Protease inhibitors of pigeonpea (Cajanus cajan) and its wild relatives

Plant protease inhibitors have been implicated in defense against insect pests. Podborer and pod fly are major pests of developing seeds of pigeonpea ( Cajanus cajan L. Millsp.). Therefore, we studied the presence of protease inhibitors in seeds of pigeonpea and its wild relatives. Seed extracts were analyzed for protease inhibitor activities by caseinolytic assay, and the number of protease inhibitors determined by polyacrylamide gel electrophoresis. Besides trypsin and chymotrypsin inhibitors, seed extracts contained weak papain inhibitor(s) but no bromelain inhibitor. Treatment of seed extract with bromelain generated new active forms of trypsin inhibitors. The relative amounts of different trypsin inhibitors and the total trypsin inhibitor activity varied with different extraction media. Trypsin inhibitors were not detectable in pigeonpea leaves. The profiles of trypsin and chymotrypsin inhibitors in almost all the cultivars of pigeonpea analyzed were similar; however, those in wild relatives were quite variable.     

Occurrence and Heterogeneity of Chymotrypsin Inhibitors in Vegetative Tissues of Barley

Inhibitors of chymotrypsin and the alkaline proteinase of Aspergillus oryzae were present in the shoots of barley seedlings and weak activities were also detected in the shoot tops of 6-week-old plants. Treatments which induce inhibitor formation in tomato and potato leaves had no effect when tested on mature leaves, seedlings, or young tillers of barley. Fractionation experiments with isoelectric focusing showed that the barley leaves contained several proteinase inhibitors acting on both chymotrypsin and the Aspergillus proteinase, and one inhibitor which acted only on the Aspergillus enzyme. All of these inhibitors were different from the five Aspergillus proteinase inhibitors which are abundant in the endosperm of resting seeds. Two chymotrypsin inhibitors with weaker activity on the Aspergillus proteinase were present in rootlets and also in embryos of resting seeds. These inhibitors were different from both the endospermal inhibitors and the inhibitors present in young leaves.     

Purification and characterization of beta-glucuronidase inhibitor from Mycobacterium tuberculosis

Factors inhibitory to beta-glucuronidase were found in the culture filtrate and in a bacillary extract of Mycobacterium tuberculosis H37Rv grown for 6 weeks on Sauton medium. The inhibitors were purified by ammonium sulfate fractionation, treatment with n-butanol and streptomycin, and chromatography on DEAE-Sepharose CL-6B. Two inhibitors were obtained from the culture filtrate. The molecular weights were estimated to be 25,500 and 15,500 by gel filtration on a Sephadex G-75 column. Three inhibitors were purified from the bacillary extract, two of which were similar to those from the culture filtrate. The molecular weight of the third inhibitor was 21,000. However, the molecular weight of all the denatured inhibitors was 8,600 in the presence of sodium dodecyl sulfate. The inhibitors contained extremely high amounts of glutamic and aspartic acids and had a highly acidic isoelectric point of pH 2.5. The inhibitors acted noncompetitively against beta-glucuronidase of guinea pig origin at an optimal pH 4.5. beta-Glucuronidases from human peripheral leukocytes and beef liver were partially sensitive to the inhibitors; all the other enzymes tested for sensitivity were unaffected by the inhibitors.     

Genetic variants of protease inhibitors against fungal protease and α-chymotrypsin from hemolymph of the silkworm,Bombyx mori

Many electrophoretic variants of hemolymph inhibitors of proteases from Aspergillus melleus and pancreatic alpha-chymotrypsin were found using 126 silkworm strains. Six inhibitors of the fungal protease were detected and eight of chymotrypsin; the distribution of inhibitors among Japanese, Chinese, and European races was investigated. Comparison of electrophoretic patterns from F1 hybrids and parents showed that the offspring produce inhibitors of both parental types. Segregation in F2 and backcrossing suggest that the expression of each inhibitor is controlled in most cases by a pair of alleles which are responsible for strong and null bands. Two bands of fungal protease inhibitors C and D were controlled by codominant alleles. These results suggest that polymorphism of hemolymph protease inhibitors in the silkworm would be a useful experimental system for the study of the genetic control of protease inhibitors.     

Molecular docking and pharmacophore model studies of Rho kinase inhibitors

Molecular docking and pharmacophore model approaches were used to characterise the binding features of four different series of Rho kinase (ROCK) inhibitors. Docking simulation of 20 inhibitors with ROCK was performed. The binding conformations and binding affinities of these inhibitors were obtained using AutoDock 4.0 software. The predicted binding affinities correlate well with the activities of these inhibitors (R ² = 0.904). 3D pharmacophore models were generated for ROCK based on highly active inhibitors implemented in Catalyst 4.11 program. The best pharmacophore model consists of one hydrogen bond acceptor feature and two hydrophobic features, and they all seemed to be essential for inhibitors in terms of their binding activities. It is anticipated that the findings reported in this paper may provide very useful information for designing new ROCK inhibitors.     

Wheat germ trypsin inhibitors. Isolation and structural characterization of single-headed and double-headed inhibitors of the Bowman-Birk type

A number of trypsin inhibitors were isolated from wheat germs by affinity chromatography on immobilized trypsin, gel-filtration, and ion-exchange and reverse-phase chromatography. These inhibitors were classified into two groups, inhibitors I (Mr = 14,500) and II (Mr = 7,000), based on their molecular sizes. Inhibitors I and II inhibited bovine trypsin stoichiometorically at an enzyme to inhibitor ratio of 2 and 1, respectively. Sequence analysis of these inhibitors indicated a high degree of homology and that inhibitors I had a duplicated structure of inhibitors II. They are highly homologous to double-headed proteinase inhibitors (Bowman-Birk inhibitors) of Leguminosae plants. Inhibitors II are the first example of single-headed inhibitor corresponding to one inhibitory domain of the Bowman-Birk type double-headed inhibitors, which suggests that inhibitors II are relic of an ancestral single-headed inhibitor before the gene-duplication that led to the formation of present-day Bowman-Birk type inhibitors.     

Proteinase inhibitors as antistress proteins in higher plants

Physicochemical and functional characteristics of plant protein proteinase inhibitors as antistress biopolymers were studied to determine the mechanisms for plant resistance to phytopathogens and to obtain disease-resistant cereal and leguminous cultures. The activity of trypsin, chymotrypsin, and subtilisin inhibitors varied in monocotyledonous and dicotyledonous cultures. Study varieties of leguminous and cereal cultures were shown to contain endogenous inhibitors specific to proteinases of phytopathogenic fungi Fusarium, Colletotrichum, Helminthosporium, and Botrytis. These inhibitors were characterized by species specificity and variety specificity. Protease inhibitors from buckwheat seeds inhibited proteases of fungal pathogens and suppressed germination of spores and growth of the fungal mycelium. Our results suggest that proteinaceous inhibitors of proteinases are involved in the protective reaction of plants under stress conditions.     

甘薯和花生胰蛋白酶抑制剂的初步研究

许多植物蛋白制成品均含有抑制动物消化的蛋白酶抑制剂。目前已从某些豆类及蔬菜种子中分离出多种对胰蛋白酶具有抑制作用的活性物质。该实验以花生、甘薯等为原料,通过DEAE-Sepharose4BFF阴离子交换柱层析分离胰蛋白酶抑制剂,以N-苯甲酰-L-精氨酸乙酯(BAEE)为底物测定其对胰蛋白酶的抑制活性;将具有抑制活性的组分通过SDS-PAGE测定蛋白质相对分子质量(Mr);以聚丙烯酰胺凝胶等电聚焦电泳测定蛋白质等电点(pI)。结果显示,甘薯中至少有4种胰蛋白酶抑制剂组分,相对分子质量为20～25kD、等电点在pH5.0～6.6之间;花生中至少有三种胰蛋白酶抑制剂组分,相对分子质量为30～70kD、等电点在pH5.0～5.8之间。     

Inhibitors of lipase activities in soybean and other oil seeds

In the cotyledon extracts of seedlings of many oil seeds, including soybean, sunflower, cucumber, and peanut, the in vitro lipase activity was too low to account for the observed in vivo lipolysis. The low in vitro lipase activity was due to the presence of lipase inhibitors in the extracts. The inhibitors from soybean were characterized based on their effects on the hydrolysis of trilinolein by corn, pancreatic, and Rhizopus lipases. The inhibitors were not dialyzable and unaltered by RNase and β-galactosidase treatment. However, they were sensitive to heating and protease digestion. The inhibitory effect of the inhibitors was expressed irrespective of the sequence of the addition of lipase, substrate, and inhibitors to the assay medium. The inhibitory effect was equally expressed when the inhibitors were added either before or after the lipase reaction had been in progress. The inhibitory effect of the inhibitors was independent of the amount of lipase present in the assay, but was dependent on the amount of substrate added. High substrate concentration eliminated totally the inhibitory effect of the inhibitors. Most of the inhibitors were recovered in the soluble fraction in subcellular fractionation. They were present in the 2-4S and not in the 7S, and 11S (storage proteins) protein fraction. There was a gradual decrease of the inhibitors in the cotyledons in the postgerminative growth. We suggest that the inhibitors are proteins which bind to the surface of the substrate micelles. The binding prevents the normal functioning of lipase which acts on the interfacial area between the aqueous medium and the micelle surface.     

Comparative metabolic profiling of parental and inhibitors-tolerant yeasts during lignocellulosic ethanol fermentation

The metabolic responses of parental and inhibitors-tolerant yeasts in presence of the combination of three inhibitors (furfural, phenol and acetic acid) during ethanol fermentation were investigated by comparative metabolic profiling. Samples of parental and tolerant yeasts with/without three inhibitors in fermentation medium represented significantly different metabolic states. Further investigation on the specific responses of two strains revealed that the levels of most amino acids, inositol, and phenethylamine were dramatically increased in presence of inhibitors in parental yeast, while they kept relatively stable in tolerant yeast. It suggested that the protein degradation was increased and oxygen stress was induced by combined inhibitors in parental yeast. In addition, carbon metabolism (glycolysis and TCA) and pyrimidine ribonucleotides pathway (uracil and cytosine) were reduced in both strains in presence of combined inhibitors, which was considered as the general stress response. Higher levels of pyridimines in tolerant yeast suggested that they were responsible for counteracting the stress of combined inhibitors. These findings provided new insights into underlying mechanisms of yeast in resistance to the synergistic effects of inhibitors in lignocellulose hydrolysates.     

Safety of Resuming Tumor Necrosis Factor Inhibitors in Ankylosing Spondylitis Patients Concomitant with the Treatment of Active Tuberculosis: A Retrospective Nationwide Registry of the Korean Society of Spondyloarthritis Research

BackgroundsPatients who develop an active tuberculosis infection during tumor necrosis factor (TNF) inhibitor treatment typically discontinue TNF inhibitor and receive standard anti-tuberculosis treatment. However, there is currently insufficient information on patient outcomes following resumption of TNF inhibitor treatment during ongoing anti- tuberculosis treatment. Our study was designed to investigate the safety of resuming TNF inhibitors in ankylosing spondylitis (AS) patients who developed tuberculosis as a complication of the use of TNF inhibitors.MethodsThrough the nationwide registry of the Korean Society of Spondyloarthritis Research, 3929 AS patients who were prescribed TNF inhibitors were recruited between June 2003 and June 2014 at fourteen referral hospitals. Clinical information was analyzed about the patients who experienced tuberculosis after exposure to TNF inhibitors. The clinical features of resumers and non-resumers of TNF inhibitors were compared and the outcomes of tuberculosis were surveyed individually.FindingsFifty-six AS patients were treated for tuberculosis associated with TNF inhibitors. Among them, 23 patients resumed TNF inhibitors, and these patients were found to be exposed to TNF inhibitors for a longer period of time and experienced more frequent disease flare-up after discontinuation of TNF inhibitors compared with those who did not resume. Fifteen patients resumed TNF inhibitors during anti-tuberculosis treatment (early resumers) and 8 after completion of anti-tuberculosis treatment (late resumers). Median time to resuming TNF inhibitor from tuberculosis was 3.3 and 9.0 months in the early and late resumers, respectively. Tuberculosis was treated successfully in all resumers and did not relapse in any of them during follow-up (median 33.8 [IQR; 20.8–66.7] months).ConclusionsInstances of tuberculosis were treated successfully in our AS patients, even when given concomitantly with TNF inhibitors. We suggest that early resumption of TNF inhibitors in AS patients could be safe under effective coverage of tuberculosis.     

Large Scale Purification of Isoinhibitors of Trypsin from Swine Colostrum Using Zinc Chelate Chromatography and Chromatofocusing

Low molecular weight trypsin inhibitors were purified from swine colostrum on a large scale under mild conditions. Ammonium sulfate fractionation and metal chelate chromatography on zinc chelate Sepharose and phenyl Sepharose were used for removal of the bulk of proteins. The inhibitors showed only a weak hydrophobic interaction with phenyl Sepharose even in the presence of 1 M (Nll₄)₂SO4, and advantage was taken of this property to remove the inhibitors from contaminating colostrum proteins which remained tightly adsorbed to phenyl Sepharose under these conditions. The low and high molecular weight inhibitors were then separated by gel filtration on Bio-Gel P-300. The low molecular weight material was eluted in three major inhibitor fractions on DEAE-Sepharose.

Chromatofocusing of these fractions provided greater resolution of the inhibitors, and several previously unreported inhibitor peaks were detected. The six major inhibitors purified by chromatofocusing were homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. These inhibitors were composed of a single polypeptide chain with a molecular weight of 18,000 as determined by Sephacryl S-200 gel filtration and polyacrylamide qel electrophoresis in the presence of sodium dodecyl sulfate and e-mercaptoethanol. The specific activities of the pure inhibitors were approximately 30% higher than those previously reported.     

Studies on the effect of glycoprotein processing inhibitors on fusion of L6 myoblast cell lines

The effect of oligosaccharide processing inhibitors on the fusion of L6 myoblasts was studied. The glucosidase inhibitors, castanospermine, 1-deoxynojirimycin and N-methyl-deoxynojirimycin were potent inhibitors of myoblast fusion, as was the mannosidase II inhibitor, swainsonine. Inhibition of fusion was reversed when inhibitors were removed. However, the mannosidase I inhibitor, 1-deoxymannojirimycin did not inhibit fusion. Changes in cell membrane oligosaccharide structure were followed by monitoring the binding of concanavalin A (conA) and wheat germ agglutinin (WGA) to cell surface membranes in cells treated with processing inhibitors. All the processing inhibitors resulted in increased binding of conA and decreased binding of WGA; this is consistent with the known mechanisms of inhibition of the inhibitors used in the study. Inhibition of fusion by the processing inhibitors also resulted in reduced activities of creatine phosphokinase, an enzyme used as a marker for biochemical differentiation during fusion. Treatment of a non-differentiating conA-resistant cell line with processing inhibitors did not induce fusion, but the cells did show altered lectin-binding properties. The main conclusion drawn from these studies is that cell surface glycoproteins probably containing the mannose (Man)9 structure are important for the fusion reaction.     

Sodium transport inhibition by selective mitochondrial inhibitors in the urinary bladder of the toad

The effects of selective mitochondrial inhibitors on the short-circuit current and oxygen consumption displayed by the isolated urinary bladder of the toad was studied. Three types of compounds were used: (a) electron transfer inhibitors, Amytal, Cyanide and Antimycin A; (b) energy transfer inhibitors Guanidine, Oligomycin and Rutamycin; and (c) uncoupling agents, Carbonyl cyanide m-chlorophenylhydrazone and 2–4 dinitrophenol. The kinetics of inhibition of oxygen consumption indicated that the inhibitors tested were effectively reaching the mitochondria of the bladder cells. Different kinetics of inhibition of short-circuit current were obtained with the various inhibitors tested. Uncouplers and electron transfer inhibitors rapidly blocked the short-circuit current; energy transfer inhibitors only produced a slow and partial inhibition. A site of energy-coupling, tentatively identified with the intermediate formed in the energy transfer reactions closest to the electron transfer chain, is proposed.     

Characterization of three new Kunitz-type inhibitors from bovine spleen: preparation of specific antibodies

Antibodies to bovine spleen inhibitors I, II and III were elicited and their effect on the antiproteolytic activity of these Kunitz type inhibitors was tested. The immunoglobulins contain antibodies common to the four inhibitors in agreement with the great structural similarity of these antigens. Specific antibodies, which only react with the related inhibitor, were also isolated with the aim of localizing and quantifying these inhibitors "in vivo".     

Characterization of Three New Kunitz-Type Inhibitors from Bovine Spleen: Preparation of Specific Antibodies

Antibodies to bovine spleen inhibitors I, II and III were elicited and their effect on the antiproteolytic activity of these Kunitz type inhibitors was tested. The immunoglobulins contain antibodies common to the four inhibitors in agreement with the great structural similarity of these antigens. Specific antibodies, which only react with the related inhibitor, were also isolated with the aim of localizing and quantifying these inhibitors “in vivo”.     

Proteinase inhibitors from Vigna unguiculata subsp. cylindrica: II. Inhibitors of subtilisin and trypsin

Two subtilisin inhibitors and two trypsin-chymotrypsin inhibitors were purified from seeds of Vigna unguiculata subsp. cylindrica. A third subtilisin inhibitor was partially purified. The subtilisin isoinhibitors were present in very small amounts in the seeds and the degree of purification of the three inhibitors was 20,000- to 48,000-fold. The purified inhibitors were found to be homogeneous on ultracentrifugation and polyacrylamide gel electrophoresis with or without dodecyl sulfate. The subtilisin inhibitors had no action on papain, ficin, chymopapain, bromelain, trypsin, chymotrypsin, or papain and the trypsin-chymotrypsin inhibitors were also inactive with other enzymes.     

Design, synthesis, and evaluation of trifluoromethyl ketones as inhibitors of SARS-CoV 3CL protease

A series of trifluoromethyl ketones as SARS-CoV 3CL protease inhibitors was developed. The inhibitors were synthesized in four steps from commercially available compounds. Three different amino acids were explored in the P1-position and in the P2-P4 positions varying amino acids and long alkyl chain were incorporated. All inhibitors were evaluated in an in vitro assay using purified enzyme and fluorogenic substrate peptide. One of the inhibitors showed a time-dependent inhibition, with a K(i) value of 0.3 microM after 4h incubation.     

SAR directed design and synthesis of novel beta(1-4)-glucosyltransferase inhibitors and their in vitro inhibition studies

This paper describes SAR directed design and synthesis of novel beta(1-4)-glucosyltransferase (BGT) inhibitors. The designed inhibitors 1-5 provide conformational mimicry of the transition-state in glucosyltransfer reactions. The compounds were tested for in vitro inhibitory activity against (BGT) and the inhibition kinetics were examined. Three of the designed molecules were found to be potential inhibitors of BGT having IC50 values in micromolar (microM) range. Useful structure-activity relationships were established, which provide guidelines for the design of future generations of inhibitors of BGT.     

Serine proteinase inhibitors of seminal plasma of teleost fish: distribution of activity, electrophoretic profiles and relation to proteinase inhibitors of blood

Anti-proteinase activity has been found in seminal plasma of eight teleost fish species: brown trout, rainbow trout, brook trout, lake whitefish, bream, northern pike, Danube salmon and burbot. This activity correlated with seminal plasma protein and sperm concentrations. Using a mammalian (bovine) trypsin for detecting proteinase inhibitors it was found for the first time that there are species-specific electrophoretic profiles of anti-proteinase activity. One to three bands could be identified by this method. However, additional proteinase inhibitors could be identified by using fish (cod) trypsin. These inhibitors were detected in seminal plasma of salmonids and coregonids and have a slow migration rate. Fast-migrating proteinase inhibitors were present in rainbow, brown and brook trout, northern pike, whitefish and burbot. These inhibitors could be detected in brook and brown trout by using either trypsins. However, they were detected only with bovine trypsin in rainbow trout, northern pike, whitefish and burbot. These results suggest that multiple forms of serine proteinase inhibitors exist in seminal plasma of teleost fish and they differ in their affinity toward serine proteinases. Seminal plasma serine proteinase inhibitors of rainbow trout migrated during electrophoresis similarly to blood plasma proteinase inhibitors, and suggests that the two inhibitors may be similar or the same. Anti-proteinase specific activity was similar in blood and seminal plasma. Proteinase inhibitors of fish seminal plasma seem to be an important part of sperm physiology, possibly related to protection of spermatozoa. Staining for detection of serine proteinase inhibitors also allowed detection of presence of nonspecific esterase in seminal plasma of most species.