Digestive proteinases of the larger black flour beetle, Cynaeus angustus (Coleoptera: Tenebrionidae)

2006 ◽  
Vol 96 (2) ◽  
pp. 167-172 ◽  
Author(s):  
B. Oppert ◽  
P. Walters ◽  
M. Zuercher
Keyword(s):  
Proteinase Inhibitors ◽  
Control Strategies ◽  
Cysteine Proteinase ◽  
Serine Proteinase ◽  
Cysteine Proteinase Inhibitor ◽  
Flour Beetle ◽  
Reducing Conditions ◽  
Caseinolytic Activity ◽  
Hydrolysis Of

AbstractDigestion in the larger black flour beetle, Cynaeus angustus (LeConte), was studied to identify new control methods for this pest of stored grains and grain products. The physiological pH of the larval gut, as measured with extracts in water, was approximately 6.1, and the pH for optimal hydrolysis of casein by gut extracts was 6.2 when buffers were reducing. However, under non-reducing conditions, hydrolysis of casein and synthetic serine proteinase substrates was optimal in alkaline buffer. Three major proteinase activities were observed in zymograms using casein or gelatin. Caseinolytic activity of C. angustus gut extracts was inhibited by inhibitors that target aspartic and serine proteinase classes, with minor inhibition by a cysteine proteinase inhibitor. In particular, soybean trypsin and trypsin/chymotrypsin inhibitors were most effective in reducing the in vitro caseinolytic activity of gut extracts. Based on these data, further studies are suggested on the effects of dietary soybean inhibitors of serine proteinases, singly and in combination with aspartic and cysteine proteinase inhibitors, on C. angustus larvae. Results from these studies can be used to develop new control strategies to prevent damage to grains and stored products by C. angustus and similar coleopteran pests.

Involvement of cysteine proteinases in excystment of Paragonimus ohirai metacercariae induced by sodium cholate and A23187

2003 ◽  
Vol 77 (1) ◽  
pp. 21-26 ◽  
Author(s):  
T. Ikeda
Keyword(s):  
Proteinase Inhibitor ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Serine Proteinase ◽  
Sodium Cholate ◽  
Cysteine Proteinase Inhibitor ◽  
Cysteine Proteinases ◽  
Serine Proteinase Inhibitor ◽  
Similar Activity

AbstractThe involvement of intrinsic proteinases in the excystment of Paragonimus ohirai metacercariae was studied in in vitro excystment induced by sodium (Na) cholate, a bile salt and A23187, a Ca2+ ionophore. The effects of various proteinase inhibitors on the in vitro excystment were examined and similar inhibitory profiles were obtained. Benzyloxycarbonyl-L-leucyl-L-leucinal (Z-Leu-Leu-H), a cysteine proteinase inhibitor and 4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc SC), a serine proteinase inhibitor completely inhibited excystment, while L-3-carboxy-2,3-trans-epoxypropionyl-leucylamido (4-guanidino)-butane (E-64), a cysteine proteinase inhibitor and leupeptin, a cysteine/serine proteinase inhibitor permitted partial excystment at a lower rate, but inhibited it from proceeding from the partial excystment stage. In secretions released from metacercariae during excystment, proteinase activities detected towards various fluorogenic peptidyl substrates were almost completely inhibited by Z-Leu-Leu-H and E-64, but not by Pefabloc SC. Sodium cholate induced a higher secretion of cysteine proteinases and a higher rate of excystment than A23187. Profiles of cysteine proteinase activities towards five peptidyl substrates detected were markedly different among the two secretions and the lysate of newly excysted juveniles. Newly excysted juveniles released cysteine proteinases with similar activity profiles and levels to metacercariae induced by Na cholate-incubation, whereas the release of cysteine proteinases was reduced compared with metacercariae induced by A23187-incubation. These results provide valuable information about the involvement of intrinsic proteinases in metacercarial excystment.

Influence of a Cysteine Proteinase Inhibitor on Alfalfa Weevil (Coleoptera: Curculionidae) Growth and Development Over Successive Generations

2000 ◽  
Vol 35 (1) ◽  
pp. 70-76 ◽  
Author(s):  
T. C. Elden
Keyword(s):  
Proteinase Inhibitor ◽  
Growth And Development ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Serine Proteinase ◽  
Adult Survival ◽  
Cysteine Proteinase Inhibitor ◽  
Alfalfa Weevil ◽  
Hypera Postica ◽  
Successive Generations

The influence of leupeptin, a cysteine and serine proteinase inhibitor, on alfalfa weevil, Hypera postica (Gyllenhal), growth and development was investigated over nine successive generations. Concern that ingestion of proteinase inhibitors by phytophagous insects could induce production of inhibitor-insensitive proteinase activity initiated this investigation. The percent alfalfa weevil larval, pupal and adult survival, and defoliation was significantly lower on alfalfa foliage treated with leupeptin than on untreated foliage in all nine generations tested. Main effects for generations and treatment times generation were nonsignificant for all variables. This study demonstrates that after nine generations leupeptin, when compared to an untreated control, does not lose its ability to significantly inhibit alfalfa weevil growth and development. This suggests that the alfalfa weevil did not utilize or induce other proteinases (digestive enzymes) to compensate for inhibition of one of its major proteinases.

scholarly journals Binding of FAD to 6-hydroxy-d-nicotine oxidase apoenzyme prevents degradation of the holoenzyme

1989 ◽  
Vol 258 (1) ◽  
pp. 187-192 ◽  
Author(s):  
R Brandsch ◽  
V Bichler ◽  
B Krauss
Keyword(s):  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Cysteine Proteinase Inhibitor ◽  
Western Blots ◽  
E Coli ◽  
Cell Extracts ◽  
Arthrobacter Oxidans ◽  
Cloned Gene

Expression of the 6-hydroxy-D-nicotine oxidase (6-HDNO) gene from Arthrobacter oxidans cloned into Escherichia coli showed a marked temperature-dependence. Transformed E. coli cells grown at 30 degrees C exhibited a several-fold higher 6-HDNO activity than did cells grown at 37 degrees C. This effect did not depend on the promoter used for expression of the cloned gene in E. coli, nor was it an effect of 6-HDNO mRNA instability at 37 degrees C. Studies performed in vivo and in vitro revealed that an increased susceptibility of apo-6-HDNO to proteolytic attack at 37 degrees C was responsible for the observed phenomenon. Extracts from cells grown at 37 degrees C showed on Western blots a decrease in immunologically detectable 6-HDNO polypeptide when compared with extracts from cells grown at 30 degrees C. The 6-HDNO polypeptide is covalently modified by attachment of the cofactor FAD to a histidine residue. It could be shown that covalent flavinylation of the apoenzyme in vitro, i.e. formation of holoenzyme, by incubation of cell extracts with FAD and phosphoenolpyruvate protected the 6-HDNO polypeptide from degradation at 37 degrees C. Of a variety of proteinase inhibitors tested only the cysteine-proteinase inhibitor L-3-trans-carboxyoxiran-2-carbonyl-L-leucylagmatine (E64) prevented degradation, by up to 70%, of the apoenzyme.

Cysteine proteinase inhibitor in eccrine sweat is derived from sweat gland

1991 ◽  
Vol 260 (2) ◽  
pp. R314-R320 ◽  
Author(s):  
H. Yokozeki ◽  
T. Hibino ◽  
T. Takemura ◽  
K. Sato
Keyword(s):  
Liquid Chromatography ◽  
Molecular Mass ◽  
Sweat Gland ◽  
Proteinase Inhibitors ◽  
Sweat Rate ◽  
Cysteine Proteinase ◽  
Sweat Glands ◽  
Cysteine Proteinase Inhibitor ◽  
Eccrine Sweat

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

scholarly journals Inhibition of human liver cathepsin L by α2 cysteine-proteinase inhibitor and the low-Mr cysteine proteinase inhibitor from human serum

1984 ◽  
Vol 220 (1) ◽  
pp. 147-155 ◽  
Author(s):  
M Pagano ◽  
F Esnard ◽  
R Engler ◽  
F Gauthier
Keyword(s):  
Human Serum ◽  
Proteinase Inhibitor ◽  
Human Liver ◽  
Physiological Function ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Cysteine Proteinase Inhibitor ◽  
Lysosomal Proteinases

The inhibition of human liver cathepsin L by two specific proteinase inhibitors present in human serum, namely alpha 2 cysteine-proteinase inhibitor and the low-Mr cysteine-proteinase inhibitor, was studied. Kinetic parameters, including inhibition constants (Ki) and rate constants for association and dissociation (k+1 and K-1), were determined. The values found are consistent with a possible physiological function of these inhibitors to control cathepsin L activity. Furthermore, a transfer of active proteinase from the complex with either cysteine-proteinase inhibitor species to alpha 2-macroglobulin was demonstrated in vitro. Given the rate of dissociation of both cathepsin-L-cysteine-proteinase inhibitor complexes, a function of transitory inhibitor can therefore be hypothesized for these proteins and might then provide an explanation of the clearance of lysosomal proteinases.

Effect of Cysteine Proteinase Inhibitors on Murine B16 Melanoma Cell Invasion in vitro

2002 ◽  
Vol 383 (5) ◽  
pp. 839-842 ◽  
Author(s):  
Natasa Sever ◽  
Metka Filipic ◽  
Joze Brzin ◽  
Tamara T. Lah
Keyword(s):  
Cell Invasion ◽  
Cathepsin B ◽  
Proteinase Inhibitor ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Cysteine Proteinase Inhibitor ◽  
Cysteine Proteinases ◽  
Cysteine Proteinase Inhibitors

Abstract Various types of proteinases are implicated in the malignant progression of human and animal tumors. Proteinase inhibitors may therefore be useful as therapeutic agents in antiinvasive and antimetastatic treatment. The aims of this study were (1) to estimate the relative importance of proteinases in B16 cell invasion in vitro using synthetic, classspecific proteinase inhibitors and (2) to assess the inhibitory effect of some naturally occurring cysteine proteinase inhibitors. Serine proteinase inhibitor reduced invasiveness by up to 24%, whereas inhibition of aspartic proteinases reduced invasion by 11%. Synthetic inhibitors of cysteine proteinases markedly impaired invasion: cathepsin B inhibitors, particularly Ca 074Me, inhibited invasion from 20 40%, whereas cathepsin L inhibitor Clik 148 reduced invasion by 11%. The potato cysteine proteinase inhibitor PCPI 8.7 inhibited invasion by 21%, whereas another potato inhibitor, PCPI 6.6, and the mushroom cysteine proteinase inhibitor clitocypin had no effects. As the inhibitors that inhibited cathepsin B were in general more efficient at impairing the invasiveness, we conclude that of the two cysteine proteinases, cathepsin B plays a more important role than cathepsin L in murine melanoma cell invasion.

scholarly journals Inhibition of early but not late proteolytic processing events leads to the missorting and oversecretion of precursor forms of lysosomal enzymes in Dictyostelium discoideum.

1988 ◽  
Vol 107 (6) ◽  
pp. 2097-2107 ◽  
Author(s):  
J M Richardson ◽  
N A Woychik ◽  
D L Ebert ◽  
R L Dimond ◽  
J A Cardelli
Keyword(s):  
Dictyostelium Discoideum ◽  
Lysosomal Enzymes ◽  
Proteinase Inhibitors ◽  
Cysteine Proteinase ◽  
Proteolytic Processing ◽  
Cysteine Proteinase Inhibitor ◽  
Intracellular Location ◽  
Cell Extracts ◽  
Intracellular Compartments ◽  
Beta Glucosidase

Lysosomal enzymes are initially synthesized as precursor polypeptides which are proteolytically cleaved to generate mature forms of the enzymatically active protein. The identification of the proteinases involved in this process and their intracellular location will be important initial steps in determining the role of proteolysis in the function and targeting of lysosomal enzymes. Toward this end, axenically growing Dictyostelium discoideum cells were pulse radiolabeled with [35S]methionine and chased in fresh growth medium containing inhibitors of aspartic, metallo, serine, or cysteine proteinases. Cells exposed to the serine/cysteine proteinase inhibitors leupeptin and antipain and the cysteine proteinase inhibitor benzyloxycarbonyl-L-phenylalanyl-L-alanine-diazomethyl ketone (Z-Phe-AlaCHN2) were unable to complete proteolytic processing of the newly synthesized lysosomal enzymes, alpha-mannosidase and beta-glucosidase. Antipain and leupeptin treatment resulted in both a dramatic decrease in the efficiency of proteolytic processing, as well as a sevenfold increase in the secretion of alpha-mannosidase and beta-glucosidase precursors. However, leupeptin and antipain did not stimulate secretion of lysosomally localized mature forms of the enzymes suggesting that these inhibitors prevent the normal sorting of lysosomal enzyme precursors to lysosomes. In contrast to the results observed for cells treated with leupeptin or antipain, Z-Phe-AlaCHN2 did not prevent the cleavage of precursor polypeptides to intermediate forms of the enzymes, but greatly inhibited the production of the mature enzymes. The accumulated intermediate forms of the enzymes, however, were localized to lysosomes. Finally, fractionation of cell extracts on Percoll gradients indicated that the processing of radiolabeled precursor forms of alpha-mannosidase and beta-glucosidase to intermediate products began in cellular compartments intermediate in density between the Golgi complex and mature lysosomes. The generation of the mature forms, in contrast, was completed immediately upon or soon after arrival in lysosomes. Together these results suggest that different proteinases residing in separate intracellular compartments may be involved in generating intermediate and mature forms of lysosomal enzymes in Dictyostelium discoideum, and that the initial cleavage of the precursors may be critical for the proper localization of lysosomal enzymes.

scholarly journals The Heparin Inhibiting Property of Brain Thromboplastin

1977 ◽  
Author(s):  
E.D. Gomperts ◽  
M. Zucker
Keyword(s):  
Prothrombin Time ◽  
Antithrombin Iii ◽  
Proteinase Inhibitors ◽  
Serine Proteinase ◽  
Weight Fraction ◽  
High Molecular Weight Fraction ◽  
Thrombin Time ◽  
Factor X ◽  
Heparin Resistance

Antithrombin III is one of the serine proteinase inhibitors of the plasma which has been shown to specifically inhibit thrombin as well as Factor X. Heparin acts via antithrombin III, the heparin cofactor, hence it is difficult to explain the relative insensitivity of the prothrombin time to the presence of heparin in plasma as both thrombin, ana Factox Xa are associated functionally with the prothrombin time. This insensitivity becomes more obvious on appreciating the extreme sensitivity to heparin of the activated partial thromboplastin time as well as the thrombin time. This communication reports the demonstration of heparin inhibiting action of brain thromboplastin. The response of the prothrombin time to heparin under various conditions, and the effect of brain thromboplastin obtained from various sources and by different preparative techniques on the action of heparin in vitvo have been studied. The heparin inhibiting activity was shown to parallel the tissue factor activity. It is heat labile, non-dialysable, destroyed by detergent activity and lies in a high molecular weight fraction of the brain thromboplastin preparation (>300,000). In addition to explaining certain in vitro phenomena, these observations may explain the previously observed heparin resistance in the generalised Schwartzman phenomenon.

scholarly journals Rapid Purification and Procoagulant and Platelet Aggregating Activities of Rhombeobin: A Thrombin-Like/Gyroxin-Like Enzyme fromLachesis muta rhombeataSnake Venom

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Frank Denis Torres-Huaco ◽  
Cláudio C. Werneck ◽  
Cristina Pontes Vicente ◽  
Talita Vassequi-Silva ◽  
Ana Cláudia Coelho Nery-Diez ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Proteinase Inhibitors ◽  
Ex Vivo ◽  
Serine Proteinase ◽  
Dependent Manner ◽  
Amidolytic Activity ◽  
Venom Component ◽  
Purification Method ◽  
Rapid Purification

We report a rapid purification method using one-step chromatography of SVSP Rhombeobin (LMR-47) fromLachesis muta rhombeatavenom and its procoagulant activities and effects on platelet aggregation. The venom was fractionated by a single chromatographic step in RP-HPLC on a C8 Discovery BIO Wide Pore, showing high degree of molecular homogeneity with molecular mass of 47035.49 Da. Rhombeobin showed amidolytic activity upon BAρNA, with a broad optimum pH (7–10) and was stable in solution up to 60°C. The amidolytic activity was inhibited by serine proteinase inhibitors and reducing agents, but not chelating agents. Rhombeobin showed high coagulant activity on mice plasma and bovine fibrinogen. The deduced amino acid sequence of Rhombeobin showed homology with other SVSPs, especially with LM-TL (L. m. muta) and Gyroxin (C. d. terrificus). Rhombeobin acts,in vitro, as a strong procoagulant enzyme on mice citrated plasma, shortening the APTT and PT tests in adose-dependent manner. The protein showed, “ex vivo”, a strong defibrinogenating effect with 1 µg/animal. Lower doses activated the intrinsic and extrinsic coagulation pathways and impaired the platelet aggregation induced by ADP. Thus, this is the first report of a venom component that produces a venom-induced consumptive coagulopathy (VICC).

197 INHIBITION OF CYSTEINE PROTEINASES DURING IN VITRO OOCYTE MATURATION IMPROVES DEVELOPMENT OF PORCINE CUMULUS - OOCYTE COMPLEXES

2012 ◽  
Vol 24 (1) ◽  
pp. 211
Author(s):  
A. M. Lichtenauer ◽  
L. D. Spate ◽  
R. S. Prather ◽  
J. A. Green
Keyword(s):  
Oocyte Maturation ◽  
Proteolytic Enzymes ◽  
Cysteine Proteinase ◽  
Polar Body ◽  
Morphological Characteristics ◽  
Cumulus Cells ◽  
Cysteine Proteinase Inhibitor ◽  
Cysteine Proteinases ◽  
Developmental Potential

Biochemical differences exist between oocytes that give rise to viable blastocysts and oocytes that give rise to embryos that are developmentally compromised. For example, specific proteolytic enzymes (e.g. cathepsin B) are transcriptionally abundant in in vitro-matured bovine oocytes from prepubertal heifers that have diminished developmental potential. The effects of the cysteine proteinase inhibitor, E-64, was recently investigated in bovine cumulus–oocyte complexes (COC) that represented both poor- and good-quality oocytes. Those reports revealed that the addition of E-64 promoted both oocyte maturation and subsequent embryo development. This project sought to determine if similar results would be obtained in a porcine oocyte/embryo culture system. Inclusion of 10 and 20 μM E-64 in maturation medium was performed. Maturation rates of porcine COC in 20 μM E-64 were elevated compared to those incubated in 10 μM E-64 (74% vs 53%; P < 0.05) or without E-64 (55%; P < 0.05: N = 1750 oocytes tested). Successful maturation to metaphase II was based on the presence of a polar body and a uniform cytoplasm 44 h after follicular aspiration. Based on these preliminary results and the earlier bovine work, it was hypothesized that the E-64 was having little influence on normal oocytes, but was promoting maturation of low-quality oocytes, possibly those that were beginning to degenerate. Consequently, 20 μM of E-64 was added to the maturation media of COC segregated based on morphological characteristics of the oocytes. Good COC had a homogeneous cytoplasm and greater than 3 layers of cumulus cells; the COC were considered poor if they displayed a nonhomogeneous cytoplasm and 1 layer or less of cumulus cells, yet were still considered fertilizable. Without E-64, an increase in maturation was measured when good oocytes were compared to poor oocytes (52% vs 29%; P < 0.05: N = 1600). No significant differences in maturation were observed between good oocytes incubated in the presence or absence of E-64. Likewise, no significant differences were observed between poor oocytes incubated in the presence or absence of E-64. The percentage of maturation of good oocytes cultured in E-64 was significantly higher than that of poor oocytes cultured with E-64 (67% vs 43%; P < 0.05). Maturation with the inhibitor did not significantly affect the subsequent cleavage or blastocyst rates of embryos that arose from these oocyte groups after fertilization. These experiments suggest that inhibition of cysteine proteinases significantly promotes oocyte maturation, as was seen in previous bovine work. Our data did not support the hypothesis that cysteine proteinase inhibition was selectively improving maturation of poor oocytes within the pool. It remains possible that increased maturation in good oocytes is a result of cysteine inhibition on juvenile oocytes that morphologically appeared good and the effect was less on already degenerated oocytes that appeared poor. Differences between treatments were determined by ANOVA with post-test by Tukey's multiple comparison test.

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