Search

Rationale: The chemokine interleukin-8 is implicated in the development of bronchopulmonary dysplasia in preterm infants. The 77-amino acid isoform of interleukin-8 (interleukin-877) is a less potent chemoattractant than other shorter isoforms. Although interleukin-877 is abundant in the preterm circulation, its regulation in the preterm lung is unknown. Objectives: To study expression and processing of pulmonary interleukin-877 in preterm infants who did and did not develop bronchopulmonary dysplasia. Methods: Total interleukin-8 and interleukin-877 were measured in bronchoalveolar lavage fluid from preterm infants by immunoassay. Neutrophil serine proteases were used to assess processing. Neutrophil chemotaxis assays and degranulation of neutrophil matrix metalloproteinase-9 were used to assess interleukin-8 function. Main Results: Peak total interleukin-8 and interleukin-877 concentrations were increased in infants who developed bronchopulmonary dysplasia compared to those who did not. Shorter forms of interleukin-8 predominated in the preterm lung (96.3% No-bronchopulmonary dysplasia vs 97.1% bronchopulmonary dysplasia, p>0.05). Preterm bronchoalveolar lavage fluid significantly converted exogenously added interleukin-877 to shorter isoforms (p<0.001). Conversion was greater in bronchopulmonary dysplasia infants (p<0.05). This conversion was inhibited by α-1 antitrypsin and antithrombin III (p<0.01). Purified neutrophil serine proteases efficiently converted interleukin-877 to shorter isoforms in a time- and dose-dependent fashion; shorter interleukin-8 isoforms were primarily responsible for neutrophil chemotaxis (p<0.001). Conversion by proteinase-3 resulted in significantly increased interleukin-8 activity in vitro (p<0.01). Conclusions: Shorter, potent, isoforms interleukin-8 predominate in the preterm lung, and are increased in infants developing bronchopulmonary dysplasia, due to conversion of interleukin-877 by neutrophil serine proteases and thrombin. Processing of interleukin-8 provides an attractive therapeutic target to prevent development of bronchopulmonary dysplasia. [ABSTRACT FROM AUTHOR]

One of the most important roles of proteins in cellular milieu is recognition of other biomolecules including other proteins. Protein–protein complexes are involved in many essential cellular processes. Interfaces of protein–protein complexes are traditionally known to be conserved in evolution and less flexible than other solvent interacting tertiary structural surface. But many examples are emerging where these features do not hold good. An understanding of inter-play between flexibility and sequence conservation is emerging, providing a fresh dimension to the paradigm of sequence–structure–function relationship. The functional manifestation of the inter-relation between sequence conservation and flexibility of interface is exemplified in this review using proteinase–inhibitor protein complexes. [ABSTRACT FROM AUTHOR]

The molecular interactions between pancreatic lipase (PL) and four tea polyphenols (EGCG analogs), like (−)-epigallocatechin gallate (EGCG), (−)-gallocatechin gallate (GCG), (−)-epicatechin gallate (ECG), and (−)-epigallocatechin (EC), were studied from PL activity, conformation, kinetics and thermodynamics. It was observed that EGCG analogs inhibited PL activity, and their inhibitory rates decreased by the order of EGCG>GCG>ECG>EC. PL activity at first decreased rapidly and then slowly with the increase of EGCG analogs concentrations. α-Helix content of PL secondary structure decreased dependent on EGCG analogs concentration by the order of EGCG>GCG>ECG>EC. EGCG, ECG, and EC could quench PL fluorescence both dynamically and statically, while GCG only quenched statically. EGCG analogs would induce PL self-assembly into complexes and the hydrodynamic radii of the complexes possessed a close relationship with the inhibitory rates. Kinetics analysis showed that EGCG analogs non-competitively inhibited PL activity and did not bind to PL catalytic site. DSC measurement revealed that EGCG analogs decreased the transition midpoint temperature of PL enzyme, suggesting that these compounds reduced PL enzyme thermostability. In vitro renaturation through urea solution indicated that interactions between PL and EGCG analogs were weak and non-covalent. [ABSTRACT FROM AUTHOR]

Background: Malaria remains a major global health concern. The development of novel therapeutic strategies is critical to overcome the selection of multiresistant parasites. The subtilisin-like protease (SUB1) involved in the egress of daughter Plasmodium parasites from infected erythrocytes and in their subsequent invasion into fresh erythrocytes has emerged as an interesting new drug target. Findings: Using a computational approach based on homology modeling, protein–protein docking and mutation scoring, we designed protein–based inhibitors of Plasmodium vivax SUB1 (PvSUB1) and experimentally evaluated their inhibitory activity. The small peptidic trypsin inhibitor EETI-II was used as scaffold. We mutated residues at specific positions (P4 and P1) and calculated the change in free-energy of binding with PvSUB1. In agreement with our predictions, we identified a mutant of EETI-II (EETI-II-P4LP1W) with a Ki in the medium micromolar range. Conclusions: Despite the challenges related to the lack of an experimental structure of PvSUB1, the computational protocol we developed in this study led to the design of protein-based inhibitors of PvSUB1. The approach we describe in this paper, together with other examples, demonstrates the capabilities of computational procedures to accelerate and guide the design of novel proteins with interesting therapeutic applications. [ABSTRACT FROM AUTHOR]

Targeting allosteric sites is gaining increasing recognition as a strategy for modulating the activity of enzymes, especially in drug design. Here we investigate the mechanisms of allosteric regulation of cathepsin K as a representative of cysteine cathepsins and a promising drug target for the treatment of osteoporosis. Eight novel modifiers are identified by computational targeting of predicted allosteric sites on the surface of the enzyme. All act via hyperbolic kinetic mechanisms in presence of low molecular mass substrates, as expected for allosteric effectors. Two compounds have sizable effects on enzyme activity using interstitial collagen as a natural substrate of cathepsin K and four compounds show a significantly stabilizing effect on cathepsin K. The concept of activity modification space is introduced to obtain a global perspective of the effects elicited by the modifiers. Analysis of the activity modification space reveals that the activity of cathepsin K is regulated via multiple, different allosteric mechanisms. [ABSTRACT FROM AUTHOR]

Although SW-AT-1, a serpin-type trypsin inhibitor from silkworm (Bombyx mori), was identified in previous study, its structure-function relationship has not been studied. In this study, SW-AT-1 was cloned from the body wall of silkworm and expressed in E. coli. rSW-AT-1 inhibited both trypsin and chymotrypsin in a concentration-dependent manner. The association rate constant for rSW-AT-1 and trypsin is 1.31×10−5 M−1s−1, for rSW-AT-1 and chymotrpsin is 2.85×10−6 M−1s−1. Circular dichroism (CD) assay showed 33% α-helices, 16% β-sheets, 17% turns, and 31% random coils in the secondary structure of the protein. Enzymatic and CD analysis indicated that rSW-AT-1 was stable at wide pH range between 4–10, and exhibited the highest activity at weakly acidic or alkaline condition. The predicted three-dimensional structure of SW-AT-1 by PyMOL (v1.4) revealed a deductive reactive centre loop (RCL) near the C-terminus, which was extended from the body of the molecule. In addition to trypsin cleavage site in RCL, matrix-assisted laser desorption ionization time of flight mass spectrometry indicated that the chymotrypsin cleavage site of SW-AT-1 was between F336 and T337 in RCL. Directed mutagenesis indicated that both the N- and C-terminal sides of RCL have effects on the activity, and G327 and E329 played an important role in the proper folding of RCL. The physiological role of SW-AT-1 in the defense responses of silkworm were also discussed. [ABSTRACT FROM AUTHOR]

Experiment was conducted to estimate the protein content and inhibitory potential of different genotypes of okra and its wild relatives towards trypsin and chymotrypsin in in vitro. The wild relatives of okra possessed high protein content as compared to the cultivated genotypes. From wild relatives A. tuberculatus (90396 and 90515) contained high protein content up to 3.31 mg g-1 and 3.27 mg g-1 seed flour, respectively. The wild relatives of okra PIs from A. tuberculatus (90396 and 90515) inhibited the maximum trypsin activity i.e. upto 68.1 and 66.9 per cent, whereas, the chymotrypsin activity was found to be 68.4 and 65.8 per cent respectively. On the other hand VRO-3 and AKO-102 exhibited 63.3 and 62.2 per cent trypsin activity, also least chymotrypsin activity found upto 62.4 and 61.1 per cent. Since the proteinase inhibitors of wild relatives of okra possessed higher trypsin inhibitory potential than cultivated ones. [ABSTRACT FROM AUTHOR]

Helicoverpa armigerais a polyphagous pest damaging vast numbers of different crops leading to decrease in total production. Use ofBttransgenic to controlH. armigerahas worked well but has increased resistance againstBtinH. armigeraand controversies about theBttransgenic making it imperative to find another strategy to control attack. Soybean is a nonhost plant forH. armigera; reason could be laid in the defense system of the soybean. Proteinase Inhibitor (PIs) have been extensively studied for development of resistance against insect pest. Two cultivars developed by our university were investigated for the presence of proteinase inhibitors namely, MAUS-158 and MAUS-61. Partially purified inhibitors were showed inhibition of total protease activity of gut extract by 91.34±1.49 and 89.95±0.96% by MAUS-158 and MAUS-61, respectively. While inhibition of trypsin like proteases were found between 65 and 71% and inhibition of chymotrypsin like proteases ranges between 40 and 42%. The partial purification study shows stability of PIs up to 60°C. Soybean PIs are also showing more prominent inhibition pattern against trypsin than chymotrypsin. [ABSTRACT FROM AUTHOR]

The article discusses a study on the role of the hydroxyl group of Ser6 in the inhibitor-enzyme interaction with the goal of obtaining serine proteinase inhibitors with a simplified structure when compared with that of the wild trypsin inhibitor STFI-1. N-substituted glycine derivatives were introduced into the peptide chain through the submonomeric approach. The result of the study shows that in linear SFTI-1 analogues, the conservative residue present in P1 position inhibitors is important in preserving chymotrypsin inhibitory activity.

Protease inhibitors (PIs) retard growth and development and cause mortality in a range of insect pests belonging to various orders. Some transgenic plants expressing PI genes have also been effective against insect pest attack. The effects of two purified soybean PIs, i.e. trypsin inhibitor (Kunitz type; SBTI) and trypsin–chymotrypsin inhibitor (Bowman-Birk type; SBBI), were investigated on first and third instar larvae of the melon fruit fly Bactrocera cucurbitae (Coquillett) in laboratory feeding bioassays. The larval period of both the first and third instar larvae was prolonged when the larvae were treated with various concentrations of SBTI and SBBI. The total development period prolonged significantly in both the instars of larvae treated with SBTI, but in SBBI-treated larvae, the development period was delayed significantly only in the third instar larvae. A decrease in percentage pupation and adult emergence was observed in the first and third instar larvae treated with different concentrations of both SBTI and SBBI. The present studies showed that SBTI and SBBI have a potential in inhibiting the growth and development of melon fruit fly, but further detailed and molecular level studies are required before their use in transgenics. [ABSTRACT FROM AUTHOR]

Screening of genetically enhanced cotton varieties with a high content of protein markers, proteinase inhibitors, was carried out for the first time. It was found that only 5 of 68 studied cotton selection lines were resistant to wilt, black root rot, and gummosis. [ABSTRACT FROM AUTHOR]

Experiments were conducted to investigate the potential induction of plant defenses by Myzus persicae Sulzer (Homoptera: Aphididae) feeding on five lupin, Lupinus spp. (Leguminosae), varieties with well-characterized levels of aphid resistance. Myzus persicae feeding on L. angustifolius and L. luteus varieties induced genotype-specific changes in their host that were not consistent with the level of aphid resistance or the plant species. The plant responses were systemically detected by apterous and alate forms of the aphids. Chemical assays revealed no induction of oxidizing enzyme (catalase, peroxidase, or polyphenol oxidase) activity, serine or cystein proteinase inhibitors, or soluble phenolics in any of the five varieties tested following 3 days of feeding by 10 or 30 aphids. However, there were significant differences among the five lupin varieties in the levels of peroxidase and polyphenol oxidase activity, proteinase inhibitors, and soluble phenolics. [ABSTRACT FROM AUTHOR]

The effect of ritonavir and saquinavir, HIV proteinase inhibitors, on the secreted aspartyl proteinase (Sap) activity of Candida parapsilosis was studied. In a proteinase-inducing medium (yeast carbon base-bovine serum albumin), Sap activity in all clinical isolates of C. parapsilosis (n = 20) was observed at 37°C but not at 22°C. The presence of ritonavir at a concentration of 8 μg/ml produced an inhibition close to 50% albumin consumption and also delayed yeast growth; however, saquinavir did not have any effect on growth or on Sap activity. In Sabouraud broth, which does not induce Sap production, no effect was shown on yeast growth by either of the two HIV proteinase inhibitors studied. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Mucosal secretions contain a range of defense effector molecules including antimicrobial peptides and proteinase inhibitors. These molecules play a central role in host defense against infection, and in a variety of immune and inflammatory reactions. The aim of this study was to analyze the levels of neutrophil defensins, the cathelicidin hCAP-18/LL-37, and the proteinase inhibitors secretory leukocyte proteinase inhibitor, SKALP/elafin and cystatin M/E in various mucosal secretions and urine. We show here that especially seminal plasma is characterized by high concentrations of hCAP-18/LL-37, SLPI, SKALP/elafin and cystatin M/E. The results of this study demonstrate that each mucosal secretion is characterized by a unique profile of effector molecules, which may supply individual mucosal secretions with specific properties related to the control of local infection and inflammation. [ABSTRACT FROM AUTHOR]

This review analyzes the literature on various mechanisms of proteolytic enzyme inhibitors involved in plant defense against attack by phytopathogenic microorganisms. The action of proteinase inhibitors from plants upon the enzymes from pathogenic microorganisms and viruses is reviewed. Considerable attention is given to the induction of proteinase inhibitors in plants in response to the invasion of pathogens. Some aspects of application of proteinase inhibitors in biotechnology for production of transgenic plants with enhanced resistance to diseases are discussed. [ABSTRACT FROM AUTHOR]

The inhibitory activities of the new gossypol-free cotton variety Bukhara-9 and varieties UzFA-705 and Mirishkor were compared. It was found that Bukhara-9 and UzFA-705 had the same inhibitory activity as the resistant regionalized variety An-Bayaut-2. The inhibitory activity of Mirishkor was greater than that of variety Turon, which was developed for growing in arid zones. It was shown that Mirishkor proteinase inhibitors suppressed practically fully growth in vitro of the phytopathogenic fungus Fusarium oxysporum. [ABSTRACT FROM AUTHOR]

Wounding induces expression of genes encoding defense-related proteins involved in wound healing. An intensive survey has been carried out to clarify the initial signal transduction pathways that mediate this stress to expression of genes. In this context, signal molecules that intermediate in the wound signal to cellular response have been actively searched for. Jasmonic acid (JA) has been considered to be a key signal molecule in this pathway. Systemin, ABA, ethylene, and electrical current have been suggested to function by transmitting the wound signal to JA. A mitogen-activated protein kinase has been shown to respond rapidly to wounding, and proposed to function as one of the key enzymes involved in JA biosynthesis. Transgenic plants overexpressing a gene encoding a Rab-type, small GTP-binding protein contained 6-fold higher levels of cytokinins than wild-type plants, and responded to wounding by rapidly producing JA and, uncommonly, accumulating salicylic acid (SA), a pathogenic signal. These phenomena observed in the transgenic plants were reproduced when wild-type plants were wounded in the presence of the synthetic cytokinin, benzylaminopurine, suggesting that cytokinins are indispensable in the control of endogenous levels of JA and SA. [ABSTRACT FROM AUTHOR]

α&verbar;-antitrypsin (α&verbar;AT) is a serum inhibitor of several neutrophil-derived proteolytic enzymes, the serine proteinases. In certain individuals, a deficiency of this enzyme inhibitor is present, whereby only 10 to 20% of the normal concentration is present in the circulation. The purpose of this study was to evaluate the influence of this α&verbar;AT deficiency on the periodontal condition in a case control study. 10 patients aged 20-50 years with a mean age of 32 and showing a deficiency of α&verbar;AT were randomly selected from a national data bank. which contains persons known to be affected with the α&verbar;AT deficiency state. Control subjects were matched for age. gender and socioeconomic status. Full periodontal charting on all subjects was performed on 6 sites per tooth, including probing attachment level, probing depth, bleeding on probing and a plaque score. Analysis of the data revealed no differences with regard to attachment loss between the two groups. The deficiency group showed 9.3% pockets ≥5 mm and the matched control group 5.9%. When these results were covariate with plaque scores, the deficiency group appeared to have a higher number of sites with probing depths ≥5 mm. [ABSTRACT FROM AUTHOR]