 receptors, protons strongly affected the current time course due to a down regulation of binding and desensitization rates. This effect was qualitatively similar to that described in neurons. Protons strongly influenced the amplitude of alpha1beta2 receptor-mediated currents but the effect on their kinetics was weak suggesting a predominant direct non-competitive inhibition with a minor allosteric modulation. In conclusion, we provide evidence that extracellular protons strongly affect GABAA receptors and that, depending on the presence of the gamma2 subunit, the modulatory mechanisms show profound quantitative and qualitative differences.

  20. Blood pressure, risk of ischemic cerebrovascular and ischemic heart disease, and longevity in alpha(1)-antitrypsin deficiency

    DEFF Research Database (Denmark)

    Dahl, Morten; Tybjaerg-Hansen, Anne; Sillesen, Henrik


    Because elastase in alpha(1)-antitrypsin deficiency may attack elastin in the arterial wall, we tested whether alpha(1)-antitrypsin deficiency is associated with reduced blood pressure, risk of ischemic cerebrovascular (ICVD) and ischemic heart disease (IHD), and longevity.......Because elastase in alpha(1)-antitrypsin deficiency may attack elastin in the arterial wall, we tested whether alpha(1)-antitrypsin deficiency is associated with reduced blood pressure, risk of ischemic cerebrovascular (ICVD) and ischemic heart disease (IHD), and longevity....

  1. [Inactivation of T4 phage in water environment using proteinase]. (United States)

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


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

  2. Production of a heterologous proteinase A by Saccharomyces kluyveri

    DEFF Research Database (Denmark)

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


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

  3. Binding and functional characterization of alpha1-adrenoceptor subtypes in the rat prostate. (United States)

    Hiraoka, Y; Ohmura, T; Oshita, M; Watanabe, Y; Morikawa, K; Nagata, O; Kato, H; Taniguchi, T; Muramatsu, I


    The alpha1-adrenoceptor subtypes of rat prostate were characterized in binding and functional experiments. In binding experiments, [3H]tamsulosin bound to a single class of binding sites with an affinity (pKD) of 10.79+/-0.04 and Bmax of 87+/-2 fmol mg(-1) protein. This binding was inhibited by prazosin, 2-(2,6-dimethoxy-phenoxyethyl)-aminomethyl-1,4-benzodioxane hydrochloride (WB4101), 5-methylurapidil, alpha-ethyl-3,4,5,-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)-amin o)-propyl)benzeneacetonitrile fumarate (HV723) and oxymetazoline with high efficacy, resulting in a good correlation with the binding characteristics of cloned alpha1a but not alpha1b and alpha1d-adrenoceptor subtypes. In functional studies, noradrenaline and oxymetazoline produced concentration-dependent contractions. These contractions were antagonized by tamsulosin, prazosin, WB4101 and 5-methylurapidil with an efficacy lower than that exhibited by these agents for inhibition of [3H]tamsulosin binding. The relationship between receptor occupancy and contractile amplitude revealed the presence of receptor reserve for noradrenaline, but the contraction induced by oxymetazoline was not in parallel with receptor occupation and developed after predicted receptor saturation. From these results, it is suggested that alpha1A-adrenoceptors are the dominant subtype in the rat prostate which can be detected with [3H]tamsulosin, but that the functional subtype mediating adrenergic contractions has the characteristics of the alpha1L-adrenoceptor subtype, having a lower affinity for prazosin and some other drugs than the alpha1A-adrenoceptor subtype.

  4. Enhanced alpha 1(I) mRNA expression in frozen shoulder and dupuytren tissue. (United States)

    Kilian, Olaf; Pfeil, U; Wenisch, S; Heiss, C; Kraus, R; Schnettler, R


    The purpose of this study has been to investigate collagen I and III synthesis during the fibrosing stage of frozen shoulder and Dupuytren samples in comparison to normal capsule tissue. - By using the quantitative PCR significantly increased levels of alpha 1(I) mRNA transcription in samples of frozen shoulder (p = 0.016) and Duypuytren (p = 0.041) could be demonstrated, whereas alpha 2(I) and alpha 1(III) chains have shown the same mRNA levels as in normal capsule tissue. - Despite an enhancement of alpha 1(I) mRNA transcription in frozen shoulder and Dupuytren samples the intracellular precursor procollagen I and extracellular mature collagen I was detected immunohistochemically in reduced levels. - The structural alteration of collagen I assembly might be caused by disturbed post-translation from the polypeptide chains into the triple helices procollagen I though alpha 1(I) mRNA transcription was significantly increased and alpha 2(I) mRNA transcription was in normal range. Fibroblasts might release high quantities of free alpha 1(I) polypeptide chains or (alpha 1(I)) 3 homotrimer into the extracellular space during the fibrosing stage of frozen shoulder and Dupuytren disease. - In all samples neither differences of alpha 1(III) mRNA transcription nor differences of immunohistochemical staining intensity of collagen III could be seen. This might result from apoptosis of myofibroblasts in the final phase of the fibrosing processes. - The stimulating effect of insulin-like growth factor type I (IGF-I) to induce fibrosis in connective tissue such as scarlet is known. In all patients suffering from frozen shoulder and Dupuytren disease the serum IGF-I level was in a normal range and the IGF-I receptor - (IGFR-I) mRNA transcription in the samples was also in the same level compared with normal capsule tissue.

  5. Separation of the subtypes of type V collagen molecules, [alpha 1(V)]2 alpha 2(V) and alpha 1(V) alpha 2(V) alpha 3(V), by chain composition-dependent affinity for heparin: single alpha 1(V) chain shows intermediate heparin affinity between those of the type V collagen subtypes composed of [alpha 1(V)]2 alpha 2(V) and of alpha 1(V) alpha 2(V) alpha 3(V). (United States)

    Mizuno, K; Hayashi, T


    The heparin affinities of heat-treated type V collagen alpha-chains and the triple-helical molecules were evaluated in terms of the NaCl concentration required for prevention of binding to a heparin-Sepharose column. After heat treatment, alpha 1(V) chain required approximately two-fold higher NaCl concentration to pass through the column than the other two chains, alpha 2(V) and alpha 3(V). Thus, the heparin affinity of alpha 1(V) may be approximately two-fold higher than those of the other alpha (V)-chains. The type V collagen molecules in triple-helical conformation were separated into two fractions at 170 mM NaCl in 20 mM phosphate buffer, pH 7.2, containing 2 M urea; bound and non-bound. The ratio of the three alpha-chains, alpha 1(V): alpha 2(V): alpha 3(V) was 2 : 1 : 0 and 1 : 1 : 1 in the bound and flow-through fractions, respectively, on analysis by SDS-PAGE. The differential affinity of the two fractions could be accounted for by the number of alpha 1(V) chains in the triple-helical molecule, if these fractions contained triple-helical subtypes with the chain compositions of [alpha 1(V)]2 alpha 2(V) and alpha 1(V) alpha 2(V) alpha 3(V), respectively. From the comparison of the NaCl concentration required for prevention of the binding, [alpha 1(V)]2, alpha 2(V) had about two-fold higher affinity than alpha 1(V) alpha 2(V) alpha 3(V), and the separated alpha 1(V) chain showed an intermediate affinity. A possible explanation for difference in heparin affinity among the subtypes of molecules and the separated alpha-chains is that the heparin affinity of type V collagen molecule is governed by the number of alpha 1(V) chains contained in the molecule and that steric restraint in a triple-helical conformation weakens the binding of alpha 1(V) chain to heparin.

  6. Purification of human leucocyte DNA: proteinase K is not necessary. (United States)

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


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

  7. Collagen polymorphism: characterization of molecules with the chain composition (alpha 1 (3)03 in human tissues. (United States)

    Chung, E; Miller, E J


    Collagen moleculess with the chain comizposition [alpha1(III)](3), have been isolated from pepsin-solubilized collagen of dermis, aorta, and leiomlyoma of the uterus by differential salt precipitation. On denaturation, approximately 90 percent of this collagen is recovered as a gamma component (300,000 daltons). Reduction and alkylation of the high-molecular-weight component yields alpha1(III) chains (95,000 daltons). In addition to containing cysteine, alpha1(III) chains exhibit several other compositional differences when compared to alpha1(I), alpha1(II), or alpha2 chains from human tissues.

  8. The effect of calciums on molecular motions of proteinase K. (United States)

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


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

  9. The possible involvement of D-amino acids or their metabolites in Arabidopsis cysteine proteinase/cystatin N-dependent proteolytic pathway. (United States)

    Gholizadeh, A


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

  10. Appearance and cellular distribution of lectin-like receptors for alpha 1-acid glycoprotein in the developing rat testis

    DEFF Research Database (Denmark)

    Andersen, U O; Bøg-Hansen, T C; Kirkeby, S


    glycoprotein glycoforms to their receptors is inhibited by steroids. Testosterone, oestradiol and progesterone inhibited the binding of alpha 1-acid glycoprotein glycoform A to its receptor. Cortisone, aldosterone, oestradiol and progesterone inhibited the binding of alpha 1-acid glycoprotein glycoforms B......A histochemical avidin-biotin technique with three different alpha 1-acid glycoprotein glycoforms showed pronounced alterations in the cellular localization of two alpha 1-acid glycoprotein lectin-like receptors during cell differentiation in the developing rat testis. The binding of alpha 1-acid...... and C to their receptor. A difference in the cellular content of alpha 1-acid glycoprotein glycoforms and alpha 1-acid glycoprotein receptors separates the spermatocytes and the early spermatids from the late spermatids. The difference in receptor composition implies a difference in the effect...

  11. Zonal variation in the distribution of an alpha 1-acid glycoprotein glycoform receptor in human adrenal cortex

    DEFF Research Database (Denmark)

    Andersen, U O; Bøg-Hansen, T C; Kirkeby, S


    Using a histochemical technique with three different alpha 1-acid glycoprotein glycoform one glycoform specific receptor has been identified in human adrenal cortex. The receptor is associated to alpha 1-acid glycoprotein glycoform B and alpha 1-acid glycoprotein glycoform C. The glycoform specific...... specific receptor. The binding of alpha 1-acid glycoprotein glycoform B and alpha 1-acid glycoprotein glycoform C to the glycoform specific receptor is inhibited by the steroid hormones cortisone, aldosterone, estradiol and progesterone but not by testosterone. The pronounced changes in the distribution....... The binding activity was dependent on the presence of calcium ions and not on thiol reagents. Thus the lectin-like receptor may belong to the C-type lectin family. Using an antibody to alpha 1-acid glycoprotein the presence of alpha 1-acid glycoprotein was observed in the same location as the glycoform...

  12. Alpha-1 antitrypsin Pi*Z gene frequency and Pi*ZZ genotype numbers worldwide: an update

    Directory of Open Access Journals (Sweden)

    Blanco I


    Africa, 32,154 in Asia, 4,126 in Australia, and 2,216 in New Zealand. In addition, the IDW-interpolation maps predicted Pi*Z frequencies throughout the world even in some areas that lack real data. In conclusion, the inclusion of new well-designed studies and the exclusion of the low-quality ones have significantly improved the reliability of results, which may be useful to plan strategies for future research and diagnosis and to rationalize the therapeutic resources available. Keywords: SERPINA1, alpha-1 antitrypsin deficiency, protease inhibitor, genetic epidemiology, inverse distance weighted interpolation, geographic information system

  13. Intracellular Localization and Trafficking of Serine Proteinase AhSub and Cysteine Proteinase AhCP of Acanthamoeba healyi


    Moon, E.-K.; Lee, S.-T.; Chung, D.-I.; Kong, H.-H.


    Proteinases have been proposed to play important roles in pathogenesis and various biologic actions in Acanthamoeba. Although genetic characteristics of several proteases of Acanthamoeba have been reported, the intracellular localization and trafficking of these enzymes has yet to be studied. In the present study, we analyzed the intracellular localization and trafficking of two proteinases, AhSub and AhCP, of Acanthamoeba healyi by transient transfection. Full-length AhSub-enhanced green flu...

  14. Alpha1-antitrypsin deficiency with fatal intracranial hemorrhage in a newborn. (United States)

    Israels, S J; Gilfix, B M


    A 4-week-old boy had a fatal intracranial hemorrhage resulting from vitamin K deficiency. The infant had received no vitamin K prophylaxis and was exclusively breastfed. At autopsy, examination of the liver showed cholestasis and fibrosis. DNA was isolated from a blood spot on a Gutherie sample card obtained from the infant for routine metabolic screening. This DNA was used for alpha1-antitrypsin genotyping studies. Genotyping studies identified homozygosity for the point mutation 9989G-->A, confirming a diagnosis of alpha1-antitrypsin deficiency (ZZ phenotype), and resulted in appropriate screening of siblings born after this child's death. Alpha1-antitrypsin deficiency should be considered in the differential diagnosis of infants with late hemorrhagic disease of the newborn. Use of blood from the metabolic screening card as a source of DNA allowed confirmation of this diagnosis after the infant's death.

  15. Neuronal changes resulting in up-regulation of alpha-1 adrenoceptors after peripheral nerve injury

    Institute of Scientific and Technical Information of China (English)

    Peter D.Drummond


    Under normal conditions, the sympathetic neurotransmitter noradrenaline inhibits the pro-duction and release of pro-inlfammatory cytokines. However, after peripheral nerve and tissue injury, pro-inflammatory cytokines appear to induce the expression of the alpha1A-adreno-ceptor subtype on immune cells and perhaps also on other cells in the injured tissue. In turn, noradrenaline may act on up-regulated alpha1-adrenoceptors to increase the production of the pro-inflammatory cytokine interleukin-6. In addition, the release of inflammatory mediators and nerve growth factor from keratinocytes and other cells may augment the expression of al-pha1-adrenoceptors on peripheral nerve ifbers. Consequently, nociceptive afferents acquire an abnormal excitability to adrenergic agents, and inlfammatory processes build. These mechanisms could contribute to the development of sympathetically maintained pain in conditions such as post-herpetic neuralgia, cutaneous neuromas, amputation stump pain and complex regional pain syndrome.

  16. Five different profiles of dihydropyridines in blocking T-type Ca(2+) channel subtypes (Ca(v)3.1 (alpha(1G)), Ca(v)3.2 (alpha(1H)), and Ca(v)3.3 (alpha(1I))) expressed in Xenopus oocytes. (United States)

    Furukawa, Taiji; Nukada, Toshihide; Namiki, Yoshiko; Miyashita, Yoriko; Hatsuno, Kento; Ueno, Yasunari; Yamakawa, Takeshi; Isshiki, Takaaki


    1,4-dihydropyridine (DHP) Ca(2+) antagonists have recently been shown to block T-type Ca(2+) channels, which may render favorable actions on cardiovascular systems. However, this evaluation remains to be done systematically for each T-type Ca(2+) channel subtype except for the Ca(v)3.1 (alpha(1G)) subtype. To address this issue at the molecular level, blocking effects of 14 kinds of DHPs (amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, efonidipine, felodipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nitrendipine), which are clinically used for treatments of hypertension, on 3 subtypes of T-type Ca(2+) channels [Ca(v)3.2 (alpha(1H)), Ca(v)3.3 (alpha(1I)), and Ca(v)3.1 (alpha(1G))] were investigated in the Xenopus oocyte expression system using the two-microelectrode voltage-clamp technique. These 3 kinds (alpha(1H), alpha(1I) and alpha(1G)) of T-type channels were blocked by amlodipine, manidipine and nicardipine. On the other hand, azelnidipine, barnidipine, benidipine and efonidipine significantly blocked alpha(1H) and alpha(1G), but not alpha(1I) channels, while nilvadipine and nimodipine apparently blocked alpha(1H) and alpha(1I), but not alpha(1G) channels. Moreover, aranidipine blocked only alpha(1H) channels. By contrast, cilnidipine, felodipine, nifedipine and nitrendipine had little effects on these subtypes of T-type channels. The result indicates that the blockade of T-type Ca(2+) channels by derivatives of DHP Ca(2+) antagonist was selective for the channel subtype. Therefore, these selectivities of DHPs in blocking T-type Ca(2+) channel subtypes would provide useful pharmacological and clinical information on the mode of action of the drugs including side-effects and adverse effects.

  17. Rapid component I(Kr) of cardiac delayed rectifier potassium currents in guinea-pig is inhibited by alpha(1)-adrenoreceptor activation via protein kinase A and protein kinase C-dependent pathways. (United States)

    Wang, Sen; Xu, Dong-Jie; Cai, Jing-Bo; Huang, Yuan-Zhu; Zou, Jian-Gang; Cao, Ke-Jiang


    Ventricular tachyarrhythmias are often precipitated by physical or emotional stress, indicating a link between increased adrenergic stimulation and cardiac ion channel activity. Human ether-a-go-go related gene (hERG) potassium channels conduct the rapid component of delayed rectifier potassium current, I(kr), a crucial component for action potential repolarization. To evaluate the correlation between increased alpha(1)-adrenergic activity and the rapid component of cardiac I(kr), whole-cell patch-clamp recording was performed in isolated guinea-pig ventricular myocytes. Stimulation of alpha(1)-adrenoceptors using phenylephrine (0.1 nM-100 microM) reduced I(kr) current in a dose-dependent manner at 37 degrees C. Phenylephrine (0.1 microM) reduced I(kr) current to 66.83+/-3.16%. Chelerythrine (1 microM), a specific inhibitor of protein kinase C (PKC) completely inhibited the changes in I(kr) trigged by 0.1 microM phenylephrine. KT5720 (2.5 microM), a specific inhibitor of protein kinase A (PKA) partially inhibited the current decrease induced by 0.1 microM phenylephrine. Both chelerythrine and KT5720 drastically reduced the phenylephrine-induced effects, indicating possible involvement of PKC and PKA in the alpha(1)-adrenergic inhibition of I(kr). Our data suggest a link between I(kr) and the alpha(1)-adrenoceptor, involving activation of PKC and PKA in arrhythmogenesis.

  18. Molecular determinants of desensitization and assembly of the chimeric GABA(A) receptor subunits (alpha1/gamma2) and (gamma2/alpha1) in combinations with beta2 and gamma2

    DEFF Research Database (Denmark)

    Elster, L; Kristiansen, U; Pickering, D S


    2 and the remainder of the gamma2 or alpha1 subunits, respectively, were expressed with beta2 and beta2gamma2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (alpha1/gamma2)beta2 and (alpha1/gamma2)beta2gamma2 but not the (gamma2/alpha1)beta2 and (gamma2/alpha1......)beta2gamma2 subunit combinations formed functional receptor complexes as shown by whole-cell patch-clamp recordings and [3H]muscimol and [3H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (alpha1/gamma2)-containing receptors was pronounced...

  19. [Purification and properties of serine proteinases from European catfish Silurus glanis L. pancreas]. (United States)

    Ulitina, N N; Khabliuk, V V; Proskuriakov, M T


    Three trypsin isoforms (designated as T1, T2, and T3), three chymotrypsin isoforms (Kh1, Kh2, and Kh3), and two elastase isoforms (E1 and E2) were isolated from the pancreas of European catfish Silurus glanis L. by salting out with (NH4)2SO4, gel chromatography on Sephadex G-75, and ion exchange chromatography on DEAE cellulose. Isoelectric points of the enzymes, determined by isoelectric focusing, amounted to 4.42 for T1, 5.64 for T2, 6.90 for T3, 4.93 for Khl, 5.23 for Kh2, 6.18 for Kh3, 6.17 for E1, and 8.48 for E2. Molecular weights of proteinases within each group were close and amounted to 30100 Da for trypsins, 39800 Da for chymotrypsins, and 24000 Da for elastases. The enzymes isolated displayed maximal activities at alkaline pH values. Inhibitor analysis demonstrated that all the proteinases isolated from European catfish pancreas belonged to the serine type.

  20. 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


    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

  1. Developing a rapid throughput screen for detection of nematicidal activity of plant cysteine proteinases: the role of Caenorhabditis elegans cystatins. (United States)

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


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

  2. In vitro digestibility of globulins from sapucaia (Lecythis pisonis Camb. nuts by mammalian digestive proteinases Digestibilidade in vitro de globulinas das amêndoas de sapucaia (Lecythis pisonis Camb. por proteinases digestivas de mamíferos

    Directory of Open Access Journals (Sweden)

    Sandra Maria Silveira Denadai


    Full Text Available Sapucaia (Lecythis pisonis Camb. raw nuts collected from Brazil were analyzed to determine the proximate composition, amino acid profile of protein fractions, in vitro protein digestibility and antinutritional factors in order to evaluate their potential as a protein alimentary complement. The nuts contained adequate amounts of essential amino acids, fatty acids and minerals. In the present study, no hemagglutinating or inhibitory activities were observed in any of the samples investigated, indicating low or non-detectable levels of proteinase inhibitors or lectins in the samples. In vitro digestibility of in natura and heated nut globulins by mammalian digestive proteinases was carried out using trypsin + chymotrypsin + peptidase, with resulting mean values of approximately 70.30 and 71.35%, respectively. Taken together, the results suggest that sapucaia nuts may provide a new source of protein to use as a potential nutritional agent.Amêndoas cruas de Sapucaia (Lecythis pisonis Camb. colhidas no Brasil foram analisadas para se determinar a composição centesimal, o perfil de aminoácidos de suas proteínas, a digestibilidade protéica in vitro e a presença de fatores antinutricionais, para avaliar o seu potencial como complemento alimentar protéico. As amêndoas apresentaram quantidades adequadas de aminoácidos essenciais, ácidos graxos e minerais; no entanto, baixo teor de fibra foi observado. No presente estudo, a presença de lectinas ou inibidores de proteinases, quando detectada, apresentou baixos níveis. A digestibilidade in vitro de globulinas, in natura ou aquecidas, por proteinases digestivas de mamíferos foi realizada utilizando-se tripsina + quimotripsina + peptidase, obtendo-se valores aproximados de 71,5 e 73,5%, respectivamente. Estes resultados sugerem que as amêndoas de sapucaia podem ser utilizadas como complemento alimentar de proteínas, sendo um potencial agente nutricional.

  3. 21 CFR 866.5580 - Alpha-1-lipoprotein immuno-logical test system. (United States)


    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alpha-1-lipoprotein immuno-logical test system. 866.5580 Section 866.5580 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN...-lipoprotein may aid in the diagnosis of Tangier disease (a hereditary disorder of fat metabolism)....

  4. Deformation and recursion for the $N = 2 \\; \\alpha = 1$ supersymmetric KdV hierarchy

    NARCIS (Netherlands)

    Sorin, A.S.; Kersten, P.H.M.


    A detailed description is given for the construction of the deformation of the $N=2$ supersymmetric $\\alpha=1$ KdV-equation, leading to the recursion operator for symmetries and the zero-th Hamiltonian structure; the solution to a longstanding problem.

  5. Bi-Hamiltonian structure of the $N=2$ supersymmetric $\\alpha = 1$ KdV hierarchy

    NARCIS (Netherlands)

    Kersten, P.H.M.; Sorin, A.S.


    The $N=2$ supersymmetric ${\\alpha}=1$ KdV hierarchy in $N=2$ superspace is considered and its rich symmetry structure is uncovered. New nonpolynomial and nonlocal, bosonic and fermionic symmetries and Hamiltonians, bi-Hamiltonian structure as well as a recursion operator connecting all symmetries an

  6. Alpha-1 adrenergic receptor: Binding and phosphoinositide breakdown in human myometrium

    Energy Technology Data Exchange (ETDEWEB)

    Breuiller-Fouche, M.; Doualla-Bell Kotto Maka, F.; Geny, B.; Ferre, F. (INSERM U.166 Groupe de recherches sur l' Endocrinologie de la Reproduction, Maternite Baudelocque, Paris (France))


    Alpha-1 adrenergic receptors were examined in both inner and outer layers of human pregnant myometrium using radioligand binding of (3H)prazosin. (3H)prazosin bound rapidly and reversibly to a single class of high affinity binding sites in myometrial membrane preparations. Scatchard analysis gave similar values of equilibrium dissociation constants in both myometrial layers. In contrast, more alpha-1 adrenergic receptors were detected in the outer layer than in the inner layer. Antagonist inhibited (3H)prazosin binding with an order of potency of prazosin greater than phentolamine greater than idazoxan. Competition experiments have also revealed that a stable guanine nucleotide decreases the apparent affinity of norepinephrine for myometrial (3H)prazosin binding sites. The functional status of these alpha-1 adrenergic receptors was also assessed by measuring the norepinephrine-induced accumulation of inositol phosphates in myometrial tissue. Norepinephrine produced a concentration-dependent accumulation of inositol phosphates in both myometrial layers. However, norepinephrine-induced increases in inositol 1,4,5-triphosphate were only observed in the outer layer. These results indicate that alpha-1 adrenergic receptors in human myometrium at the end of pregnancy are linked to phosphoinositide hydrolysis and that this response occurs mainly in the outer layer.

  7. Biochemical analysis and solubilization of central alpha 1-adrenoceptors in the rat

    Energy Technology Data Exchange (ETDEWEB)

    Guicheney, P.; Rappaport, A.; Meyer, P.


    To study the structure and the molecular mechanisms of action of brain alpha-adrenoceptors, their solubilization was undertaken. alpha 1-Adrenoceptors were first successfully solubilized from fresh rat brain membranes by treatment with 0.3 percent deoxycholate, after prelabeling of the binding site by the highly specific tritiated antagonist /sup 3/H-prazosin. The complex thus solubilized underwent a rapid loss of activity at 25 degrees C. Direct solubilization of brain alpha 1-adrenoceptors was obtained by treatment with a new zwitterionic derivative of cholic acid (CHAPS) at a concentration of 5 to 10 mM. The soluble complex was detected by precipitation by polyethylene glycol 6,000 with gamma globulin as a carrier. Binding of /sup 3/H-prazosin at 25 degrees C was rapid; at 4 degrees C the steady state was obtained within two hours and remained unchanged for at least six hours. The affinity of the soluble binding site, determined by Scatchard analysis (congruent to 0.6 nM), varied with the concentration of detergent. Specificity of the membrane-bound receptor was preserved as demonstrated by incubation in the presence of alpha 1- and alpha 2-antagonists at various concentrations (by order of potency: prazosin greater than phentolamine greater than yohimbine). Stereoselectivity was also retained in the solubilized binding protein. The solubilization of an active brain alpha 1-adrenoceptor will allow further investigation at the molecular level.

  8. Alpha 1-acid glycoprotein has immunomodulatory effects in neonatal swine adipose tissue (United States)

    Alpha 1-acid glycoprotein (AGP) is the most abundant protein in serum of neonatal swine. This protein functions as an immunomodulator in the pig. Recent work has demonstrated that adipose tissue can express AGP mRNA, as well as numerous cytokine mRNA. The present study was designed to determine i...

  9. Vitamin K deficiency bleeding in cholestatic infants with alpha-1-antitrypsin deficiency.

    NARCIS (Netherlands)

    Hasselt, P.M. van; Kok, K.F.; Vorselaars, A.D.; Vlerken, L. van; Nieuwenhuys, E.; Koning, T.J. de; Vries, R.A. de; Houwen, R.H.J.


    OBJECTIVE: Exclusively breastfed infants with unrecognised cholestatic jaundice are at high risk of a vitamin K deficiency (VKD) bleeding. It is presently unknown whether (the size of) this risk depends on the degree of cholestasis. Since alpha-1-antitrypsin deficiency (A1AD) induces a variable degr

  10. Human CRISP-3 binds serum alpha(1)B-glycoprotein across species

    DEFF Research Database (Denmark)

    Udby, Lene; Johnsen, Anders H; Borregaard, Niels


    CRISP-3 was previously shown to be bound to alpha(1)B-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular...

  11. Efficacy of alpha1-antitrypsin augmentation therapy in conditions other than pulmonary emphysema

    Directory of Open Access Journals (Sweden)

    de Serres Frederick


    Full Text Available Abstract Up to now alpha 1-antitrypsin (AAT augmentation therapy has been approved only for commercial use in selected adults with severe AAT deficiency-related pulmonary emphysema (i.e. PI*ZZ genotypes as well as combinations of Z, rare and null alleles expressing AAT serum concentrations

  12. Pharmacological tolerance to alpha 1-adrenergic receptor antagonism mediated by terazosin in humans. (United States)

    Vincent, J; Dachman, W; Blaschke, T F; Hoffman, B B


    Chronic administration of alpha 1-receptor antagonists is associated with loss of clinical efficacy, especially in congestive heart failure, although the mechanism is uncertain. To evaluate changes in venous alpha 1-adrenoceptor responsiveness during chronic alpha 1-adrenoceptor blockade, dose-response curves to phenylephrine and angiotensin II were constructed in 10 healthy subjects before, during, and after administration of terazosin 1 mg orally for 28 d. Terazosin initially shifted the dose-response curve of phenylephrine to the right, with a significant increase in ED50 for phenylephrine from a control value of 102 to 759 ng/min on day 1 of terazosin (P < 0.001). However, by day 28, the dose-response curve had shifted back towards baseline with an ED50 of 112 ng/min. After discontinuing terazosin, the ED50 for phenylephrine remained near the baseline value, indicating no evidence of supersensitivity to phenylephrine. There was no change in responsiveness to angiotensin II during the course of treatment with terazosin. Plasma terazosin concentrations were stable throughout the period of drug administration. The mean Kd of terazosin was estimated as 11 +/- 15 nM in the first few days of treatment. This study demonstrates that pharmacological tolerance to the alpha 1-adrenoceptor blocking action of terazosin occurs in man and may be responsible for loss in efficacy with chronic therapy. PMID:1358918

  13. The pharmacology of spontaneously open alpha 1 beta 3 epsilon GABA A receptor-ionophores. (United States)

    Maksay, Gábor; Thompson, Sally A; Wafford, Keith A


    Human alpha(1)beta(3) epsilon GABA(A) receptors were expressed in Xenopus oocytes and examined using the conventional two-electrode voltage-clamp technique and compared to alpha(1)beta(3)gamma(2) receptors. The effects of several GABA(A) agonists were studied, and the allosteric modulation of the channel by a number of GABAergic modulators investigated. The presence of the epsilon subunit increased the potency and efficacy of direct activation by partial GABA(A) agonists (piperidine-4-sulphonic acid and thio-4-PIOL), pentobarbital and neuro-steroids. Direct activation by 3-hydroxylated neurosteroids was restricted to 3alpha epimers, while chirality at C5 was indifferent. The 3beta-sulfate esters of pregnenolone and dehydroepiandrosterone inhibited the spontaneous currents with efficacies higher, while bicuculline methiodide and SR 95531 did so lower than picrotoxin and TBPS. Furosemide, fipronil, triphenylcyanoborate and Zn(2+) blocked the spontaneous currents of alpha(1)beta(3) epsilon receptors with different efficacies. Flunitrazepam and 4'-chlorodiazepam inhibited the spontaneous currents with micromolar potencies. In conclusion, spontaneously active alpha(1)beta(3) epsilon GABA(A) receptors can be potentiated and blocked by GABAergic agents within a broad range of efficacy.

  14. Lower lid entropion secondary to treatment with alpha-1a receptor antagonist: a case report

    Directory of Open Access Journals (Sweden)

    Simcock Peter


    Full Text Available Abstract Introduction The use of alpha-1a receptor antagonists (tamsulosin is widely accepted in the treatment of benign prostatic hypertrophy (BPH. It has previously been implicated as a causative agent in intra-operative floppy iris syndrome due to its effects on the smooth muscle. We report a case of lower lid entropion that may be related to a patient commencing treatment of tamsulosin. Case presentation A 74-year-old Caucasian man was started on alpha 1-a receptor antagonist (Tamsulosin treatment for benign prostatic hypertrophy. Eight days later, he presented to the ophthalmology unit with a right lower lid entropion which was successfully treated surgically with a Weiss procedure. Conclusion We report a case of lower lid entropion that may be secondary to the recent use of an alpha-1a blocker (tamsulosin. This can be explained by considering the effect of autonomic blockade on alpha-1 receptors in the Muller's muscle on a patient that may already have an anatomical predisposition to entropion formation due to a further reduction in muscle tone.

  15. 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.


    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

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

    NARCIS (Netherlands)

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


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

  17. Involvement of central alpha1-adrenoceptors on renal responses to central moxonidine and alpha-methylnoradrenaline. (United States)

    de Andrade, Carina A F; de Andrade, Glaucia M F; De Paula, Patricia M; De Luca, Laurival A; Menani, José V


    Moxonidine (alpha2-adrenoceptor/imidazoline receptor agonist) injected into the lateral ventricle induces diuresis, natriuresis and renal vasodilation. Moxonidine-induced diuresis and natriuresis depend on central imidazoline receptors, while central alpha1-adrenoceptors are involved in renal vasodilation. However, the involvement of central alpha1-adrenoceptors on diuresis and natriuresis to central moxonidine was not investigated yet. In the present study, the effects of moxonidine, alpha-methylnoradrenaline (alpha2-adrenoceptor agonist) or phenylephrine (alpha1-adrenoceptor agonist) alone or combined with previous injections of prazosin (alpha1-adrenoceptor antagonist), yohimbine or RX 821002 (alpha2-adrenoceptor antagonists) intracerebroventricularly (i.c.v.) on urinary sodium, potassium and volume were investigated. Male Holtzman rats (n = 5-18/group) with stainless steel cannula implanted into the lateral ventricle and submitted to gastric water load (10% of body weight) were used. Injections of moxonidine (20 nmol) or alpha-methylnoradrenaline (80 nmol) i.c.v. induced natriuresis (196 +/- 25 and 171 +/- 30, respectively, vs. vehicle: 101 +/- 9 microEq/2 h) and diuresis (9.0 +/- 0.4 and 12.3 +/- 1.6, respectively, vs. vehicle: 5.2 +/- 0.5 ml/2 h). Pre-treatment with prazosin (320 nmol) i.c.v. abolished the natriuresis (23 +/- 4 and 76 +/- 11 microEq/2 h, respectively) and diuresis (5 +/- 1 and 7.6 +/- 0.8 ml/2 h, respectively) produced by i.c.v. moxonidine or alpha-methylnoradrenaline. RX 821002 (320 nmol) i.c.v. abolished the natriuretic effect of alpha-methylnoradrenaline, however, yohimbine (320 nmol) did not change renal responses to moxonidine. Phenylephrine (80 nmol) i.c.v. induced natriuresis and kaliuresis that were blocked by prazosin. Therefore, the present data suggest that moxonidine and alpha-methylnoradrenaline acting on central imidazoline receptors and alpha2-adrenoceptors, respectively, activate central alpha1-adrenergic mechanisms to

  18. Antagonism of apomorphine-enhanced startle by alpha 1-adrenergic antagonists. (United States)

    Davis, M; Kehne, J H; Commissaris, R L


    The present study investigated the possible involvement of central noradrenergic neurons in mediating the excitatory effect of the dopamine agonist apomorphine on the acoustic startle response in rats. Experiment 1 assessed the effects of intraperitoneal (i.p.) administration of adrenergic antagonists on apomorphine-enhanced startle. The excitation of startle produced by apomorphine (1.0-3.0 mg/kg i.p.) was blocked by the alpha 1-adrenergic antagonists prazosin (0.03-1.0 mg/kg) and WB-4101 (1.0 mg/kg). Prazosin was very potent in this regard, having an ED50 of 0.03 mg/kg. Blockade of beta-adrenergic receptors with propranolol (20 mg/kg) or blockade of peripheral alpha-adrenergic receptors with phentolamine (10 mg/kg) failed to alter the effect of apomorphine. Prazosin did not block the enhancement of startle produced by other drugs (5-methoxy-N,N-dimethyltryptamine, strychnine), nor did it alter the entry of apomorphine into the brain. The alpha 1-adrenergic antagonists piperoxane (0.03 mg/kg), yohimbine (0.03 mg/kg) or RX781094 (0.07 mg/kg) markedly potentiated apomorphine excitation. These data indicated that specific blockade of central alpha 1-adrenergic receptors prevents apomorphine-enhanced startle. In contrast to the effects of alpha 1-adrenergic antagonists, Experiment 2 found that other drugs that produce an acute (clonidine, 0.040 mg/kg) or chronic (intraventricular 6-hydroxydopamine, 2 X 200 micrograms; DSP4, 50 mg/kg i.p.) disruption of noradrenergic transmission failed to affect apomorphine excitation. Thus, the ability of alpha 1-adrenergic antagonists to block apomorphine's excitation of startle cannot be explained by a simple dopamine-norepinephrine interaction. Alternative hypothesis are discussed.

  19. Characterization of Peptides from Capsicum annuum Hybrid Seeds with Inhibitory Activity Against α-Amylase, Serine Proteinases and Fungi. (United States)

    Vieira Bard, Gabriela C; Nascimento, Viviane V; Ribeiro, Suzanna F F; Rodrigues, Rosana; Perales, Jonas; Teixeira-Ferreira, André; Carvalho, André O; Fernandes, Katia Valevski S; Gomes, Valdirene M


    Over the last several years, the activity of antimicrobial peptides (AMPs), isolated from plant species, against different microorganisms has been demonstrated. More recently, some of these AMPs have been described as potent inhibitors of α-amylases and serine proteinases from insects and mammals. The aim of this work was to obtain AMPs from protein extracts of a hybrid Capsicum (Ikeda × UENF 1381) seeds and to evaluate their microbial and enzyme inhibitory activities. Initially, proteins were extracted from the Capsicum hybrid seeds in buffer (sodium phosphate pH 5.4,) and precipitated with ammonium sulfate (90% saturated). Extract of hybrid seeds was subjected to size exclusion chromatography, and three fractions were obtained: S1, S2 and S3. The amino acid sequence, obtained by mass spectrometry, of the 6 kDa peptide from the S3 fraction, named HyPep, showed 100% identity with PSI-1.2, a serine protease inhibitor isolated from C. annuum seeds, however the bifunctionality of this inhibitor against two enzymes is being shown for the first time in this work. The S3 fraction showed the highest antifungal activity, inhibiting all the yeast strains tested, and it also exhibited inhibitory activity against human salivary and Callosobruchus maculatus α-amylases as well as serine proteinases.

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

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


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

  1. Inhibition of human ether-a-go-go-related gene potassium channels by alpha 1-adrenoceptor antagonists prazosin, doxazosin, and terazosin. (United States)

    Thomas, Dierk; Wimmer, Anna-Britt; Wu, Kezhong; Hammerling, Bettina C; Ficker, Eckhard K; Kuryshev, Yuri A; Kiehn, Johann; Katus, Hugo A; Schoels, Wolfgang; Karle, Christoph A


    Human ether-a-go-go-related gene (HERG) potassium channels are expressed in multiple tissues including the heart and adenocarcinomas. In cardiomyocytes, HERG encodes the alpha-subunit underlying the rapid component of the delayed rectifier potassium current, I(Kr), and pharmacological reduction of HERG currents may cause acquired long QT syndrome. In addition, HERG currents have been shown to be involved in the regulation of cell proliferation and apoptosis. Selective alpha 1-adrenoceptor antagonists are commonly used in the treatment of hypertension and benign prostatic hyperplasia. Recently, doxazosin has been associated with an increased risk of heart failure. Moreover, quinazoline-derived alpha 1-inhibitors induce apoptosis in cardiomyocytes and prostate tumor cells independently of alpha1-adrenoceptor blockade. To assess the action of the effects of prazosin, doxazosin, and terazosin on HERG currents, we investigated their acute electrophysiological effects on cloned HERG potassium channels heterologously expressed in Xenopus oocytes and HEK 293 cells.Prazosin, doxazosin, and terazosin blocked HERG currents in Xenopus oocytes with IC(50) values of 10.1, 18.2, and 113.2 microM respectively, whereas the IC(50) values for HERG channel inhibition in human HEK 293 cells were 1.57 microM, 585.1 nM, and 17.7 microM. Detailed biophysical studies revealed that inhibition by the prototype alpha 1-blocker prazosin occurred in closed, open, and inactivated channels. Analysis of the voltage-dependence of block displayed a reduction of inhibition at positive membrane potentials. Frequency-dependence was not observed. Prazosin caused a negative shift in the voltage-dependence of both activation (-3.8 mV) and inactivation (-9.4 mV). The S6 mutations Y652A and F656A partially attenuated (Y652A) or abolished (F656A) HERG current blockade, indicating that prazosin binds to a common drug receptor within the pore-S6 region. In conclusion, this study demonstrates that HERG

  2. Prevalence of S and Z alpha 1-antitrypsin mutations in patients with pancreatic diseases in Serbian population

    Directory of Open Access Journals (Sweden)

    Nikolić Aleksandra


    Full Text Available One of the key points in research of pancreatic disease pathology is further elucidation of the role of proteases and antiproteases, since their imbalance can lead to pancreatic injury. Alpha 1-antitrypsin (AAT is one of the most important serum inhibitors of proteolytic enzymes, including pancreatic enzymes trypsin, chymotrypsin and elastase. It is speculated that mutations in the AAT gene may influence the onset and the development of pancreatic disease. The presence of the most common AAT mutations Z and S was analyzed in 160 patients with pancreatic diseases (50 patients with pancreatic cancer, 50 patients with chronic pancreatitis and 60 patients with type 2 diabetes mellitus and 129 healthy individuals by PCR-mediated site-directed mutagenesis (PSM method. One patient with pancreatic cancer was found to be a carrier of Z mutation, as well as one patient with type 2 diabetes mellitus. One patient with chronic pancreatitis was found to be a carrier of S mutation. The common AAT mutations were statistically significantly over-represented in patients with pancreatic diseases (3 of 160 patients, allelic frequency 0.9% than in the control group (1 of 129 individuals, allelic frequency 0.4%. The results of this study, requiring confirmation, suggest that common AAT mutations Z and S may be associated with a modest increase in susceptibility to the development of pancreatic disease.

  3. Constraints on the Preferred-Frame {\\alpha}1, {\\alpha}2 parameters from Solar System planetary precessions

    CERN Document Server

    Iorio, Lorenzo


    Analytical expressions for the orbital precessions affecting the relative motion of the components of a local binary system induced by Lorentz-violating Preferred Frame Effects (PFE) are explicitly computed in terms of the PPN parameters {\\alpha}1, {\\alpha}2. A linear combination of the supplementary perihelion precessions of all the inner planets of the Solar System, able to remove the a-priori bias of unmodelled/mismodelled standard effects such as the general relativistic Lense-Thirring precessions and the classical rates due to the Sun's oblateness J2, allows to infer {\\alpha}1 <= 10^-6, {\\alpha}2 <= 10^-5. Such bounds should be improved in the near future after processing the data that are being collected by the MESSENGER spacecraft, currently orbiting Mercury. Further improvements may come in the mid-future from the approved BepiColombo mission to Mercury (Abridged).

  4. Apoptosis induction by doxazosin and other quinazoline alpha(1)-adrenoceptor antagonists: a new mechanism for cancer treatment?

    NARCIS (Netherlands)

    N. Kyprianou; T.B. Vaughan; M.C. Michel


    Doxazosin and related, quinazoline-based alpha(1)-adrenoceptor antagonists can induce apoptosis in prostate and various other normal, benign, smooth muscle, endothelial and malignant cells. Such apoptosis-inducing effects occur independently of alpha(1)-adrenoceptor antagonism and typically require

  5. Investigation of the genes for RET and its ligand complex, GDNF/GFR alpha-1, in small cell lung carcinoma

    NARCIS (Netherlands)

    Mulligan, LM; Timmer, T; Ivanchuk, SM; Campling, BG; Young, LC; Rabbitts, PH; Sundaresan, [No Value; Hofstra, RMW; Eng, C


    RET is a receptor tyrosine kinase expressed in neuroendocrine cells and in tumors of these cell types. RET activation may be mediated by a ligand complex comprising glial cell line-derived neurotrophic factor (GDNF) and GDNF family receptor alpha-1 (GFR alpha-1). Activating RET mutations are found i

  6. Change in lung function and morbidity from chronic obstructive pulmonary disease in alpha1-antitrypsin MZ heterozygotes

    DEFF Research Database (Denmark)

    Dahl, Morten; Tybjaerg-Hansen, Anne; Lange, Peter


    A deteriorating effect of severe alpha(1)-antitrypsin deficiency (ZZ genotype) on lung function is well known, whereas the role of intermediate deficiency (MZ genotype) remains uncertain.......A deteriorating effect of severe alpha(1)-antitrypsin deficiency (ZZ genotype) on lung function is well known, whereas the role of intermediate deficiency (MZ genotype) remains uncertain....

  7. Functional characterisation of the human alpha1 glycine receptor in a fluorescence-based membrane potential assay

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Kristiansen, Uffe


    be suppressed by pre-incubation with GlyR antagonists. Agonists and antagonists displayed EC50 and Ki values in good agreement with previously reported values from studies of recombinant alpha1 GlyRs and native alpha1beta GlyRs. The rank orders of potencies was glycine > beta-alanine > taurine for the agonists...

  8. Localization of type II collagen, long form alpha 1(IX) collagen, and short form alpha 1(IX) collagen transcripts in the developing chick notochord and axial skeleton. (United States)

    Swiderski, R E; Solursh, M


    In this study we compare, by in situ hybridization, the spatial and temporal expression patterns of transcripts of avian type II collagen and the long and short forms of the (alpha 1) chain of type IX collagen during the development of the notochord and axial skeleton. We observed type II collagen and short form type IX collagen transcripts in the developing (stage 25-28) nonchondrogenic notochord. Conversely, long form type IX transcripts were not detectable in the notochord or perinotochordal sheath. Interestingly, all three transcripts colocalized in the developing chondrogenic vertebrae of the axial skeleton as well as in the chondrocranium and Meckel's cartilage. The expression of the short form of type IX collagen in these regions was more restricted than that of the long form. This report provides additional support for a complex regulatory pathway of cartilage marker gene expression in chondrogenic vs. nonchondrogenic tissues during avian embryogenesis.

  9. Relationship between alpha-1 receptors and cations in rat liver plasma membranes

    Energy Technology Data Exchange (ETDEWEB)

    Smart, J.L.


    The influence of cations on binding of (/sup 3/H)-prazosin (PRZ), an alpha-1 specific antagonist, to alpha receptor sites in rat liver plasma membranes was examined. All cations tested were able to produce dose-dependent shifts to lower affinity binding sites for PRZ. The maximum number of binding sites was also observed to be altered. Inclusion of cations resulted in a slower observed rate constant for association as well as a delay in the dissociation of specifically bound PRZ following the addition of phentolamine. In contrast, the ability of (-)-norepinephrine to displace PRZ was enhanced by the addition of cations. The influence of alpha-1 receptor stimulation on Na/sup +//K/sup +/-ATPase activity in rat liver was examined by two methods - rat liver plasma membrane Na/sup +//K/sup +/-ATPase activity following liver perfusion in situ and /sup 86/Tb uptake in rat liver slices. The activity of the Na/sup +/ pump was found to be biphasic following exposure to phenylephrine (PE), an alpha-1 agonist. Stimulation (35%) was present over the first two minutes, while activity was inhibited over the interval of 5 to 10 minutes of continued PE exposure. Both phases were blocked by prazosin. The influence of DAG and protein kinase C (PKC) in alpha-1 receptor modulation of the Na/sup +/ pump was studied by employing 4-beta-phorbol (PMA), a phorbol ester which activates PKC. Perfusion of livers with PMA in situ or incubation with slices yielded inhibition of ATPase activity in membranes and /sup 86/Rb uptake in that was qualitatively and quantitatively similar to PE. These results suggest cations may influence receptor function in vivo and in vitro and the inhibitory effects of PE on the sodium pump may be mediated through PKC.

  10. The alpha1-adrenergic antagonist prazosin improves sleep and nightmares in civilian trauma posttraumatic stress disorder. (United States)

    Taylor, Fletcher; Raskind, Murray A


    Heightened noradrenergic reactivity may be a contributing factor in the pathophysiology of posttraumatic stress disorder (PTSD). Prazosin is an alpha1-adrenoceptor antagonist commonly used as an antihypertensive agent. Because alpha1-adrenergic activity has been associated with fear and startle responses, a drug that blocks central alpha1-adrenergic activity may be useful in the treatment of PTSD symptoms. An outpatient who had been exposed to civilian trauma and had subsequent chronic refractory PTSD was thus prescribed prazosin. The marked reduction in PTSD symptoms, particularly sleep and nightmares, prompted the following open-label feasibility trial. Five outpatients with non-combat-related PTSD were consecutively identified and received prazosin in a 6-week open-label trial. In each case, the prazosin doses were slowly increased until optimal benefit was achieved. Change was assessed with the Clinician-Administered PTSD Scale for DSM-IV, One Week Symptom Version (CAPS-SX), the Clinical Global Impression of Change Scale (CGIC), and the Clinical Impression of Change-Nightmares (CIC-Nightmares) score. All five patients experienced moderate to marked improvement on the CGIC. The CAPS-SX PTSD nightmare and sleep PTSD categories showed at least a four-point reduction of those symptoms. All patients reported at least moderate improvement on the CIC-Nightmare score. Optimal doses of prazosin ranged from 1 to 4 mg/day. The drug was reasonably tolerated, and there were no drug discontinuations. These preliminary findings provide a rationale for blind placebo-controlled efficacy trials of the alpha 1 antagonist prazosin for PTSD.

  11. The aspartic proteinase family of three Phytophthora species

    NARCIS (Netherlands)

    Kay, J.; Meijer, H.J.G.; Have, ten A.; Kan, van J.A.L.


    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

  12. An increased expression of Ca(2+) channel alpha(1A) subunit immunoreactivity in deep cerebellar neurons of rolling mouse Nagoya. (United States)

    Sawada, K; Sakata-Haga, H; Ando, M; Takeda, N; Fukui, Y


    Rolling mouse Nagoya (RMN) is an ataxic mutant and carries a mutation in the gene coding for the alpha(1A) subunit of the P/Q-type Ca(2+) channel. We examined the immunohistochemical expression of the alpha(1A) subunit in deep cerebellar nuclei of RMN. The antibody used recognized residues 865-883 of the mouse alpha(1A) subunit not overlapping the altered sequences in RMN. In RMN, many neurons exhibited definite alpha(1A) subunit-staining in the medial nucleus, interposed nucleus, and lateral nucleus of deep cerebellar nuclei. The number of positive neurons in these nuclei was significantly higher in RMN than in controls. Increased expression of the alpha(1A) subunit in deep cerebellar neurons might compensate for the altered function of the P/Q-type Ca(2+) channel of RMN.

  13. Issues in pharmaceutical development of thymosin alpha1 from preclinical studies through marketing. (United States)

    Tuthill, Cynthia


    SciClone Pharmaceuticals licensed the commercial and patent rights to thymosin alpha1, for geographical regions of the world excluding the United States and Europe, in the early 1990s. With this license, SciClone embarked on global drug development, and the issues encountered for thymosin alpha1 are reflective of the roller coaster of modern approval of pharmaceuticals. Most of the required toxicology studies had been completed prior to licensure, but some newer studies had to be conducted to obtain approvals in certain countries. The recent development of the "International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use" (ICH) guidelines allows for a clearer definition of the required battery of toxicology studies, although some countries still have not adopted these guidelines, and the local regulations have had to be understood and followed. Other hurdles include the complications that manufacturing requirements can differ between countries, and certain countries require local clinical experience trials in addition to SciClone's cumulative clinical data. A further obstacle was the pleiotropic nature of the mechanism of action of thymosin alpha1, with the resulting difficulty in the unraveling of its pharmacologic effects. With close attention to these regulatory details, SciClone has obtained approvals in more than 30 countries and has successfully begun commercial sales.

  14. Modification of certain pharmacological effects of ethanol by lipophilic alpha-1 adrenergic agonists

    Energy Technology Data Exchange (ETDEWEB)

    Menon, M.K.; Dinovo, E.C.; Haddox, V.G.


    The influence of four centrally-acting alpha-1 adrenoceptor agonists, namely, 2(2-chloro-5-trifluoromethylphenylimino) imidazolidine (St 587), cirazoline, (-) 1,2,3,4-tetrahydro-8-methoxy-5-methylthio-2-naphthalenamine ((-)SKF 89748A) and 2-(2-methylindazol-4-imino)imidazolidine (Sgd 101/75) on the pharmacological effects of ethanol was investigated. All four drugs reduced the duration of ethanol-induced hypnosis in C57B1/6 mice, this effect being proportional to their relative potencies to exert central alpha-1 agonism. In prazosin-pretreated mice, St 587 failed to reduce the hypnotic effect of ethanol, which provided strong evidence for the role of alpha-1 agonism for the hypnosis reducing effect of St 587. Hyperactivity induced in C57B1/6 mice by a subhypnotic dose of ethanol and St 587 was reported earlier. In the present study, St 587, cirazoline and (-)SKF 89748A produced similar response, but no correlation between this effect and ethanol hypnosis blockade could be established. 19 references, 8 figures, 2 tables.

  15. Alpha-1 adrenergic antagonists in aircrew for the treatment of benign prostatic hypertrophy. (United States)

    Matthies, Andrew K; Tachikawa, Nina J


    Benign prostatic hypertrophy (BPH) affects the majority of men later in life. Other than surgery, finasteride (Proscar) is currently the only pharmacologic option available for U.S. Air Force (USAF) aircrew. This article will evaluate the current literature regarding the treatment of benign prostatic hypertrophy with FDA approved tamsulosin (Flomax) and alfuzosin (Uroxatrol), third-generation alpha-1 adrenergic antagonists. Current literature supports the fact that some third-generation alpha blockers limit the side effects of hypotension when compared to other alpha blockers as a result of the specificity of subtype binding of the receptors and the sustained release formulation. Alpha blockers are currently used almost universally for the treatment of BPH; however, they are currently not approved for USAF aircrew. This article will review the aeromedical implications of the side effects of alpha-1 adrenergic antagonists (alfuzosin, tamsulosin), which affect aircrew while performing aeronautical duties, and examine whether alpha-1 adrenergic antagonists should be acceptable medications in certain situations depending on airframe and aeronautical duties.

  16. In silico analysis of alpha1-antitrypsin variants: the effects of a novel mutation

    Directory of Open Access Journals (Sweden)

    Sabri Denden


    Full Text Available Alpha1-antitrypsin (AAT is a highly polymorphic protein with more than 120 variants that are classified as normal (normal protein secretion, deficient (reduced circulating AAT level caused by defective secretion or null (no protein secretion. Alpha1-antitrypsin deficiency, one of the most common genetic disorders, predisposes adults to pulmonary emphysema and, to a lesser extent, chronic liver disease and cirrhosis. In this report, we provide additional sequence data for alpha1-antitrypsin based on the characterization of a novel variant detected in a 53-year-old heterozygous patient with chronic obstructive pulmonary disease. The mutation occurred on a PI*M2 base allele and was characterized by a T → C transition at nt 97 in exon II that led to the replacement of phenylalanine by leucine (F33L. Since the mutation was found in the heterozygous state with the expression of a normally secreted variant (PI*M1 it was not possible to assess the pattern of F33L secretion. However, computational analyses based on evolutionary, structural and functional information indicated a reduction of 23 ų in the side chain volume and the creation of a cavity in the protein hydrophobic core that likely disturbed the tridimensional structure and folding of AAT. The accuracy of the in silico prediction was confirmed by testing known mutations.

  17. Biosynthesis of mycobacterial arabinogalactan: identification of a novel alpha(1-->3) arabinofuranosyltransferase. (United States)

    Birch, Helen L; Alderwick, Luke J; Bhatt, Apoorva; Rittmann, Doris; Krumbach, Karin; Singh, Albel; Bai, Yu; Lowary, Todd L; Eggeling, Lothar; Besra, Gurdyal S


    The cell wall mycolyl-arabinogalactan-peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis and is the target of several antitubercular drugs. For instance, ethambutol targets arabinogalactan biosynthesis through inhibition of the arabinofuranosyltransferases Mt-EmbA and Mt-EmbB. A bioinformatics approach identified putative integral membrane proteins, MSMEG2785 in Mycobacterium smegmatis, Rv2673 in Mycobacterium tuberculosis and NCgl1822 in Corynebacterium glutamicum, with 10 predicted transmembrane domains and a glycosyltransferase motif (DDX), features that are common to the GT-C superfamily of glycosyltransferases. Deletion of M. smegmatis MSMEG2785 resulted in altered growth and glycosyl linkage analysis revealed the absence of AG alpha(1-->3)-linked arabinofuranosyl (Araf) residues. Complementation of the M. smegmatis deletion mutant was fully restored to a wild-type phenotype by MSMEG2785 and Rv2673, and as a result, we have now termed this previously uncharacterized open reading frame, arabinofuranosyltransferase C (aftC). Enzyme assays using the sugar donor beta-d-arabinofuranosyl-1-monophosphoryl-decaprenol (DPA) and a newly synthesized linear alpha(1-->5)-linked Ara(5) neoglycolipid acceptor together with chemical identification of products formed, clearly identified AftC as a branching alpha(1-->3) arabinofuranosyltransferase. This newly discovered glycosyltransferase sheds further light on the complexities of Mycobacterium cell wall biosynthesis, such as in M. tuberculosis and related species and represents a potential new drug target.

  18. The mechanism of noradrenergic alpha 1 excitatory modulation of pontine reticular formation neurons. (United States)

    Stevens, D R; McCarley, R W; Greene, R W


    The alpha 1 adrenergic receptor occurs in all major divisions of the CNS and is thought to play a role in all behaviors influenced by norepinephrine (NE). In the medial pontine reticular formation (mPRF), the proposed site of adrenergic enhancement of startle responses (Davis, 1984), alpha 1 agonists excite most neurons (Gerber et al., 1990). We here report that alpha 1 excitation results from a reduction of a voltage- and calcium-dependent potassium current, not previously recognized as ligand-modulated. The calcium sensitivity is suggested by its antagonism with Mg2+, Cd2+, Ba2+, low concentrations of tetraethylammonium, and charybdotoxin. The voltage sensitivity of this conductance falls within the membrane potential range critical to action potential generation. Based on this voltage sensitivity, the change in repetitive firing characteristics may be predicted according to a mathematical model of the mPRF neuronal electrophysiology. The predicted response to a 50% decrease in the phenylephrine (PE)-sensitive conductance is similar to the observed responses, with respect to both the current response under voltage-clamp conditions and alterations of the AHP and frequency/current curve. In contrast, modeling a reduction of a voltage-insensitive leak current predicts none of these changes. Thus, the noradrenergic reduction of this current depolarizes the membrane, increases the likelihood of an initial response to depolarizing input, and increases firing rate during sustained depolarization in a manner consistent with an NE role as an excitatory neuromodulator of the mPRF.

  19. Identification of a novel SERPINA-1 mutation causing alpha-1 antitrypsin deficiency in a patient with severe bronchiectasis and pulmonary embolism

    Directory of Open Access Journals (Sweden)

    Milger K


    Full Text Available Katrin Milger,1 Lesca Miriam Holdt,2 Daniel Teupser,2 Rudolf Maria Huber,1 Jürgen Behr,1 Nikolaus Kneidinger1 1Department of Internal Medicine V, University of Munich, Comprehensive Pneumology Center, Member of the German Center for Lung Research, 2Institute of Laboratory Medicine, University of Munich, Munich, Germany Abstract: Deficiency in the serine protease inhibitor, alpha-1 antitrypsin (AAT, is known to cause emphysema and liver disease. Other manifestations, including airway disease or skin disorders, have also been described. A 44-year-old woman presented to our emergency department with dyspnea and respiratory insufficiency. She had never smoked, and had been diagnosed with COPD 9 years earlier. Three months previously, she had suffered a pulmonary embolism. Chest computed tomography scan revealed severe cystic bronchiectasis with destruction of the lung parenchyma. The sweat test was normal and there was no evidence of the cystic fibrosis transmembrane conductance regulator (CFTR mutation. Capillary zone electrophoresis showed a decrease of alpha-1 globin band and AAT levels were below the quantification limit (<25 mg/dL. No S or Z mutation was identified, but sequencing analysis found a homozygous cytosine and adenine (CA insertion in exon 2 of the SERPINA-1 gene, probably leading to a dysfunctional protein (PI Null/Null. This mutation has not been previously identified. The atypical presentation of the patient, with severe cystic bronchiectasis, highlights AAT deficiency as a differential diagnosis in bronchiectasis. Further, awareness should be raised regarding a possible increased risk of thromboembolism associated with AAT deficiency. Keywords: alpha-1 antitrypsin deficiency, bronchiectasis, SERPINA-1 mutation, pulmonary embolism

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

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


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

  1. Trypanosoma cruzi: cruzipain and membrane-bound cysteine proteinase isoform(s) interacts with human alpha(2)-macroglobulin and pregnancy zone protein. (United States)

    Ramos, Adrián M; Duschak, Vilma G; Gerez de Burgos, Nelia M; Barboza, Mariana; Remedi, María S; Vides, Miguel A; Chiabrando, Gustavo A


    Plasmatic levels of pregnancy zone protein (PZP) increase in children with acute Chagas disease. PZP, as well as alpha2-macroglobulin (alpha2-M), are able to interact with Trypanosoma cruzi proteinases. The interaction of alpha2-M and PZP with cruzipain, the major cysteine proteinase of T. cruzi, was investigated. Several molecular changes on both alpha-M inhibitors under reaction with cruzipain were found. PAGE analysis showed: (i) formation of complexes of intermediate mobility and tetramerization of native alpha2-M and PZP, respectively; (ii) limited proteolysis of bait region in alpha2-M and PZP, and (iii) covalent binding of cruzipain to PZP and alpha2-M. Conformational and structural changes experimented by alpha-Ms correlate with modifications of the enzyme electrophoretic mobility and activity. Cruzipain-alpha-M complexes were also detected by gelatin SDS-PAGE and immunoblotting using polyclonal anti-cruzipain antibodies. Concomitantly, alpha2-M and PZP impaired the activity of cruzipain towards Bz-Pro-Phe-Arg-pNA substrate. In addition, alpha-Ms were able to form covalent complexes with membrane isoforms of cysteine proteinases cross-reacting with cruzipain. The present study suggests that both human alpha-macroglobulin inhibitors could prevent or minimize harmful action of cruzipain on host's molecules and hypothetically regulate parasite functions controlled by cruzipain.

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

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    Gustavo Coelho Correa


    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

  3. Sequestration of mutated alpha1-antitrypsin into inclusion bodies is a cell-protective mechanism to maintain endoplasmic reticulum function. (United States)

    Granell, Susana; Baldini, Giovanna; Mohammad, Sameer; Nicolin, Vanessa; Narducci, Paola; Storrie, Brian; Baldini, Giulia


    A variant alpha1-antitrypsin with E342K mutation has a high tendency to form intracellular polymers, and it is associated with liver disease. In the hepatocytes of individuals carrying the mutation, alpha1-antitrypsin localizes both to the endoplasmic reticulum (ER) and to membrane-surrounded inclusion bodies (IBs). It is unclear whether the IBs contribute to cell toxicity or whether they are protective to the cell. We found that in hepatoma cells, mutated alpha1-antitrypsin exited the ER and accumulated in IBs that were negative for autophagosomal and lysosomal markers, and contained several ER components, but not calnexin. Mutated alpha1-antitrypsin induced IBs also in neuroendocrine cells, showing that formation of these organelles is not cell type specific. In the presence of IBs, ER function was largely maintained. Increased levels of calnexin, but not of protein disulfide isomerase, inhibited formation of IBs and lead to retention of mutated alpha1-antitrypsin in the ER. In hepatoma cells, shift of mutated alpha1-antitrypsin localization to the ER by calnexin overexpression lead to cell shrinkage, ER stress, and impairment of the secretory pathway at the ER level. We conclude that segregation of mutated alpha1-antitrypsin from the ER to the IBs is a protective cell response to maintain a functional secretory pathway.

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

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


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

  5. Characterization of a cloned subtilisin-like serine proteinase from a psychrotrophic Vibrio species. (United States)

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


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

  6. Evaluation of alpha 1-antitrypsin and the levels of mRNA expression of matrix metalloproteinase 7, urokinase type plasminogen activator receptor and COX-2 for the diagnosis of colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Luis Bujanda

    Full Text Available BACKGROUND: Colorectal cancer (CRC is the second most common cause of death from cancer in both men and women in the majority of developed countries. Molecular tests of blood could potentially provide this ideal screening tool. AIM: Our objective was to assess the usefulness of serum markers and mRNA expression levels in the diagnosis of CRC. METHODS: In a prospective study, we measured mRNA expression levels of 13 markers (carbonic anhydrase, guanylyl cyclase C, plasminogen activator inhibitor, matrix metalloproteinase 7 (MMP7, urokinase-type plasminogen activator receptor (uPAR, urokinase-type plasminogen activator, survivin, tetranectin, vascular endothelial growth factor (VEGF, cytokeratin 20, thymidylate synthase, cyclooxygenase 2 (COX-2, and CD44 and three proteins in serum (alpha 1 antitrypsin, carcinoembryonic antigen (CEA and activated C3 in 42 patients with CRC and 33 with normal colonoscopy results. RESULTS: Alpha 1-antitrypsin was the serum marker that was most useful for CRC diagnosis (1.79 ± 0.25 in the CRC group vs 1.27 ± 0.25 in the control group, P<0.0005. The area under the ROC curve for alpha 1-antitrypsin was 0.88 (0.79-0.96. The mRNA expression levels of five markers were statistically different between CRC cases and controls: those for which the ROC area was over 75% were MMP7 (0.81 and tetranectin (0.80, COX-2 (0.78, uPAR (0.78 and carbonic anhydrase (0.77. The markers which identified early stage CRC (Stages I and II were alpha 1-antitrypsin, uPAR, COX-2 and MMP7. CONCLUSIONS: Serum alpha 1-antitrypsin and the levels of mRNA expression of MMP7, COX-2 and uPAR have good diagnostic accuracy for CRC, even in the early stages.

  7. Oral phentolamine: an alpha-1, alpha-2 adrenergic antagonist for the treatment of erectile dysfunction. (United States)

    Goldstein, I


    Phentolamine mesylate is an alpha-1 and alpha-2 selective adrenergic receptor antagonist which has undergone clinical trials for erectile dysfunction treatment. Biochemical and physiological studies in human erectile tissue have revealed a high affinity of phentolamine for alpha-1 and alpha-2 adrenergic receptors. Based on pharmacokinetic studies, it is suggested that 30-40 min following oral ingestion of 40 or 80 mg of phentolamine (Vasomax), the mean plasma phentolamine concentrations are sufficient to occupy the alpha-1 and -2 adrenergic receptors in erectile tissue and thereby result in inhibition of adrenergic-mediated physiologic activity. In large multi-center, placebo-controlled pivotal phase III clinical trials, the mean change in the erectile function domain of the International Index of Erectile Function scores (Questions 1-5 and 15) from screening to the end of treatment was significantly higher following use of active drug (40 mg and 80 mg) compared to placebo. Three to four times as many patients receiving phentolamine reported being satisfied or very satisfied compared with those receiving placebo. At doses of 40 mg and 80 mg respectively, 55% and 59% of men were able to achieve vaginal penetration with 51% and 53% achieving penetration on 75% of attempts. The correction of erectile dysfunction or improvement to a less severe category of dysfunction was experienced by 53% of men with the 80 mg dose and 40% with the 40 mg dose of phentolamine. All trends of response were the same regardless of any concomitant medication. There were no severe adverse events. At 40 mg, 7.7% experienced rhinitis and fewer than 3.1% experienced any other side effect of treatment. Phentolamine is safe, well tolerated and efficacious for the treatment of erectile dysfunction.

  8. (/sup 125/Iodo)BE 2254, a new radioligand for alpha 1-adrenoceptors

    Energy Technology Data Exchange (ETDEWEB)

    Engel, G.; Hoyer, D.


    (/sup 125/cIodo)2-(beta-(14-hydroxyphenyl)-ethyl-aminomethyl)-tetralone((/sup 125/Iodo)BE 2254 or IBE 2254), a new iodinated radioligand of high specific radioactivity (2175 Ci/mmol), was synthesized and used to characterize alpha 1-adrenoceptors in rat lung and cerebral cortex membranes. The binding constants of IBE 2254, using rat lung and cortex membranes, were Kd . 53 +/- 10 pM, Bmax . 53 +/- 8 fmol/mg; and Kd . 78 +/- 14 pM, Bmas . 210 +/- 26 fmol/mg, respectively (Kd . dissociation constant of IBE 2254 determined in saturation experiments). In equilibrium binding experiments with IBE 2254, at concentrations of the free ligand up to 1.2 nM, only one class of binding sites could be detected. In kinetic experiments, the association and dissociation rate constants were 2.3 X 10(9) M-1 min-1 and 0.10 min-1, respectively. In rat cerebral cortex membranes, alpha-adrenoceptor antagonists competed for IBE 2254 binding in the following order: prazosin greater than BE 2254 greater than WB 4101 greater than phentolamine greater than corynanthine greater than yohimbine greater than rauwolscine, indicating strongly that IBE 2254 binds to alpha 1-adrenoceptors. The calculated affinities of different alpha-adrenoceptor blocking agents from inhibition of IBE 2254 binding were nearly identical in rat lung and cerebral cortex. The low dissociation constant of the ligand together with its high specific radioactivity allows binding studies to be carried out with tissue samples where only small densities of alpha 1-adrenoceptors are present.

  9. Diaphragm arterioles are less responsive to alpha1- adrenergic constriction than gastrocnemius arterioles. (United States)

    Aaker, Aaron; Laughlin, M H


    The sympathetic nervous system has greater influence on vascular resistance in low-oxidative, fast-twitch skeletal muscle than in high-oxidative skeletal muscle (17). The purpose of this study was to test the hypothesis that arterioles isolated from low-oxidative, fast-twitch skeletal muscle [the white portion of gastrocnemius (WG)] possess greater responsiveness to adrenergic constriction than arterioles isolated from high-oxidative skeletal muscle [red portion of the gastrocnemius muscle (RG) and diaphragm (Dia)]. Second-order arterioles (2As) were isolated from WG, RG, and Dia of rats and reactivity examined in vitro. Results reveal that Dia 2As constrict less to norepinephrine (NE) (10(-9) to 10 (-4) M) than 2As from RG and WG, which exhibited similar NE-induced constrictions. This difference was not endothelium dependent, because responses of denuded 2As were similar to those of intact arterioles. The blunted NE-induced constrictor response of Dia 2As appears to be the result of differences in alpha1-receptor effects because 1) arterioles from Dia also responded less to selective alpha1-receptor stimulation with phenylephrine than RG and WG arterioles; 2) arterioles from Dia, RG, and WG dilated similarly to isoproterenol (10(-9) to 10(-4) M) and did not respond to selective alpha2-receptor stimulation with UK-14304; and 3) endothelin-1 produced similar constriction in 2As from Dia, RG, and WG. We conclude that differences in oxidative capacity and/or fiber type composition of muscle tissue do not explain different NE responsiveness of Dia 2As compared with 2As from gastrocnemius muscle. Differences in alpha1-adrenergic constrictor responsiveness among arterioles in skeletal muscle may contribute to nonuniform muscle blood flow responses observed during exercise and serve to maintain blood flow to Dia during exercise-induced increases in sympathetic nerve activity.

  10. Indications for active case searches and intravenous alpha-1 antitrypsin treatment for patients with alpha-1 antitrypsin deficiency chronic pulmonary obstructive disease: an update. (United States)

    Casas, Francisco; Blanco, Ignacio; Martínez, María Teresa; Bustamante, Ana; Miravitlles, Marc; Cadenas, Sergio; Hernández, José M; Lázaro, Lourdes; Rodríguez, Esther; Rodríguez-Frías, Francisco; Torres, María; Lara, Beatriz


    The effect of hereditary alpha-1 antitrypsin (AAT) deficiency can manifest clinically in the form of chronic obstructive pulmonary disease (COPD). AAT deficiency (AATD) is defined as a serum concentration lower than 35% of the expected mean value or 50 mg/dl (determined by nephelometry). It is associated in over 95% of cases with Pi*ZZ genotypes, and much less frequently with other genotypes resulting from combinations of Z, S, rare and null alleles. A systematic qualitative review was made of 107 articles, focusing mainly on an active search for AATD in COPD patients and intravenous (iv) treatment with AAT. On the basis of this review, the consultant committee of the Spanish Registry of Patients with AATD recommends that all COPD patients be screened for AATD with the determination of AAT serum concentrations, and when these are low, the evaluation must be completed with phenotyping and, on occasions, genotyping. Patients with severe AATD COPD should receive the pharmacological and non-pharmacological treatment recommended in the COPD guidelines. There is enough evidence from large observational studies and randomized placebo-controlled clinical trials to show that the administration of iv AAT reduces mortality and slows the progression of emphysema, hence its indication in selected cases that meet the inclusion criteria stipulated in international guidelines. The administration of periodic infusions of AAT is the only specific treatment for delaying the progression of emphysema associated with AATD.

  11. Renal and cardiac function during alpha1-beta-blockade in congestive heart failure

    DEFF Research Database (Denmark)

    Heitmann, M; Davidsen, U; Stokholm, K H


    The kidney and the neurohormonal systems are essential in the pathogenesis of congestive heart failure (CHF) and the physiologic response. Routine treatment of moderate to severe CHF consists of diuretics, angiotensin-converting enzyme (ACE) inhibition and beta-blockade. The need for control...... of renal function during initiation of ACE-inhibition in patients with CHF is well known. The aim of this study was to investigate whether supplementation by a combined alpha1-beta-blockade to diuretics and ACE-inhibition might improve cardiac function without reducing renal function....

  12. A Tumor-Penetrating Peptide Modification Enhances the Antitumor Activity of Thymosin Alpha 1


    Xingzhen Lao; Meng Liu; Jiao Chen; Heng Zheng


    A serious limitation of numerous antitumor drugs is the incapacity to penetrate solid tumors. However, addition of an RGD fragment to peptide drugs might solve this problem. In this study, we explored whether the introduction of a permeability-enhancing sequence, such as iRGD (CRGDK/RGPD/EC) fragments, would enhance the activity of thymosin alpha 1 (Tα1). The modified Tα1 (Tα1-iRGD) was successfully expressed and purified, and the in vitro assay showed that Tα1-iRGD presented a similar activi...

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

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    Araki Hiromasa


    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

  14. 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


    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.

  15. The catalytic mechanism of an aspartic proteinase explored with neutron and X-ray diffraction

    Energy Technology Data Exchange (ETDEWEB)

    Kovalevsky, Andrey [Los Alamos National Laboratory (LANL); Erskine, Peter T. [University of Southampton, England; Cooper, Jon [University of Southampton, England


    Hydrogen atoms play key roles in enzyme mechanism, but as this study shows, even high-quality X-ray data to a resolution of 1 {angstrom} cannot directly visualize them. Neutron diffraction, however, can locate deuterium atoms even at resolutions around 2 {angstrom}. Both neutron and X-ray diffraction data have been used to investigate the transition state of the aspartic proteinase endothiapepsin. The different techniques reveal a different part of the story, revealing the clearest picture yet of the catalytic mechanism by which the enzyme operates. Room temperature neutron and X-ray diffraction data were used in a newly developed joint refinement software package to visualize deuterium atoms within the active site of the enzyme when a gem-diol transition state analogue inhibitor is bound at the active site. These data were also used to estimate their individual occupancy, while analysis of the differences between the bond lengths of the catalytic aspartates was performed using atomic resolution X-ray data. The two methods are in agreement on the protonation state of the active site with a transition state analogue inhibitor bound confirming the catalytic mechanism at which the enzyme operates.

  16. Cystatin F regulates proteinase activity in IL-2-activated natural killer cells. (United States)

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


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

  17. Proteinases of Streptomyces fradiae. I. Preliminary characterization and purification. (United States)

    Galas, E; Kaluzewska, T


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

  18. Dispersal of Bap-mediated Staphylococcus aureus biofilm by proteinase K. (United States)

    Kumar Shukla, Sudhir; Rao, Toleti Subba


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

  19. laminin alpha 1 gene is essential for normal lens development in zebrafish

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    Bosenko Dmitry V


    Full Text Available Abstract Background Laminins represent major components of basement membranes and play various roles in embryonic and adult tissues. The functional laminin molecule consists of three chains, alpha, beta and gamma, encoded by separate genes. There are twelve different laminin genes identified in mammals to date that are highly homologous in their sequence but different in their tissue distribution. The laminin alpha -1 gene was shown to have the most restricted expression pattern with strong expression in ocular structures, particularly in the developing and mature lens. Results We identified the zebrafish lama1 gene encoding a 3075-amino acid protein (lama1 that possesses strong identity with the human LAMA1. Zebrafish lama1 transcripts were detected at all stages of embryo development with the highest levels of expression in the developing lens, somites, nervous and urogenital systems. Translation of the lama1 gene was inhibited using two non-overlapping morpholino oligomers that were complementary to sequences surrounding translation initiation. Morphant embryos exhibited an arrest in lens development and abnormalities in the body axis length and curvature. Conclusion These results underline the importance of the laminin alpha 1 for normal ocular development and provide a basis for further analysis of its developmental roles.

  20. Impact of alpha 1-adrenergic antagonist use for benign prostatic hypertrophy on outcomes in patients with heart failure. (United States)

    Dhaliwal, Amandeep S; Habib, Gabriel; Deswal, Anita; Verduzco, Melinda; Souchek, Julianne; Ramasubbu, Kumudha; Aguilar, David; Ma, Tony S; Jneid, Hani M; Bolos, Mariana; Bozkurt, Biykem


    Previous clinical trials have shown that alpha(1)-adrenergic antagonists are not effective in subjects with heart failure (HF) and might increase HF rates when used for hypertension. However, alpha(1)-adrenergic antagonists may be prescribed to subjects with HF who have symptomatic benign prostatic hyperplasia. We sought to determine any association between alpha(1)-adrenergic antagonist use, commonly prescribed for benign prostatic hyperplasia, and the clinical outcomes of subjects with HF receiving contemporary therapy. An existing database of 388 subjects with decompensated HF admissions from 2002 to 2004 at the Veterans Affairs Hospital was analyzed according to the use of alpha(1)-adrenergic antagonists at discharge. Covariate-adjusted Cox proportional hazard models were used to examine any association with future admissions for decompensated HF and total mortality. Alpha-1-adrenergic antagonist therapy was prescribed in 25% of our HF population, predominantly for benign prostatic hyperplasia, and was not associated with significant increases in the combined risk of all-cause mortality and rehospitalization for HF (hazard ratio 1.24, 95% confidence interval 0.93 to 1.65, p = 0.14), HF hospitalization (hazard ratio 1.20, 95% confidence interval 0.85 to 1.70, p = 0.31), or all-cause mortality (hazard ratio 1.10, 95% confidence interval 0.78 to 1.56, p = 0.57). In patients not receiving beta-blocker therapy, alpha(1)-adrenergic antagonist therapy was significantly associated with increased HF hospitalizations (hazard ratio 1.94, 95% confidence interval 1.14 to 3.32, p = 0.015). In conclusion, in patients with chronic HF, the use of alpha(1)-adrenergic antagonists was significantly associated with more HF hospitalizations when prescribed without concomitant beta blockade. Thus, background beta-blocker therapy appears to be protective against the potential harmful effects of alpha(1)-adrenergic antagonist therapy in patients with HF.

  1. VaSP1, catalytically active serine proteinase from Vipera ammodytes ammodytes venom with unconventional active site triad. (United States)

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


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

  2. Elevated levels of serum antibodies against alpha-1, 6-glucan in patients with systemic lupus erythematosus or rheumatoid arthritis

    Institute of Scientific and Technical Information of China (English)

    Hui Dai; Xiao-Ming Gao


    This study was undertaken to investigate whether levels of anti-alpha-1,6-glucan antibodies in human sera correlate with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE).Serum samples were collected from patients with SLE (n =30),RA (n =30) and healthy adult volunteers.IgG,IgA and IgM levels against alpha-1,6-glucan were measured using enzyme linked immunosorbent assays.Anti-alpha-1,6-glucan IgG prevalence was raised in patients with active SLE (73.3%)and RA (60%) compared with healthy controls (13.3%).Strong correlation between anti-alpha-1,6-glucan-IgG levels and anti-perinuclear factor (r =0.642; p< 0.05) in RA patients or anti-nuclear antibodies (r =0.675; p < 0.05)in SLE patients was observed.No significant differences in anti-alpha-1,6-glucan-lgA or-lgM levels were noted between different groups.We conclude that anti-alpha1,6-glucan-lgG levels were significantly elevated in patients with SLE or RA and positively correlated with disease activity.

  3. Tertiary structure of human alpha1-acid glycoprotein (orosomucoid). Straightforward fluorescence experiments revealing the presence of a binding pocket. (United States)

    Albani, Jihad R


    Binding of hemin to alpha1-acid glycoprotein has been investigated. Hemin binds to the hydrophobic pocket of hemoproteins. The fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonate (TNS) binds to a hydrophobic domain in alpha1-acid glycoprotein with a dissociation constant equal to 60 microM. Addition of hemin to an alpha1-acid glycoprotein-TNS complex induces the displacement of TNS from its binding site. At saturation (1 hemin for 1 protein) all the TNS has been displaced from its binding site. The dissociation constant of hemin-alpha1-acid glycoprotein was found equal to 2 microM. Thus, TNS and hemin bind to the same hydrophobic site: the pocket of alpha1-acid glycoprotein. Energy-transfer studies performed between the Trp residues of alpha1-acid glycoprotein and hemin indicated that efficiency (E) of Trp fluorescence quenching was equal to 80% and the Förster distance, R0 at which the efficiency of energy transfer is 50% was calculated to be 26 A, revealing a very high energy transfer.

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

    Directory of Open Access Journals (Sweden)

    Goldman Maria Helena S.


    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.

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

    Directory of Open Access Journals (Sweden)

    Dümen Emek


    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. .

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

    Energy Technology Data Exchange (ETDEWEB)



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

  7. A new crystal form of proteinase A, a non-pepsin-type acid proteinase from Aspergillus niger var. macrosporus. (United States)

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


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

  8. Introduction of the human pro. cap alpha. 1(I) collagen gene into pro. cap alpha. 1(I)-deficient Mov-13 mouse cells leads to formation of functional mouse-human hybrid type I collagen

    Energy Technology Data Exchange (ETDEWEB)

    Schnieke, A.; Dziadek, M.; Bateman, J.; Mascara, T.; Harbers, K.; Gelinas, R.; Jaenisch, R.


    The Mov-13 mouse strain carries a retroviral insertion in the pro..cap alpha..1(I) collagen gene that prevents transcription of the gene. Cell lines derived from homozygous embryos do not express type I collagen although normal amounts of pro..cap alpha..2 mRNA are synthesized. The authors have introduced genomic clones of either the human or mouse pro..cap alpha..1(I) collagen gene into homozygous cell lines to assess whether the human or mouse pro..cap alpha..1(I) chains can associate with the endogenous mouse pro..cap alpha..2(I) chain to form stable type I collagen. The human gene under control of the simian virus 40 promoter was efficiently transcribed in the transfected cells. Protein analyses revealed that stable heterotrimers consisting of two human ..cap alpha..1 chains and one mouse ..cap alpha..2 chain were formed and that type I collagen was secreted by the transfected cells at normal rates. However, the electrophoretic migration of both ..cap alpha..1(I) and ..cap alpha..2(I) chains in the human-mouse hybrid molecules were retarded, compared to the ..cap alpha..(I) chains in control mouse cells. Inhibition of the posttranslational hydroxylation of lysine and proline resulted in comigration of human and mouse ..cap alpha..1 and ..cap alpha..2 chains, suggesting that increased posttranslational modification caused the altered electrophoretic migration in the human-mouse hybrid molecules. Amino acid sequence differences between the mouse and human ..cap alpha.. chains may interfere with the normal rate of helix formation and increase the degree of posttranslational modifications similar to those observed in patients with lethal perinatal osteogenesis imperfecta. The Mov-13 mouse system should allow the authors to study the effect specific mutations introduced in transfected pro..cap alpha..1(I) genes have on the synthesis, assembly, and function of collagen I.

  9. Involvement of central alpha1- and alpha2-adrenoceptors on cardiovascular responses to moxonidine. (United States)

    Moreira, Thiago S; Takakura, Ana C; Menani, José V; Colombari, Eduardo


    In the present study we compared the effects produced by moxonidine (alpha2-adrenoceptor/imidazoline agonist) injected into the 4th cerebral ventricle and into the lateral cerebral ventricle on mean arterial pressure, heart rate and on renal, mesenteric and hindquarter vascular resistances, as well as the possible action of moxonidine on central alpha1- or alpha2-adrenoceptors to produce cardiovascular responses. Male Holtzman rats (n=7-8) anesthetized with urethane (0.5 g/kg, intravenously-i.v.) and alpha-chloralose (60 mg/kg, i.v.) were used. Moxonidine (5, 10 and 20 nmol) injected into the 4th ventricle reduced arterial pressure (-19+/-5, -30+/-7 and -43+/-8 mmHg vs. vehicle: 2+/-4 mmHg), heart rate (-10+/-6, -16+/-7 and -27+/-9 beats per minute-bpm, vs. vehicle: 4+/-5 bpm), and renal, mesenteric and hindquarter vascular resistances. Moxonidine (5, 10 and 20 nmol) into the lateral ventricle only reduced renal vascular resistance (-77+/-17%, -85+/-13%, -89+/-10% vs. vehicle: 3+/-4%), without changes on arterial pressure, heart rate and mesenteric and hindquarter vascular resistances. Pre-treatment with the selective alpha2-adrenoceptor antagonist yohimbine (80, 160 and 320 nmol) injected into the 4th ventricle attenuated the hypotension (-32+/-5, -25+/-4 and -12+/-6 mmHg), bradycardia (-26+/-11, -23+/-5 and -11+/-6 bpm) and the reduction in renal, mesenteric and hindquarter vascular resistances produced by moxonidine (20 nmol) into the 4th ventricle. Pre-treatment with yohimbine (320 nmol) into the lateral ventricle did not change the renal vasodilation produced by moxonidine (20 nmol) into the lateral ventricle. The alpha1-adrenoceptor antagonist prazosin (320 nmol) injected into the 4th ventricle did not affect the cardiovascular effects of moxonidine. However, prazosin (80, 160 and 320 nmol) into the lateral ventricle abolished the renal vasodilation (-17+/-4, -6+/-9 and 2+/-11%) produced by moxonidine. The results indicate that the decrease in renal vascular

  10. A complete alpha1,3-galactosyltransferase gene is present in the human genome and partially transcribed. (United States)

    Lantéri, Marion; Giordanengo, Valérie; Vidal, Frédérique; Gaudray, Patrick; Lefebvre, Jean-Claude


    The synthesis of Galalpha1-3Gal-terminated oligosaccharides (alpha-Gal) epitopes has been interrupted during the course of evolution, starting with Old World primates. Partial sequences similar to the alpha1,3-galactosyltransferase (alpha1,3GalT) gene, which governs the synthesis of alpha-Gal epitopes, have been detected in the human genome and were found to correspond to pseudogenes. We completed the sequence of the human alpha1,3GalT pseudogene present on chromosome 9 and found it to be organized like the murine alpha1,3GalT gene. In human cell lines and several normal and tumor tissues we detected truncated transcripts corresponding to this pseudogene. Considering these mRNAs, translation of an open reading frame containing the first four translated exons but missing the two catalytic exons could predict a truncated alpha1,3GalT polypeptide that should be enzymatically inactive. We show that transcription of human alpha1,3GalT is prematurely terminated at the level of a strong transcriptional stop signal in the middle of intron VII. We were able to reproduce this effect in vitro by subcloning the implicated DNA region upstream from a reporter cDNA. The premature transcriptional arrest of human alpha1,3-GalT gene leads to an ectopic splicing event and to the connection of a short intronic sequence downstream from translated exons. Finally, we show that these truncated transcripts are overexpressed in cell lines with modifications of O-glycans.

  11. Alpha1A-adrenergic receptor-directed autoimmunity induces left ventricular damage and diastolic dysfunction in rats.

    Directory of Open Access Journals (Sweden)

    Katrin Wenzel

    Full Text Available BACKGROUND: Agonistic autoantibodies to the alpha(1-adrenergic receptor occur in nearly half of patients with refractory hypertension; however, their relevance is uncertain. METHODS/PRINCIPAL FINDINGS: We immunized Lewis rats with the second extracellular-loop peptides of the human alpha(1A-adrenergic receptor and maintained them for one year. Alpha(1A-adrenergic antibodies (alpha(1A-AR-AB were monitored with a neonatal cardiomyocyte contraction assay by ELISA, and by ERK1/2 phosphorylation in human alpha(1A-adrenergic receptor transfected Chinese hamster ovary cells. The rats were followed with radiotelemetric blood pressure measurements and echocardiography. At 12 months, the left ventricles of immunized rats had greater wall thickness than control rats. The fractional shortening and dp/dt(max demonstrated preserved systolic function. A decreased E/A ratio in immunized rats indicated a diastolic dysfunction. Invasive hemodynamics revealed increased left ventricular end-diastolic pressures and decreased dp/dt(min. Mean diameter of cardiomyocytes showed hypertrophy in immunized rats. Long-term blood pressure values and heart rates were not different. Genes encoding sarcomeric proteins, collagens, extracellular matrix proteins, calcium regulating proteins, and proteins of energy metabolism in immunized rat hearts were upregulated, compared to controls. Furthermore, fibrosis was present in immunized hearts, but not in control hearts. A subset of immunized and control rats was infused with angiotensin (Ang II. The stressor raised blood pressure to a greater degree and led to more cardiac fibrosis in immunized, than in control rats. CONCLUSIONS/SIGNIFICANCE: We show that alpha(1A-AR-AB cause diastolic dysfunction independent of hypertension, and can increase the sensitivity to Ang II. We suggest that alpha(1A-AR-AB could contribute to cardiovascular endorgan damage.

  12. Alpha-1 antitrypsin: a potent anti-inflammatory and potential novel therapeutic agent.

    LENUS (Irish Health Repository)

    Bergin, David A


    Alpha-1 antitrypsin (AAT) has long been thought of as an important anti-protease in the lung where it is known to decrease the destructive effects of major proteases such as neutrophil elastase. In recent years, the perception of this protein in this simple one dimensional capacity as an anti-protease has evolved and it is now recognised that AAT has significant anti-inflammatory properties affecting a wide range of inflammatory cells, leading to its potential therapeutic use in a number of important diseases. This present review aims to discuss the described anti-inflammatory actions of AAT in modulating key immune cell functions, delineate known signalling pathways and specifically to identify the models of disease in which AAT has been shown to be effective as a therapy.

  13. Alpha1-acid glycoprotein post-translational modifications: a comparative two dimensional electrophoresis based analysis

    Directory of Open Access Journals (Sweden)

    P. Roncada


    Full Text Available Alpha1-acid glycoprotein (AGP is an immunomodulatory protein expressed by hepatocytes in response to the systemic reaction that follows tissue damage caused by inflammation, infection or trauma. A proteomic approach based on two dimensional electrophoresis, immunoblotting and staining of 2DE gels with dyes specific for post-translational modifications (PTMs such as glycosylation and phosphorylation has been used to evaluate the differential interspecific protein expression of AGP purified from human, bovine and ovine sera. By means of these techniques, several isoforms have been identified in the investigated species: they have been found to change both with regard to the number of isoforms expressed under physiological condition and with regard to the quality of PTMs (i.e. different oligosaccharidic chains, presence/absence of phosphorilations. In particular, it is suggested that bovine serum AGP may have one of the most complex pattern of PTMs among serum proteins of mammals studied so far.

  14. Structure and characterisation of a duplicated human alpha 1 acid glycoprotein gene. (United States)

    Merritt, C M; Board, P G


    Human alpha 1-acid glycoprotein (AGP), also known as orosomucoid, is a major acute-phase plasma protein. The amino acid sequence of AGP, which was determined by sequencing from protein isolated from pooled plasma, contained amino acid substitutions in 21 different positions. Genomic and cDNA clones which correspond to one of the possible amino acid sequences have been previously reported. In this paper we present the complete nucleotide sequence of a second gene, AGP2 which is located approx. 3.3 kb downstream from AGP1. The derived amino acid sequence of AGP2 contains 19 of the possible alternative amino acid substitutions as well as two additional differences. It is clear from the results presented here that the AGP in human plasma is the product of two separate gene loci.

  15. Evolution of human alpha 1-acid glycoprotein genes and surrounding Alu repeats. (United States)

    Merritt, C M; Easteal, S; Board, P G


    There is a mosaic pattern of variation between the two tandemly arranged human alpha 1-acid glycoprotein genes. Both the synonymous and the nonsynonymous sites of exons 3 and 4 are more divergent than the rest of the gene, suggesting that they have had a different evolutionary history. Comparisons of the two gene sequences with rat AGP indicate that exons 3 and 4 of AGP2 have been evolving without functional constraint since their divergence from AGP1. It is proposed that the conserved region of the gene has been homogenized recently by gene conversion with the homologous regions of AGP1. The Alu sequences surrounding the genes appear to have been involved in both the gene duplication and the gene conversion events.

  16. Two lectin-like receptors for alpha 1-acid glycoprotein in mouse testis

    DEFF Research Database (Denmark)

    Andersen, U O; Kirkeby, S; Bøg-Hansen, T C


    Three glycoforms of alpha 1-acid glycoprotein (AGP) were biotinylated to examine their binding in mouse testis by light microscopy. The transition from one stage to another in the spermatogenic cycle is marked with an appearance of a receptor for the Concanavalin A (Con A) non-reactive glycoform...... AGP-A in the cytoplasm of spermatocytes, young spermatids and Sertoli cells. This receptor disappears in the late stages of the spermatids. The Con-A intermediately reactive and the Con-A reactive glycoforms, AGP-B and AGP-C, showed weak reaction in the cytoplasm of spermatocytes, spermatids....... AGP-A was inhibited by testosterone, oestradiol and progesterone, while AGP-B and AGP-C were inhibited by mannose, GlcNAc, cortisone, aldosterone, oestradiol and progesterone. The receptors and the corresponding AGP glycoforms may be adhesion molecules between Sertoli cells and the spermatogenic cells...

  17. [Purification of recombinant proteins with an example of tumor necrosis factor thymosin-alpha1]. (United States)

    Fedorov, T V; Korobov, V I; Nazarov, V G; Smolkina, A E; Shmelev, V A


    Hybrid protein, cancer necrosis factor thymosin-alpha1 (CNF-T), when synthesizing in strain-producer of Escherichia coli SG200-50 with plasmid pThy315, was a part of "inclusion bodies" mostly in the form of a high-molecular complex with other proteins due to the S-S bonds formation. An approach of purification of CNF-T has been proposed, which is based on the destruction of the complex in the presence of sodium dodecylsulfate (DDS-NA) and dithiotreitol (DDT) followed by gel-filtration on Sephadex G-100 and renaturation by ultrafiltration on hollow fibers. The method allows the isolation of electrophoretically homogeneous CNF-T containing no DDS-Na and having high cytotoxic activity against cancer cells of mouse adenocarcinome L-929. The yield of CNF-T achieved 80% relative its content in biomass and 30% relative the total protein.

  18. Production of human alpha-1-antitrypsin from transgenic rice cell culture in a membrane bioreactor. (United States)

    McDonald, Karen A; Hong, Lo Ming; Trombly, David M; Xie, Qing; Jackman, Alan P


    Transgenic plant cell cultures offer a number of advantages over alternative host expression systems, but so far relatively low product concentrations have been achieved. In this study, transgenic rice cells are used in a two-compartment membrane bioreactor (CELLine 350, Integra Biosciences) for the production of recombinant alpha-1-antitrypsin (rAAT). Expression of rAAT is controlled by the rice alpha-amylase (RAmy3D) promoter, which is induced in the absence of sugar. The extracellular product is retained in the bioreactor's relatively small cell compartment, thereby increasing product concentration. Due to the packed nature of the cell aggregates in the cell compartment, a clarified product solution can be withdrawn from the bioreactor. Active rAAT reached levels of 100-247 mg/L (4-10% of the total extracellular protein) in the cell compartment at 5-6 days postinduction, and multiple inductions of the RAmy3D promoter were demonstrated.

  19. Construction, Expression, and Characterization of Thymosin Alpha 1 Tandem Repeats in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Xiao-Chang Xue


    Full Text Available Thymosin alpha 1 (Tα1, which is composed of 28 amino acids, has been commercialized worldwide for its immune-modulatory and antitumor effects. Tα1 can stimulate T cell proliferation and differentiation from bone marrow stem cells, augment cell-mediated immune responses, and regulate homeostasis of immune system. In this study, we developed a novel strategy to produce Tα1 concatemer (Tα1 in Escherichia coli and compared its activity with chemically synthesized Tα1. Results showed that Tα1 can more effectively stimulate T cell proliferation and significantly upregulate IL-2 receptor expression. We concluded that the expression system for Tα1 concatemer was constructed successfully, which could serve as an efficient tool for the production of large quantities of the active protein.

  20. Ligand-specific conformational changes in the alpha1 glycine receptor ligand-binding domain

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Lynch, Joseph W


    indicate that channel opening is accompanied by conformational rearrangements in both beta-sheets. In an attempt to resolve ligand-dependent movements in the ligand-binding domain, we employed voltage-clamp fluorometry on alpha1 glycine receptors to compare changes mediated by the agonist, glycine......, and by the antagonist, strychnine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. In the inner beta-sheet, we labeled residues in loop 2 and in binding domain loops D and E....... At each position, strychnine and glycine induced distinct maximal fluorescence responses. The pre-M1 domain responded similarly; at each of four labeled positions glycine produced a strong fluorescence signal, whereas strychnine did not. This suggests that glycine induces conformational changes...

  1. Quantitation of alpha 1-adrenergic receptors in porcine uterine and mesenteric arteries

    Energy Technology Data Exchange (ETDEWEB)

    Farley, D.B.; Ford, S.P.; Reynolds, L.P.; Bhatnagar, R.K.; Van Orden, D.E.


    The activation of vascular alpha-adrenergic receptors may be involved in the control of uterine blood flow. A radioligand binding assay with the use of the alpha 1-adrenergic antagonist /sup 3/H-WB-4101 was established to characterize the alpha-adrenergic receptors in uterine and mesenteric arterial membranes obtained from nonpregnant pigs. Specific binding of /sup 3/H-WB-4101 was rapid, saturable, and exhibited the alpha-adrenergic agonist potency order of (-)-epinephrine inhibition constant (Ki) . 0.6 mumol/L greater than (-)-norepinephrine (Ki . 1.5 mumol/L) much greater than (-)-isoproterenol (Ki . 120 mumol/L). The alpha-adrenergic antagonist phentolamine (Ki . 6.0 nmol/L) was 200 times more potent than the beta-adrenergic antagonist (+/-)-propranolol (Ki . 1,200 nmol/L); the alpha 1-selective antagonist prazosin (Ki . 1.2 nmol/L) was 130 times more potent than the alpha 2-selective antagonist yohimbine (Ki . 160 nmol/L). Scatchard analysis, as well as iterative curve-fitting analysis, demonstrated that /sup 3/H-WB-4101 binding by arterial membranes was to a single class of binding sites. Uterine arteries exhibited greater maximal binding capacity (BMax) than that of mesenteric arteries (47.5 +/- 3.2 versus 30.9 +/- 3.6 fmol per milligram of protein, p less than 0.01), but the uterine artery dissociation constant (Kd) was higher, thus indicating a lower affinity, when compared with mesenteric artery (0.43 +/- 0.04 versus 0.33 +/- 0.04 nmol/L, p less than 0.05).

  2. Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis. (United States)

    Conrad, Curdin; Boyman, Onur; Tonel, Giulia; Tun-Kyi, Adrian; Laggner, Ute; de Fougerolles, Antonin; Kotelianski, Victor; Gardner, Humphrey; Nestle, Frank O


    Psoriasis is a common T cell-mediated autoimmune inflammatory disease. We show that blocking the interaction of alpha1beta1 integrin (VLA-1) with collagen prevented accumulation of epidermal T cells and immunopathology of psoriasis. Alpha1beta1 integrin, a major collagen-binding surface receptor, was exclusively expressed by epidermal but not dermal T cells. Alpha1beta1-positive T cells showed characteristic surface markers of effector memory cells and contained high levels of interferon-gamma but not interleukin-4. Blockade of alpha1beta1 inhibited migration of T cells into the epidermis in a clinically relevant xenotransplantation model. This was paralleled by a complete inhibition of psoriasis development, comparable to that caused by tumor necrosis factor-alpha blockers. These results define a crucial role for alpha1beta1 in controlling the accumulation of epidermal type 1 polarized effector memory T cells in a common human immunopathology and provide the basis for new strategies in psoriasis treatment focusing on T cell-extracellular matrix interactions.

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

    DEFF Research Database (Denmark)

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


    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...

  4. Causal and synthetic associations of variants in the SERPINA gene cluster with alpha1-antitrypsin serum levels.

    Directory of Open Access Journals (Sweden)

    Gian Andri Thun

    Full Text Available Several infrequent genetic polymorphisms in the SERPINA1 gene are known to substantially reduce concentration of alpha1-antitrypsin (AAT in the blood. Since low AAT serum levels fail to protect pulmonary tissue from enzymatic degradation, these polymorphisms also increase the risk for early onset chronic obstructive pulmonary disease (COPD. The role of more common SERPINA1 single nucleotide polymorphisms (SNPs in respiratory health remains poorly understood. We present here an agnostic investigation of genetic determinants of circulating AAT levels in a general population sample by performing a genome-wide association study (GWAS in 1392 individuals of the SAPALDIA cohort. Five common SNPs, defined by showing minor allele frequencies (MAFs >5%, reached genome-wide significance, all located in the SERPINA gene cluster at 14q32.13. The top-ranking genotyped SNP rs4905179 was associated with an estimated effect of β = -0.068 g/L per minor allele (P = 1.20*10(-12. But denser SERPINA1 locus genotyping in 5569 participants with subsequent stepwise conditional analysis, as well as exon-sequencing in a subsample (N = 410, suggested that AAT serum level is causally determined at this locus by rare (MAF<1% and low-frequent (MAF 1-5% variants only, in particular by the well-documented protein inhibitor S and Z (PI S, PI Z variants. Replication of the association of rs4905179 with AAT serum levels in the Copenhagen City Heart Study (N = 8273 was successful (P<0.0001, as was the replication of its synthetic nature (the effect disappeared after adjusting for PI S and Z, P = 0.57. Extending the analysis to lung function revealed a more complex situation. Only in individuals with severely compromised pulmonary health (N = 397, associations of common SNPs at this locus with lung function were driven by rarer PI S or Z variants. Overall, our meta-analysis of lung function in ever-smokers does not support a functional role of common SNPs in the SERPINA gene

  5. Causal and Synthetic Associations of Variants in the SERPINA Gene Cluster with Alpha1-antitrypsin Serum Levels (United States)

    Thun, Gian Andri; Kumar, Ashish; Obeidat, Ma'en; Zorzetto, Michele; Haun, Margot; Curjuric, Ivan; Couto Alves, Alexessander; Jackson, Victoria E.; Albrecht, Eva; Ried, Janina S.; Teumer, Alexander; Lopez, Lorna M.; Huffman, Jennifer E.; Enroth, Stefan; Bossé, Yohan; Hao, Ke; Timens, Wim; Gyllensten, Ulf; Polasek, Ozren; Wilson, James F.; Rudan, Igor; Hayward, Caroline; Sandford, Andrew J.; Deary, Ian J.; Koch, Beate; Reischl, Eva; Schulz, Holger; Hui, Jennie; James, Alan L.; Rochat, Thierry; Russi, Erich W.; Jarvelin, Marjo-Riitta; Strachan, David P.; Hall, Ian P.; Tobin, Martin D.; Dahl, Morten; Fallgaard Nielsen, Sune; Nordestgaard, Børge G.; Kronenberg, Florian; Luisetti, Maurizio; Probst-Hensch, Nicole M.


    Several infrequent genetic polymorphisms in the SERPINA1 gene are known to substantially reduce concentration of alpha1-antitrypsin (AAT) in the blood. Since low AAT serum levels fail to protect pulmonary tissue from enzymatic degradation, these polymorphisms also increase the risk for early onset chronic obstructive pulmonary disease (COPD). The role of more common SERPINA1 single nucleotide polymorphisms (SNPs) in respiratory health remains poorly understood. We present here an agnostic investigation of genetic determinants of circulating AAT levels in a general population sample by performing a genome-wide association study (GWAS) in 1392 individuals of the SAPALDIA cohort. Five common SNPs, defined by showing minor allele frequencies (MAFs) >5%, reached genome-wide significance, all located in the SERPINA gene cluster at 14q32.13. The top-ranking genotyped SNP rs4905179 was associated with an estimated effect of β = −0.068 g/L per minor allele (P = 1.20*10−12). But denser SERPINA1 locus genotyping in 5569 participants with subsequent stepwise conditional analysis, as well as exon-sequencing in a subsample (N = 410), suggested that AAT serum level is causally determined at this locus by rare (MAF<1%) and low-frequent (MAF 1–5%) variants only, in particular by the well-documented protein inhibitor S and Z (PI S, PI Z) variants. Replication of the association of rs4905179 with AAT serum levels in the Copenhagen City Heart Study (N = 8273) was successful (P<0.0001), as was the replication of its synthetic nature (the effect disappeared after adjusting for PI S and Z, P = 0.57). Extending the analysis to lung function revealed a more complex situation. Only in individuals with severely compromised pulmonary health (N = 397), associations of common SNPs at this locus with lung function were driven by rarer PI S or Z variants. Overall, our meta-analysis of lung function in ever-smokers does not support a functional role of common SNPs in

  6. The antagonistic effect of antipsychotic drugs on a HEK293 cell line stably expressing human alpha(1A1)-adrenoceptors

    DEFF Research Database (Denmark)

    Nourian, Zahra; Mulvany, Michael J; Nielsen, Karsten Bork


    challenged with phenylephrine (EC(50)=1.61x10(-8) M). From Schild analysis, prazosin, sertindole, risperidone, and haloperidol caused a concentration-dependent, rightward shift of the cumulative concentration-response curves for phenylephrine in cells expressing human recombinant alpha(1A1)-adrenoceptors...... human alpha(1A1)-adrenoceptors in competition binding studies confirmed much higher antagonist affinity of sertindole and risperidone than haloperidol for these receptors. In summary, it can be concluded that there is an approximately 10-fold higher adrenoceptor affinity of risperidone and sertindole...... for human alpha(1A1)-adrenoceptors compared to haloperidol. These findings are consistent with the observation that risperidone and sertindole have a higher incidence of orthostatic hypotension than haloperidol....

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

    Directory of Open Access Journals (Sweden)

    M.S. Genelhu


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

  8. Use of a cysteine proteinase from Carica candamarcensis as a protective agent during DNA extraction. (United States)

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


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

  9. Peptidoglycan inducible expression of a serine proteinase homologue from kuruma shrimp (Marsupenaeus japonicus). (United States)

    Rattanachai, Achara; Hirono, Ikuo; Ohira, Tsuyoshi; Takahashi, Yukinori; Aoki, Takashi


    A cDNA encoding a serine proteinase homologue of kuruma shrimp (Marsupenaeus japonicus) was cloned. The 1257 bp cDNA encodes a 339 amino acid putative peptide, with a signal sequence of 16 amino acid residues. The deduced amino acid sequence is 42-67% similar to the immune-related serine proteinases and serine proteinase homologues of arthropods. It contains catalytic triad residues in the putative catalytic domain except for one substitution of Ser by a Gly residue. The six cysteine residues that form three disulphide bridges in most serine proteinases were conserved. The M. japonicus serine proteinase homologue was mainly expressed in haemocytes, in which expression dramatically increased after 3 days feeding with peptidoglycan at 0.2 mg kg(-1) shrimp body weight per day.

  10. Protective effects of an aptamer inhibitor of neutrophil elastase in lung inflammatory injury

    DEFF Research Database (Denmark)

    Bless, N M; Smith, D; Charlton, J;


    of extracellular matrix [1-4] and has cytotoxic effects on endothelial cells [5-7] and airway epithelial cells. Three types of endogenous protease inhibitors control the activity of neutrophil elastase, including alpha-1 protease inhibitor (alpha-1PI), alpha-2 macroglobulin and secreted leukoproteinase inhibitor...... (SLPI) [8-10]. A disturbed balance between neutrophil elastase and these inhibitors has been found in various acute clinical conditions (such as adult respiratory syndrome and ischemia-reperfusion injury) and in chronic diseases. We investigated the effect of NX21909, a selected oligonucleotide (aptamer......) inhibitor of elastase, in an animal model of acute lung inflammatory disease [11-14]. This inhibitor was previously selected from a hybrid library of randomized DNA and a small-molecule irreversible inhibitor of elastase (a valine diphenyl ester phosphonate, Fig. 1), by the blended SELEX process [15]. We...

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

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


    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

  13. Foot-and-mouth disease virus leader proteinase: structural insights into the mechanism of intermolecular cleavage. (United States)

    Steinberger, Jutta; Grishkovskaya, Irina; Cencic, Regina; Juliano, Luiz; Juliano, Maria A; Skern, Tim


    Translation of foot-and-mouth disease virus RNA initiates at one of two start codons leading to the synthesis of two forms of leader proteinase L(pro) (Lab(pro) and Lb(pro)). These forms free themselves from the viral polyprotein by intra- and intermolecular self-processing and subsequently cleave the cellular eukaryotic initiation factor (eIF) 4 G. During infection, Lb(pro) removes six residues from its own C-terminus, generating sLb(pro). We present the structure of sLb(pro) bound to the inhibitor E64-R-P-NH2, illustrating how sLb(pro) can cleave between Lys/Gly and Gly/Arg pairs. In intermolecular cleavage on polyprotein substrates, Lb(pro) was unaffected by P1 or P1' substitutions and processed a substrate containing nine eIF4GI cleavage site residues whereas sLb(pro) failed to cleave the eIF4GI containing substrate and cleaved appreciably more slowly on mutated substrates. Introduction of 70 eIF4GI residues bearing the Lb(pro) binding site restored cleavage. These data imply that Lb(pro) and sLb(pro) may have different functions in infected cells.

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

    DEFF Research Database (Denmark)

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


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

  15. Discovery of Peptide-based Inhibitors against Dendrotoxin B from Black Mamba through Phage Display Screening

    DEFF Research Database (Denmark)

    O. Cob, Saioa; Munk, Andreas; Laustsen, Andreas Hougaard

    The black mamba (Dendroaspis polylepis) is Africa’s most feared snake due to its potent, rapidacting venom and its speed of attack. The most abundant toxins in D. polylepis venom are the Kunitz-type proteinase inhibitors, dendrotoxins, which are unique for mamba. Dendrotoxinsare poorly neutralized...

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

    NARCIS (Netherlands)

    Talhout, Reinskje


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

  17. How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency?

    Directory of Open Access Journals (Sweden)

    Ilaria Ferrarotti

    Full Text Available The Z deficiency in α1-antitrypsin (A1ATD is an under-recognized condition. Alpha1-antitrypsin (A1AT is the main protein in the α1-globulin fraction of serum protein electrophoresis (SPE; however, evaluation of the α1-globulin protein fraction has received very little attention. Serum Z-type A1AT manifests in polymeric forms, but their interference with quantitative immunoassays has not been reported. Here, 214 894 samples were evaluated by SPE at the G. Fracastoro Hospital of Verona, Italy. Patients with an A1AT level ≤ 0.92 g/L were recalled to complete A1ATD diagnosis. In parallel, to qualitatively and quantitatively characterize A1AT, sera samples from 10 PiZZ and 10 PiMM subjects obtained at the National Institute of Tuberculosis and Lung Diseases in Warsaw, Poland, were subjected to non-denaturing 7.5% PAGE and 7.5% SDS-PAGE followed by Western blot. Moreover, purified A1AT was heated at 60°C and analyzed by a non-denaturing PAGE and 4-15% gradient SDS-PAGE followed by Western blot as well as by isolelectrofocusing and nephelometry. A total of 966 samples manifested percentages ≤ 2.8 or a double band in the alpha1-zone. According to the nephelometry data, 23 samples were classified as severe (A1AT ≤ 0.49 g/L and 462 as intermediate (A1AT >0.49≤ 1.0 g/L A1ATD. Twenty subjects agreed to complete the diagnosis and an additional 21 subjects agreed to family screening. We detected 9 cases with severe and 26 with intermediate A1ATD. Parallel experiments revealed that polymerization of M-type A1AT, when measured by nephelometry or isolelectrofocusing, yields inaccurate results, leading to the erroneous impression that it was Z type and not M-type A1AT. We illustrate the need for confirmation of Z A1AT values by "state of the art" method. Clinicians should consider a more in-depth investigation of A1ATD in patients when they exhibit serum polymers and low α1-globulin protein levels by SPE.

  18. How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency? (United States)

    Trevisan, Maria Teresa; Dresel, Marc; Koczulla, Rembert; Ottaviani, Stefania; Baldo, Raffaele; Gorrini, Marina; Sala, Giorgia; Cavallon, Luana; Welte, Tobias; Chorostowska-Wynimko, Joanna; Luisetti, Maurizio; Janciauskiene, Sabina


    The Z deficiency in α1-antitrypsin (A1ATD) is an under-recognized condition. Alpha1-antitrypsin (A1AT) is the main protein in the α1-globulin fraction of serum protein electrophoresis (SPE); however, evaluation of the α1-globulin protein fraction has received very little attention. Serum Z-type A1AT manifests in polymeric forms, but their interference with quantitative immunoassays has not been reported. Here, 214 894 samples were evaluated by SPE at the G. Fracastoro Hospital of Verona, Italy. Patients with an A1AT level ≤ 0.92 g/L were recalled to complete A1ATD diagnosis. In parallel, to qualitatively and quantitatively characterize A1AT, sera samples from 10 PiZZ and 10 PiMM subjects obtained at the National Institute of Tuberculosis and Lung Diseases in Warsaw, Poland, were subjected to non-denaturing 7.5% PAGE and 7.5% SDS-PAGE followed by Western blot. Moreover, purified A1AT was heated at 60°C and analyzed by a non-denaturing PAGE and 4–15% gradient SDS-PAGE followed by Western blot as well as by isolelectrofocusing and nephelometry. A total of 966 samples manifested percentages ≤ 2.8 or a double band in the alpha1-zone. According to the nephelometry data, 23 samples were classified as severe (A1AT ≤ 0.49 g/L) and 462 as intermediate (A1AT >0.49≤ 1.0 g/L) A1ATD. Twenty subjects agreed to complete the diagnosis and an additional 21 subjects agreed to family screening. We detected 9 cases with severe and 26 with intermediate A1ATD. Parallel experiments revealed that polymerization of M-type A1AT, when measured by nephelometry or isolelectrofocusing, yields inaccurate results, leading to the erroneous impression that it was Z type and not M-type A1AT. We illustrate the need for confirmation of Z A1AT values by “state of the art” method. Clinicians should consider a more in-depth investigation of A1ATD in patients when they exhibit serum polymers and low α1-globulin protein levels by SPE. PMID:26270547

  19. Confirmation of mutant alpha 1 Na,K-ATPase gene and transcript in Dahl salt-sensitive/JR rats. (United States)

    Ruiz-Opazo, N; Barany, F; Hirayama, K; Herrera, V L


    As the sole renal Na,K-ATPase isozyme, the alpha 1 Na,K-ATPase accounts for all active transport of Na+ throughout the nephron. This role in renal Na+ reabsorption and the primacy of the kidney in hypertension pathogenesis make it a logical candidate gene for salt-sensitive genetic hypertension. An adenine (A)1079-->thymine (T) transversion, resulting in the substitution of glutamine276 with leucine and associated with decreased net 86Rb+ (K+) influx, was identified in Dahl salt-sensitive/JR rat kidney alpha 1 Na,K-ATPase cDNA. However, because a Taq polymerase chain reaction amplification-based reanalysis did not detect the mutant T1079 but rather only the wild-type A1079 alpha 1 Na,K-ATPase allele in Dahl salt-sensitive rat genomic DNA, we reexamined alpha 1 Na,K-ATPase sequences using Taq polymerase error-independent amplification-based analyses of genomic DNA (by polymerase allele-specific amplification and ligase chain reaction analysis) and kidney RNA (by mRNA-specific thermostable reverse transcriptase-polymerase chain reaction analysis). We also performed modified 3' mismatched correction analysis of genomic DNA using an exonuclease-positive thermostable DNA polymerase. All the confirmatory test results were concordant, confirming the A1079-->T transversion in the Dahl salt-sensitive alpha 1 Na,K-ATPase allele and its transcript, as well as the wild-type A1079 sequence in the Dahl salt-resistant alpha 1 Na,K-ATPase allele and its transcript. Documentation of a consistent Taq polymerase error that selectively substituted A at T1079 (sense strand) was obtained from Taq polymerase chain reaction amplification and subsequent cycle sequencing of reconfirmed known Dahl salt-sensitive/JR rat mutant T1079 alpha 1 cDNA M13 subclones. This Taq polymerase error results in the reversion of mutant sequence back to the wild-type alpha 1 Na,K-ATPase sequence. This identifies a site- and nucleotide-specific Taq polymerase misincorporation, suggesting that a structural

  20. Comparison of relaxation responses of cavernous and trigonal smooth muscles from rabbits by alpha1-adrenoceptor antagonists; prazosin, terazosin, doxazosin, and tamsulosin. (United States)

    Seo, K. K.; Lee, M. Y.; Lim, S. W.; Kim, S. C.


    Alpha1a-adrenergic receptor (AR) primarily mediates the contraction of the prostatic and cavernous smooth muscles. Among clinically available alpha1-AR antagonists for the medical management of benign prostatic hyperplasia (BPH), tamsulosin has a modest selectivity for alpha1A- and alpha1D- over alpha1B-ARs. To compare the effects of various alpha1-AR antagonists on relaxation responses of cavernous and trigonal smooth muscles, isometric tension studies with relatively selective (tamsulosin) and non-selective (prazosin, doxazosin, and terazosin) alpha1A-AR antagonists, were conducted in the cavernous and trigonal muscle strips of rabbits (n=10 each). Tamsulosin had the strongest inhibitory effect on contraction of trigonal smooth muscle among the various alpha1-AR antagonists, and the inhibitory activities of prazosin, doxazosin, and terazosin were not statistically different. All alpha1-AR antagonists caused concentration-dependent relaxation of the cavernous muscle strips. Tamsulosin was shown to have greater potency than prazosin (more than 100-fold), doxazosin (more than 1000-fold), and terazosin (more than 1000-fold), in relaxation of cavernous smooth muscle. In conclusion, tamsulosin might be the most effective drug among the four commonly used alpha1-AR antagonists for the medical management of BPH. Tamsulosin might be a potential substitute for phentolamine in combination with vasoactive agents as an intracavernous injection therapy for patients with erectile dysfunction. PMID:10102527

  1. Prediction of alpha1-adrenoceptor occupancy in the human prostate from plasma concentrations of silodosin, tamsulosin and terazosin to treat urinary obstruction in benign prostatic hyperplasia. (United States)

    Yamada, Shizuo; Kato, Yasuhiro; Okura, Takashi; Kagawa, Yoshiyuki; Kawabe, Kazuki


    Alpha1-adrenoceptor antagonists are clinically useful for the improvement of urinary obstruction due to benign prostatic hyperplasia (BPH), and their therapeutic effects are mediated through the blockade of prostatic alpha(1)-adrenoceptors. The present study was undertaken to predict the magnitude and duration of alpha(1)-adrenoceptor occupancy in the human prostate after oral alpha(1)-adrenoceptor antagonists. Prostatic alpha(1)-adrenoceptor-binding parameters of silodosin were estimated by measuring specific [(3)H]prazosin binding in rat prostate after oral administration of this drug. The plasma concentration of silodosin after oral administration in rats and healthy volunteers was measured using a high-performance liquid chromatographic method. The alpha(1)-adrenoceptor-binding affinities (K(i)) of silodosin, tamsulosin, and terazosin in the human prostate and plasma concentrations of tamsulosin and terazosin were obtained from the literature. Using the alpha(1)-adrenoceptor binding parameters of silodosin in rat prostate, alpha(1)-adrenoceptor occupancy in the human prostate was estimated to be around 60-70% at 1-6 h after oral administration of silodosin at doses of 3.0, 8.1, and 16.1 micromol. Thereafter, the receptor occupancy was periodically decreased, to 24% (8.1 micromol) and 54% (16.1 micromol) 24 h later. A similar magnitude and time course of alpha(1)-adrenoceptor occupancy by silodosin in the human prostate were estimated using alpha(1)-adrenoceptor-binding affinities (K(i)) in the human prostate. Despite about two orders of differences in the plasma unbound concentrations after clinically effective oral dosages of silodosin, tamsulosin, and terazosin, there was a comparable magnitude of prostatic alpha(1)-adrenoceptor occupancy by these drugs. In conclusion, the prediction of alpha(1)-adrenoceptor occupancy in the human prostate by alpha(1)-adrenoceptor antagonists may provide the rationale for the optimum dosage regimen of these drugs in the

  2. Alpha-1 antitrypsin gene polymorphism in Chronic Obstructive Pulmonary Disease (COPD

    Directory of Open Access Journals (Sweden)

    Sabri Denden


    Full Text Available Alpha-1-antitrypsin (AAT plays an important role in the pathogenesis of emphysema, the pathological lesion underlying the majority of the manifestations of Chronic Obstructive Pulmonary Disease (COPD. In this study we tested the hypothesis that common AAT polymorphisms influence the risk of developing COPDs. We investigated PiM1 (Ala213Val, PiM2 (Arg101His, PiM3 (Glu376Asp, PiS (Glu264Val and PiZ (Glu342Lys SERPINA1 alleles in 100 COPD patients and 200 healthy controls. No significant differences were observed in allele frequencies between COPD patients and controls, neither did haplotype analysis show significant differences between the two groups. A cross-sectional study revealed no significant relationship between common SERPINA1 polymorphisms (PiM1, PiM2, PiM3 and the emphysematous type of COPD. In addition, FEV1 annual decline, determined during a two-year follow up period, revealed no difference among carriers of the tested polymorphisms.

  3. Alpha-1 Antitrypsin Deficiency Targeted Testing and Augmentation Therapy: A Canadian Thoracic Society Clinical Practice Guideline

    Directory of Open Access Journals (Sweden)

    DD Marciniuk


    Full Text Available Alpha-1 antitrypsin (A1AT functions primarily to inhibit neutrophil elastase, and deficiency predisposes individuals to the development of chronic obstructive pulmonary disease (COPD. Severe A1AT deficiency occurs in one in 5000 to one in 5500 of the North American population. While the exact prevalence of A1AT deficiency in patients with diagnosed COPD is not known, results from small studies provide estimates of 1% to 5%. The present document updates a previous Canadian Thoracic Society position statement from 2001, and was initiated because of lack of consensus and understanding of appropriate patients suitable for targeted testing for A1AT deficiency, and for the use of A1AT augmentation therapy. Using revised guideline development methodology, the present clinical practice guideline document systematically reviews the published literature and provides an evidence-based update. The evidence supports the practice that targeted testing for A1AT deficiency be considered in individuals with COPD diagnosed before 65 years of age or with a smoking history of <20 pack years. The evidence also supports consideration of A1AT augmentation therapy in nonsmoking or exsmoking patients with COPD (forced expiratory volume in 1 s of 25% to 80% predicted attributable to emphysema and documented A1AT deficiency (level ≤11 μmol/L who are receiving optimal pharmacological and nonpharmacological therapies (including comprehensive case management and pulmonary rehabilitation because of benefits in computed tomography scan lung density and mortality.

  4. Alpha (1,2)-fucosyltransferase M307A polymorphism improves piglet survival. (United States)

    Kim, Kyungtae; Nguyen, Dinh Truong; Choi, Minkyung; Kim, Jin-Hoi; Seo, Han Geuk; Dadi, Hailu; Cha, Se-Yeoun; Seo, Kunho; Lee, Yun-Mi; Kim, Jong-Joo; Park, Chankyu


    To confirm the beneficial effects of alpha (1,2)-fucosyltransferase (FUT1) M307 (A) on piglet survival on commercial farms, we performed PCR-RFLP analysis of FUT1 M307 in successfully marketed (n = 245) and disease affected/deceased pigs during weaning (n = 252) at a commercial farm. We also evaluated the FUT1 genotypes of 190 healthy pigs from three different genetic backgrounds. The distribution of genotypes differed between the successfully marketed and disease affected/deceased pig groups. The frequency of the A allele, associated with resistance to edema and post-weaning diarrhea, was higher in the post-weaning survival group (0.21) than in the non-survival group (0.16, P piglet survival between AA and GG genotypes was 1.98; thus, piglet survival for individuals with the AA genotype was almost two-fold greater than for GG individuals. The FUT1 gene polymorphism can be used as an effective marker for selection programs to improve post-weaning piglet survival.

  5. Evidence for unfolded protein response activation in monocytes from individuals with alpha-1 antitrypsin deficiency.

    LENUS (Irish Health Repository)

    Carroll, Tomás P


    The hereditary disorder alpha-1 antitrypsin (AAT) deficiency results from mutations in the SERPINA1 gene and presents with emphysema in young adults and liver disease in childhood. The most common form of AAT deficiency occurs because of the Z mutation, causing the protein to fold aberrantly and accumulate in the endoplasmic reticulum (ER). This leads to ER stress and contributes significantly to the liver disease associated with the condition. In addition to hepatocytes, AAT is also synthesized by monocytes, neutrophils, and epithelial cells. In this study we show for the first time that the unfolded protein response (UPR) is activated in quiescent monocytes from ZZ individuals. Activating transcription factor 4, X-box binding protein 1, and a subset of genes involved in the UPR are increased in monocytes from ZZ compared with MM individuals. This contributes to an inflammatory phenotype with ZZ monocytes exhibiting enhanced cytokine production and activation of the NF-kappaB pathway when compared with MM monocytes. In addition, we demonstrate intracellular accumulation of AAT within the ER of ZZ monocytes. These are the first data showing that Z AAT protein accumulation induces UPR activation in peripheral blood monocytes. These findings change the current paradigm regarding lung inflammation in AAT deficiency, which up until now was derived from the protease-anti-protease hypothesis, but which now must include the exaggerated inflammatory response generated by accumulated aberrantly folded AAT in circulating blood cells.

  6. Lung clearance index for monitoring early lung disease in alpha-1-antitrypsin deficiency. (United States)

    Fuchs, Susanne I; Schwerk, Nicolaus; Pittschieler, Klaus; Ahrens, Frank; Baden, Winfried; Bals, Robert; Fähndrich, Sebastian; Gleiber, Wolfgang; Griese, Matthias; Hülskamp, Georg; Köhnlein, Thomas; Reckling, Ludmilla; Rietschel, Ernst; Staab, Doris; Gappa, Monika


    Patients with alpha-1-antitrypsin deficiency (AATD) and a PI-ZZ genotype are at high risk to develop severe emphysema during adulthood. However, little is known about early stages of emphysema and disease manifestation in other PI-types. Spirometry is commonly used for monitoring although early manifestation of emphysema is suspected within the peripheral airways that are not accessible by forced expiratory manoeuvres. We hypothesized that the Lung Clearance Index (LCI) derived from multiple breath nitrogen-washout (N2-washout) is useful to bridge this diagnostic gap. Patients from age 4 years onward and different PI-types performed N2-washout and spirometry. Results were compared to controls. 193 patients (4-79 years, 75% PI-ZZ) and 33 controls (8-60 years) were included. Mean (SD) LCI in patients was 9.1 (3.1) and 6.3 (0.6) in controls (p ≤ 0.001). 47% of adult patients with other than PI-ZZ genotypes and 39% of all patients with normal spirometry had abnormal LCIs. The LCI measured by N2-washout discriminates between patients with AATD and controls, reflects AATD related lung disease in all stages and appears to identify early peripheral lung changes in younger age than spirometry. We conclude that a normal spirometry does not exclude presence of AATD related lung disease even in genotypes other than PI-ZZ.

  7. Selenoprotein S/SEPS1 modifies endoplasmic reticulum stress in Z variant alpha1-antitrypsin deficiency.

    LENUS (Irish Health Repository)

    Kelly, Emer


    Z alpha(1)-antitrypsin (ZAAT) deficiency is a disease associated with emphysematous lung disease and also with liver disease. The liver disease of AAT deficiency is associated with endoplasmic reticulum (ER) stress. SEPS1 is a selenoprotein that, through a chaperone activity, decreases ER stress. To determine the effect of SEPS1 on ER stress in ZAAT deficiency, we measured activity of the grp78 promoter and levels of active ATF6 as markers of the unfolded protein response in HepG2 cells transfected with the mutant form of AAT, a ZAAT transgene. We evaluated levels of NFkappaB activity as a marker of the ER overload response. To determine the effect of selenium supplementation on the function of SEPS1, we investigated glutathione peroxidase activity, grp78 promoter activity, and NFkappaB activity in the presence or absence of selenium. SEPS1 reduced levels of active ATF6. Overexpression of SEPS1 also inhibited grp78 promoter and NFkappaB activity, and this effect was enhanced in the presence of selenium supplementation. This finding demonstrates a role for SEPS1 in ZAAT deficiency and suggests a possible therapeutic potential for selenium supplementation.

  8. Is PiSS Alpha-1 Antitrypsin Deficiency Associated with Disease?

    Directory of Open Access Journals (Sweden)

    Dawn McGee


    Full Text Available Background. Alpha-1 antitrypsin deficiency (AAT is an inherited condition that predisposes to lung and/or liver disease. Objective. The current study examined the clinical features of the PiSS genotype. Methods. Nineteen study participants (PiSS and 29 matched control participants (PiMM were telephone interviewed using a standardized questionnaire. Demographic features, cigarette smoking, vocation, medication history, and clinical diagnoses were compared. Statistical analysis was performed. Finally, a comprehensive literature review was performed by two investigators. Results. 12/19 (63.2% study participants reported the presence of lung and/or liver disease compared to 12/29 (41.4% control participants. There trended toward having a higher frequency of medication allergies in the study population (42.11% versus 20.69%. Conclusions. The PiSS genotype was associated with a similar incidence of obstructive lung disease to controls. Selective bias intrinsic in testing for AAT deficiency and the rarity of the PiSS genotype will make future study of this association dependent on population-based tests.

  9. Characterization of genomic rearrangements of the alpha1-acid glycoprotein/orosomucoid gene in Ghanaians. (United States)

    Yuasa, I; Nakamura, H; Henke, L; Henke, J; Nakagawa, M; Irizawa, Y; Umetsu, K


    In this study, the structure of the alpha1-acid glycoprotein (AGP), or orosomucoid (ORM), gene was investigated in a Ghanaian mother and her child, who shared an unusual variant, ORM1 S2(C), found by isoelectric focusing. Three remarkable changes of nucleotide sequence were observed: (1) The two ORM1 alleles, ORMI*S and ORMI*S2(C), had the AGP2 gene-specific sequence at one and three regions, respectively, in exon 5 to intron 5. The variant allele originating from ORMi*S was characterized by a G-to-A transition, resulting in an amino acid change from valine to methionine, which is also detected in ORM1 F2, a form that is common in Europeans. (2) The AGP2 gene of the child, inherited from the father, was duplicated, as revealed by long-range polymerase chain reaction. (3) Three new mutations were observed in two exons of the AGP2 genes of the mother and child. All of these novel genomic rearrangements, which were not observed in Japanese subjects, may have arisen through point mutation, gene conversion, and unequal crossover events. It is likely that the rearrangement of the AGP gene has often occurred in Africans.

  10. A tumor-penetrating peptide modification enhances the antitumor activity of thymosin alpha 1.

    Directory of Open Access Journals (Sweden)

    Xingzhen Lao

    Full Text Available A serious limitation of numerous antitumor drugs is the incapacity to penetrate solid tumors. However, addition of an RGD fragment to peptide drugs might solve this problem. In this study, we explored whether the introduction of a permeability-enhancing sequence, such as iRGD (CRGDK/RGPD/EC fragments, would enhance the activity of thymosin alpha 1 (Tα1. The modified Tα1 (Tα1-iRGD was successfully expressed and purified, and the in vitro assay showed that Tα1-iRGD presented a similar activity as Tα1 in promoting proliferation of mouse splenocytes. Meanwhile, cell adhesion analysis revealed that Tα1-iRGD exhibited more specific and greater binding with tumor cells compared with Tα1. Furthermore, the iRGD fragment evidently enhanced the basal ability of Tα1 to inhibit proliferation of cancer cells in vitro, particularly of mouse melanoma cell line B16F10 and human lung cancer cell line H460. Our findings indicated that the addition of an iRGD fragment increased the anti-proliferative activity of Tα1 against cancer cells by improving the ability of Tα1 to penetrate the tumor cells. This study highlighted the important roles of an iRGD sequence in the therapeutic strategy of Tα1-iRGD. Thus, Tα1-iRGD could be a novel drug candidate for cancer treatment.

  11. A tumor-penetrating peptide modification enhances the antitumor activity of thymosin alpha 1. (United States)

    Lao, Xingzhen; Liu, Meng; Chen, Jiao; Zheng, Heng


    A serious limitation of numerous antitumor drugs is the incapacity to penetrate solid tumors. However, addition of an RGD fragment to peptide drugs might solve this problem. In this study, we explored whether the introduction of a permeability-enhancing sequence, such as iRGD (CRGDK/RGPD/EC) fragments, would enhance the activity of thymosin alpha 1 (Tα1). The modified Tα1 (Tα1-iRGD) was successfully expressed and purified, and the in vitro assay showed that Tα1-iRGD presented a similar activity as Tα1 in promoting proliferation of mouse splenocytes. Meanwhile, cell adhesion analysis revealed that Tα1-iRGD exhibited more specific and greater binding with tumor cells compared with Tα1. Furthermore, the iRGD fragment evidently enhanced the basal ability of Tα1 to inhibit proliferation of cancer cells in vitro, particularly of mouse melanoma cell line B16F10 and human lung cancer cell line H460. Our findings indicated that the addition of an iRGD fragment increased the anti-proliferative activity of Tα1 against cancer cells by improving the ability of Tα1 to penetrate the tumor cells. This study highlighted the important roles of an iRGD sequence in the therapeutic strategy of Tα1-iRGD. Thus, Tα1-iRGD could be a novel drug candidate for cancer treatment.

  12. Alpha-1 adrenergic receptors gate rapid orientation-specific reduction in visual discrimination. (United States)

    Treviño, Mario; Frey, Sebastian; Köhr, Georg


    Prolonged imbalance in sensory experience leads to dramatic readjustments in cortical representation. Neuromodulatory systems play a critical role in habilitating experience-induced plasticity and regulate memory processes in vivo. Here, we show that a brief period of intense patterned visual stimulation combined with systemic activation of alpha-1 adrenergic neuromodulator receptors (α(1)-ARs) leads to a rapid, reversible, and NMDAR-dependent depression of AMPAR-mediated transmission from ascending inputs to layer II/III pyramidal cells in the visual cortex of young and adult mice. The magnitude of this form of α(1)-AR long-term depression (LTD), measured ex vivo with miniature EPSC recordings, is graded by the number of orientations used during visual experience. Moreover, behavioral tests of visual function following the induction of α(1)-AR LTD reveal that discrimination accuracy of sinusoidal drifting gratings is selectively reduced at high spatial frequencies in a reversible, orientation-specific, and NMDAR-dependent manner. Thus, α(1)-ARs enable rapid cortical synaptic depression which correlates with an orientation-specific decrease in visual discrimination. These findings contribute to our understanding of how adrenergic receptors interact with neuronal networks in response to changes in active sensory experience to produce adaptive behavior.

  13. Collagen Type XI Alpha 1 Expression in Intraductal Papillomas Predicts Malignant Recurrence

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    Javier Freire


    Full Text Available Despite the progress achieved in the treatment of breast cancer, there are still many unsolved clinical issues, being the diagnosis, prognosis, and treatment of papillary diseases, one of the highest challenges. Because of its unpredictable clinical behavior, treatment of intraductal papilloma has generated a great controversy. Even though considered as a benign lesion, it presents high rate of malignant recurrence. This is the reason why there are clinicians supporting a complete excision of the lesion, while others support an only expectant follow-up. Previous results of our group suggested that procollagen 11 alpha 1 (pro-COL11A1 expression correlates with infiltrating phenotype in breast lesions. We analyzed the correlation between expression of pro-COL11A1 in intraductal papilloma and their risk of malignant recurrence. Immunohistochemistry of pro-COL11A1 was performed in 62 samples of intraductal papilloma. Ten out 11 cases relapsed as carcinoma presents positive staining for COL11A1, while just 17 out of 51 cases with benign behaviour present immunostaining. There were significant differences (P<0.0001 when comparing patients with malignant recurrence versus nonmalignant relapse patients. These data suggest that pro-COL11A1 expression is a highly sensitive biomarker to predict malignant relapse of intraductal papilloma and it can be used as indicative factor for prevention programs.

  14. Conductivity in Exhaled Breath Condensate from Subjects with Emphysema and Type ZZ alpha-1-Antitrypsin Deficiency. (United States)

    Stolk, Jan; Fumagalli, Marco; Viglio, Simona; Iadarola, Paolo


    The assessment of biomarkers in biological samples from the lung has long been employed. Upon cooling water vapor present in exhaled breath, variable amounts of droplets of condensate (EBC) containing volatile and non-volatile compounds may be easily and non-invasively obtained from patients of any age.Objective of the present study was to compare the level of EBC conductivity determined for cohorts of individuals with different inflammatory lung disorders with that of healthy never-smoking individuals.The conductivity in EBC of PiZZ-Alpha-1-antitrypsin deficiency patients with a diagnosis of emphysema (PiZZ-AATD) was 3 fold lower than in spouse controls (54.5 ± 11.6 vs 165.3 ± 10.7 μS/cm). Non-PiZZ emphysema patients had conductivity in EBC of 59.6 ± 5.8 μS/cm and patients with sarcoidosis without airflow obstruction had EBC conductivity of 178,8 ± 6,2 μS/cm, 
not significantly different (p = 0.5) from healthy controls. Conductivity in serial EBC samples from patients with PiZZ-AATD emphysema and healthy controls was stable in 6 different samples collected over a period of 14 months. We conclude that conductivity values in EBC can be used as a correction factor for dilution of non-volatile components in EBC.

  15. The alpha,alpha-(1-->1) linkage of trehalose is key to anhydrobiotic preservation. (United States)

    Albertorio, Fernando; Chapa, Vanessa A; Chen, Xin; Diaz, Arnaldo J; Cremer, Paul S


    This study compares the efficacy of six disaccharides and glucose for the preservation of solid supported lipid bilayers (SLBs) upon exposure to air. Disaccharide molecules containing an alpha,alpha-(1-->1) linkage, such as alpha,alpha-trehalose and alpha,alpha-galacto-trehalose, were found to be effective at retaining bilayer structure in the absence of water. These sugars are known to crystallize in a clam shell conformation. Other saccharides, which are found to crystallize in more open structures, did not preserve the SLB structure during the drying process. These included the nonreducing sugar, sucrose, as well as maltose, lactose, and the monosaccharide, glucose. In fact, even close analogs to alpha,alpha-trehalose, such as alpha,beta-trehalose, which connects its glucopyranose rings via a (1-->1) linkage in an axial, equatorial fashion, permitted nearly complete delamination and destruction of supported bilayers upon exposure to air. Lipids with covalently attached sugar molecules such as ganglioside GM1, lactosyl phosphatidylethanolamine, and glucosylcerebroside were also ineffective at preserving bilayer structure. The liquid crystalline-to-gel phase transition temperature of supported phospholipid bilayers was tested in the presence of sugars in a final set of experiments. Only alpha,alpha-trehalose and alpha,alpha-galacto-trehalose depressed the phase transition temperature, whereas the introduction of other sugar molecules into the bulk solution caused the phase transition temperature of the bilayer to increase. These results point to the importance of the axial-axial linkage of disaccharides for preserving SLB structure.

  16. Cerebral artery alpha-1 AR subtypes: high altitude long-term acclimatization responses.

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    Ravi Goyal

    Full Text Available In response to hypoxia and other stress, the sympathetic (adrenergic nervous system regulates arterial contractility and blood flow, partly through differential activities of the alpha1 (α1 - adrenergic receptor (AR subtypes (α1A-, α1B-, and α1D-AR. Thus, we tested the hypothesis that with acclimatization to long-term hypoxia (LTH, contractility of middle cerebral arteries (MCA is regulated by changes in expression and activation of the specific α1-AR subtypes. We conducted experiments in MCA from adult normoxic sheep maintained near sea level (300 m and those exposed to LTH (110 days at 3801 m. Following acclimatization to LTH, ovine MCA showed a 20% reduction (n = 5; P<0.05 in the maximum tension achieved by 10-5 M phenylephrine (PHE. LTH-acclimatized cerebral arteries also demonstrated a statistically significant (P<0.05 inhibition of PHE-induced contractility in the presence of specific α1-AR subtype antagonists. Importantly, compared to normoxic vessels, there was significantly greater (P<0.05 α1B-AR subtype mRNA and protein levels in LTH acclimatized MCA. Also, our results demonstrate that extracellular regulated kinase 1 and 2 (ERK1/2-mediated negative feedback regulation of PHE-induced contractility is modulated by α1B-AR subtype. Overall, in ovine MCA, LTH produces profound effects on α1-AR subtype expression and function.

  17. Collagen type I alpha 1 gene polymorphism in premature ovarian failure

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