Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The role of intracellular oxyradicals in H2O2 and neutrophil-induced cytotoxicity is suggested by previous studies showing protection by inhibitors such as deferroxamine, dimethylthiourea, and dimethyl sulfoxide. In the current studies, the role of intracellular O2- is specifically examined by evaluating the effects of intracellular superoxide dismutase (SOD) supplementation on cytotoxicity of rat pulmonary artery endothelial cells induced by H2O2 and activated neutrophils. To minimize in vitro manipulation, supplementation was accomplished by incubating endothelial cells in the presence of SOD (1-20 mg/mL). Increases up to greater than 17-fold the baseline SOD activity were achievable using this approach, with uptake being maximal after 6 h of incubation. This increase was resistant to trypsin digestion, suggesting the intracellular location of SOD. Compared to controls, SOD-supplemented cells showed significantly increased resistance to killing by H2O2 and activated neutrophils. Inactive SOD failed to provide protection. The degree of protection was dependent on the dose of cytotoxic agent and the extent of SOD supplementation. The results provide new evidence that intracellular O2- participates in the killing process induced by these two stimuli. The intracellular source of O2- remains to be determined, although previous studies suggest xanthine oxidase as a likely candidate.
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PMID:Inhibition of cytotoxicity by intracellular superoxide dismutase supplementation. 212 22

Antileukoprotease or secretory leukocyte proteinase inhibitor is a potent serine proteinase inhibitor produced by exocrine glands of the human body. This monomeric protein (107 amino acids) comprises two homologous domains. It is generally thought that Leu19-Arg20-Tyr21 in the NH2-terminal domain represent the trypsin inhibitory activity, whereas Leu72-Met73-Leu74 in the COOH-domain represent the chymotrypsin and elastase inhibitory activity. Besides Met73, antileukoprotease contains three additional methionine residues all located in the COOH-terminal domain. Treatment of antileukoprotease with different amounts of methionine-selective reagents such as myeloperoxidase in the presence of H2O2 and Cl-, or cis-platinumdiammine dichloride resulted in a dose-dependent inactivation of all inhibitory activities, suggesting that methionine residues are involved in these activities. By using specific synthetic substrates, it was observed that elastase is able to displace trypsin from the inhibitor molecule, indicating that the trypsin and elastase inhibitory sites are located close to each other or at the same site. Incubation of antileukoprotease or its recombinant COOH-terminal domain with an antileukoprotease-specific monoclonal antibody (MoAb15) resulted in a strong selective increase of the trypsin inhibitory activity. The results presented reveal strong evidence that the inhibitory activities of antileukoprotease against trypsin, chymotrypsin and elastase are represented by its COOH-terminal domain, and that methionine residues are involved in interactions with these proteinases.
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PMID:Proteinase inhibitory activities of antileukoprotease are represented by its second COOH-terminal domain. 215 23

The ability of various reactive oxygen species and serine proteases to activate latent collagenase (matrix metalloproteinase-1) purified from human neutrophils was examined. Latent 70-75 kD human neutrophil collagenase (HNC) was efficiently activated by known non-proteolytic activators phenylmercuric chloride (an organomercurial compound) and gold thioglucose (Au(I)-salt). Corresponding degree of activation was achieved by reactive oxygen species including hypochlorous acid (HOCl), hydrogen peroxide (H2O2) and hydroxyl radical generated by hypoxanthine/xanthine oxidase (HX/XAO). The presence of trace amounts of iron and EDTA were necessary and even enhanced H2O2 induced activation of latent HNC. This activation could be abolished by an iron chelator desferrioxamine and a hydroxyl radical scavenger mannitol. HOCl induced activation of latent HNC was not affected by desferrioxamine and mannitol. Thus, these compounds do not inhibit the active/activated form of HNC. Latent HNC could also be activated by trypsin and chymotrypsin but not by plasmin and plasma kallikrein. The ability of mannitol and desferrioxamine to inhibit the H2O2-induced activation of HNC suggests the transition metal dependent Fenton reaction to be responsible for localized and/or site-specific generation of hydroxyl radical/hydroxyl radical -like oxidants to act as the activating oxygen species. Our results support the ability of myeloperoxidase derived HOCl to act as a direct oxidative activator of HNC and further suggest the existence of a new/alternative oxidative activation pathway of HNC involving hydroxyl radical.
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PMID:Activation of latent human neutrophil collagenase by reactive oxygen species and serine proteases. 217 13

Polymorphonuclear leucocyte (PMN) accumulation is associated with damage to airways epithelial cells in bronchitis, bronchiectasis and some forms of asthma. PMNs release several molecules which may mediate this damage, particularly proteases and oxidants. Using an in vitro model of intact human amnionic epithelial cells (EC) attached to native basement membrane (BM), we evaluated the capacity of several proteases and oxidants to induce detachment of EC from the BM. Maximum desquamation was observed with collagenase, elastase and trypsin, with minimum effective concentrations required to produce 50% EC-desquamation (MEC50) for highly purified collagenase, pancreatic elastase, human leucocyte elastase, human leucocyte cathepsin-G (Cath-G), trypsin, and kallikrein being 3616 +/- 989 U/mL, 32.3 +/- 14.7 U/mL, 85.8 +/- 26.7 U/mL, 360 +/- 20 U/mL, 340 +/- 49 BAEE U/mL and 300 +/- 23 U/mL, respectively. Urokinase (20 U/mL) and plasmin (500 U/mL) produced no desquamation in this system. Relatively high concentrations of oxidants also produced detachment (MEC50 for H2O2 and HOCl being 0.59 +/- 0.006 mol/L and 0.015 +/- 0.009 mol/L, respectively) and pretreatment of EC membranes with non-detaching concentrations of H2O2 rendered them 10-fold more susceptible to protease-induced desquamation, suggesting synergism. Reduced glutathione (GSH), N-acetyl cysteine (NAC), ethylenediamine tetra-acetic acid (EDTA) and 1,10 phenanthroline ablated collagenase induced EC-detachment. Elastase induced detachment was sensitive to inhibition by phenyl methyl sulfonyl fluoride (PMSF) and alpha 1-anti-proteinase (alpha 1-AP) and, to a lesser extent by aprotinin; trypsin-induced detachment was ablated by PMSF, alpha 1-AP and soybean trypsin inhibitor (SBTI) but not by 1,10 phenanthroline or EDTA. Cath-G induced detachment was profoundly inhibited by SBTI, GSH and NAC. These data demonstrate that human EC can be detached from intact BM by several PMN products, including collagenase, Cath-G and elastase, and that PMN-mediated detachment can be prevented by Cath-G and collagenase inhibitors. The data suggest a role for proteases, particularly Cath-G and collagenase, plus oxidants in synergism with proteases, in mediating PMN-induced EC detachment.
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PMID:Study of human epithelial cell detachment and damage: effects of proteases and oxidants. 220 Jul 49

Dimethylthiourea (DMTU) progressively disappeared following reaction with increasing amounts of hydrogen peroxide (H2O2) in vitro. DMTU disappearance following reaction with H2O2 was inhibited by addition of catalase, but not aminotriazole-inactivated catalase (AMT-catalase), superoxide dismutase (SOD), mannitol, benzoate or dimethyl sulfoxide (DMSO) in vitro. By comparison, DMTU disappearance did not occur following addition of histamine, oleic acid, elastase, trypsin or leukotrienes in vitro. Addition of DMTU also decreased H2O2-mediated injury to bovine pulmonary artery endothelial cells (as reflected by LDH release) and DMTU disappeared according to both added amounts of H2O2 and corresponding degrees of injury. DMTU disappearance was also relatively specific for reaction with H2O2 in suspensions of endothelial cells where it was prevented by addition of catalase, but not AMT-catalase or SOD and did not occur following sonication or treatment with elastase, trypsin or leukotrienes. Addition of washed human erythrocytes (RBC) also prevented both H2O2 mediated injury and corresponding DMTU decreases in suspensions of endothelial cells. In addition, phorbol myristate acetate (PMA) and normal neutrophils, but not O2 metabolite deficient neutrophils from patients with chronic granulomatous disease (CGD), caused DMTU disappearance in vitro which was decreased by simultaneous addition of catalase, but not SOD, sodium benzoate or DMSO. Finally, addition of normal neutrophils (but not CGD neutrophils) and PMA caused DMTU disappearance and increased the concentrations of the stable prostacyclin derivative (PGF1 alpha) in supernatants of endothelial cell suspensions. In parallel, DMTU also decreased PMA and neutrophil-mediated PGF1 alpha increases in supernatants from endothelial cell monolayers. Our results indicate that DMTU can decrease H2O2 or neutrophil mediated injury to endothelial cells and that simultaneous measurement of DMTU disappearance can be used to improve assessment of the presence and toxicity of H2O2 as well as the H2O2 inactivating ability of scavengers, such as RBC, in biological systems.
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PMID:Dimethylthiourea prevents hydrogen peroxide and neutrophil mediated damage to lung endothelial cells in vitro and disappears in the process. 254 52

S-S cross-linking enzyme, skin sulfhydryl oxidase (SSO), catalyzes the formation of disulfide bonds from sulfhydryl groups in skin. The activity of SSO was detected in differing amounts in each of the four layers--stratum corneum, stratum granulosum, stratum spinosum with basal cell layer, and dermis--of cow snout skin, with the highest specific activity being recorded in the stratum granulosum. SSO was stimulated to 130-150% of its initial activity by treatment with 1 mg/ml trypsin, chymotrypsin, or urokinase, but was not affected by plasmin or cathepsin D. These findings suggest that SSO may be activated by some kinds of serine proteases during the keratinocyte autolysis process in the stratum granulosum. SSO showed the highest activity with the addition of 5 microM of Cu2+. The atomic absorptive analysis of purified SSO showed 0.5 atoms of Cu in one molecule of SSO. From these findings, it was determined that Cu2+ was essential for the activity of SSO. The molar ratio of the disappearance of DTT, consumption of O2, and production of H2O2 during the enzyme reaction was 1:1.05:0.89. From these findings, the reactions catalyzed by SSO is suggested to be represented by the following equation: (table; see text).
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PMID:[Localization in skin, activation and reaction mechanisms of skin sulfhydryl oxidase]. 258 80

1. Pretreatment of some proteins (albumin, immunoglobulin G, elastin and fibrinogen) with hypochlorite or with the MPO-H2O2-Cl- system increased their susceptibility to proteolysis by trypsin, chymotrypsin or elastase. 2. The optimal activities of these three proteinases were attained at a different extent of albumin chlorination. 3. Elastase was found to develop a specially efficient activity towards chlorinated albumin or chlorinated elastin being by itself resistant to chlorinating species.
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PMID:Enhancement of proteinase-mediated degradation of proteins modified by chlorination. 266 67

Killing of rat pulmonary artery endothelial cells by activated polymorphonuclear leukocytes (PMNs), as measured at 4 hours, is catalase sensitive, iron dependent, and unaffected by addition of protease inhibitors. If the time course for exposure of endothelial cells to activated PMNs is extended to 18 hours, progressive injury occurs. Endothelial cell injury resulting at 18 hours is partially inhibited by catalase and partially inhibited by soybean trypsin inhibitor. Together, these two inhibitors function synergistically to protect the cells from injury. Exposure of endothelial cells to reagent H2O2 and purified proteolytic enzymes (trypsin, chymotrypsin, elastase, and cathepsin G) mimics the effects of activated PMNs: H2O2 alone is cytotoxic with maximal killing achieved by 4 hours; proteolytic enzymes produce cytotoxicity only at high concentrations and only after prolonged incubation (longer than 8 hours); and, in combination, H2O2 and proteolytic enzymes act synergistically. These data provide compelling evidence that PMN-mediated injury of endothelial cells involves interaction between oxygen products and proteases.
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PMID:Endothelial cell killing by neutrophils. Synergistic interaction of oxygen products and proteases. 267 21

The activation of 14C-labeled estradiol by "true" and "pseudo" peroxidases to form conjugates and other products was compared in four model systems using H2O2, glutathione, Mn2+ or irradiated riboflavin. Albumin was used as acceptor except in the glutathione system. The binding of estradiol to glutathione in the presence of the true peroxidases, lacto- or uterine peroxidase (no H2O2 added), was also examined and the conditions shown to differ from those required with the pseudoperoxidases, microperoxidase or trypsin-digested cytochrome c. The conjugates were purified by chromatography after elution from Amberlite XAD-2 and the relative amounts of these products assessed by autoradiography. The ratio of steroid to glutathione in the main water-soluble metabolite formed with lactoperoxidase was found to be approx 1:1 in a double label experiment with [14C]estradiol and [3H]glutathione. It was also shown, using estradiol labeled with 3H in different positions of the steroid molecule, that lactoperoxidase acts non-specifically in catalyzing the formation of glutathionyl conjugates as indicated by the release of 3H2O. The possible role of peroxidase and glutathione in the metabolism of estrogens and in the formation of artifactual products is discussed.
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PMID:Metabolism of estradiol by true and pseudoperoxidases. 299 58

Canine chromosomes are not only numerous (38 autosomal pairs), but they are small (compared to human chromosomes) and morphologically similar as well. Analysis of the canine karyotype by light microscopy (LM) of banded chromosomes is, thus, difficult, and the literature on the canine karyotype is scanty. In this study, we describe examination of chromosomes from normal and chronically irradiated dogs with the scanning electron microscope (SEM). Metaphase chromosomes from bone marrow aspirates were Giemsa-banded with either 0.025% trypsin alone or 0.1% trypsin preceded by 10% H2O2 and prepared for SEM. Examination of chromosomes from normal dogs revealed cylindrical chromosome profiles with well-defined chromatids and centromeres. The chromosome arms were consistently marked by periodic grooves that had complementary structures on sister chromatids and may represent the trypsin-sensitive chromatic regions. The quality of the preservation varied from preparation to preparation and depended on the concentration and time of trypsin treatment. Chromosomes from irradiated dogs revealed translocations, deletions, and gaps. We conclude that SEM produces images superior to LM images of canine chromosomes; SEM images can be used not only to identify individual chromosomes, but also to identify genetic lesions in the chromosomes of chronically irradiated dogs. We further conclude that the two Giemsa-banding protocols used in the present study produced variable results, although 0.025% trypsin alone appeared to give better and more consistent results than 0.1% trypsin preceded by 10% H2O2.
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PMID:SEM of canine chromosomes: normal structure and the effects of whole-body irradiation. 305 79


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