Elastase-like Activity Research Articles

Molecular and functional aspects of alterations in the kallikrein-kinin system activity in human blood plasma at different stages of peritonitis and chronic renal failure

We are concerned with the investigation of dynamics of the plasma kallikrein-kinin system, elastase-like activity and some serpins, α1-protease inhibitor, α2-macroglobulin and antithrombin III, in patients suffering from general peritonitis and chronic renal failure. The results indicate that activation of the kallikrein-kinin system, as well as elastase-like activity are elevated and while decreased inhibitory potential becomes more intensive with disease progression. However, sharply decreased levels of kallikrein, prekallikrein and the serpins were seen in patients a few days before death. We suggest that exhaustion of these components during the end-stage of general peritonitis and chronic renal failure (in the cases with lethal outcome) may be produced by leukocyte elastase release. Evidence is presented for the destructive action of leukocyte elastase on components of the kallikrein-kinin system.

An investigation into the use of SDS-PAGE of cell surface extracts and proteolytic activity to differentiate Prevotella nigrescens and Prevotella intermedia

By comparison of the cell surface proteins derived from the outer membrane and fibrils from 14 Prevotella intermedia and 19 Prevotella nigrescens strains using SDS and analysed by SDS-PAGE, it was possible to distinguish the two species. A polypeptide of approx. 21 kDa distinguished P. intermedia strains, whereas two polypeptides of approx. 18 and 22 kDa could be used to identify P. nigrescens strains. Four other human oral black pigmented bacterial species ( Porphyromonas gingivalis, Prevotella denticola, Prevotella loescheii and Prevotella melaninogenica) did not have the 18-, 21- or 22-kDa polypeptides shown by P. intermedia or P. nigrescens. The cell-associated proteolytic activity of eight strains of P. intermedia, 14 strains of P. nigrescens and one strain of P. gingivalis (W50) was assessed using four chromogenic substrates. The hydrolysis of the substrate GPPNA (indicative of dipeptidyl peptidase IV-like activity) and SAAAPNA (elastase-like activity) by P. intermedia strains varied from 32 to 114 units and 0.5 to 12.6 units of activity respectively, where one unit was defined as the amount of protease enzyme catalysing the formation of 1 nmol of p-nitroaniline under experimental conditions. 37.5% (3 of 8) of P. intermedia strains hydrolysed SAAPPPNA (chymotrypsin-like enzyme activity) with activities of between 7 and 12 units. The hydrolysis of GPPNA and SAAAPNA by P. nigrescens strains was 32–149 and 3–16 units, respectively. 57% (8 of 14) of P. nigrescens strains hydrolysed SAAPPPNA with activities ranging from 3 to 8 units. None of the P. intermedia or P. nigrescens strains examined were found to have trypsin-like enzyme activity (BAPNA hydrolysis). The GPPNA and SAAAPNA hydrolytic activity associated with the proteases from Porphyromonas gingivalis W50 was at least twice that of P. intermedia and P. nigrescens strains. The similar peptidase activities of P. intermedia and P. nigrescens against chromogenic substrates cannot be used to differentiate the species, but SDS-PAGE of cell surface protein extracts allowed unambiguous speciation between P. intermedia and P. nigrescens. This simple technique of cell surface protein analysis can be performed in most laboratories and offers a convenient way by which to differentiate the two species.

An investigation into the use of SDS-PAGE of cell surface extracts and proteolytic activity to differentiatePrevotella nigrescensandPrevotella intermedia

By comparison of the cell surface proteins derived from the outer membrane and fibrils from 14 Prevotella intermedia and 19 Prevotella nigrescens strains using SDS and analysed by SDS-PAGE, it was possible to distinguish the two species. A polypeptide of approx. 21 kDa distinguished P. intermedia strains, whereas two polypeptides of approx. 18 and 22 kDa could be used to identify P. nigrescens strains. Four other human oral black pigmented bacterial species (Porphyromonas gingivalis, Prevotella denticola, Prevotella loescheii and Prevotella melaninogenica) did not have the 18-, 21- or 22-kDa polypeptides shown by P. intermedia or P. nigrescens. The cell-associated proteolytic activity of eight strains of P. intermedia, 14 strains of P. nigrescens and one strain of P. gingivalis (W50) was assessed using four chromogenic substrates. The hydrolysis of the substrate GPPNA (indicative of dipeptidyl peptidase IV-like activity) and SAAPPNA (elastase-like activity) by P. intermedia strains varied from 32 to 114 units and 0.5 to 12.6 units of activity respectively, where one unit was defined as the amount of protease enzyme catalysing the formation of 1 nmol of p-nitroaniline under experimental conditions. 37.5% (3 of 8) of P. intermedia strains hydrolysed SAAPPNA (chymotrypsin-like enzyme activity) with activities of between 7 and 12 units. The hydrolysis of GPPNA and SAAAPNA by P. nigrescens strains was 32-149 and 3-16 units, respectively. 57% (8 of 14) of P. nigrescens strains hydrolysed SAAPPPNA with activities ranging from 3 to 8 units. None of the P. intermedia or P. nigrescens strains examined were found to have trypsin-like enzyme activity (BAPNA hydrolysis). The GPPNA and SAAAPNA hydrolytic activity associated with the proteases from Porphyromonas gingivalis W50 was at least twice that of P. intermedia and P. nigrescens strains. The similar peptidase activities of P. intermedia and P. nigrescens against chromogenic substrates cannot be used to differentiate the species, but SDS-PAGE of cell surface protein extracts allowed unambiguous speciation between P. intermedia and P. nigrescens. This simple technique of cell surface protein analysis can be performed in most laboratories and offers a convenient way by which to differentiate the two species.

Cathepsin G and elastase in synovial fluid and peripheral blood in reactive and rheumatoid arthritis.

The purpose of the study was to evaluate the involvement of serine proteinases cathepsin G and elastase on pathomechanisms in synovial fluid (SF) of patients with reactive (ReA) and rheumatoid, (RA) arthritis. Cathepsin G, elastase, and their endogenous inhibitors alpha1-antichymotrypsin (alpha1-ACT) and alpha1-proteinase inhibitor (alpha1-PI) were identified immunohistochemically from SF and peripheral blood (PB) of patients with ReA and RA. Cathepsin G and elastase activities in SF and PB were measured spectrophotometrically. Dot-immunostaining was used to identify cathepsin G, elastase, but also alpha1-ACT and alpha1-PI from SF and PB. Cathepsin G and elastase-like activities (IU/I) were slightly elevated in ReA SF compared to the corresponding peripheral blood values (11.4 +/- 9.2 vs 4.8 +/- 1.7, NS, and 5.1 +/- 2.8 vs 2.3 +/- 2.2, NS), which was similar to what was seen in RA (16.4 +/- 6.2 vs 0.53 +/- 0.4, p < 0.05, and 6.51 +/- 1.8 vs 1.22 +/- 0.58, p < 0.05). Although some samples did not contain cathepsin G and/or elastase-like activities, all samples contained immunoreactive enzyme, but also alpha1-ACT and alpha1-PI. In ReA SF, in contrast to monocytes, all polymorphonuclear (PMN) cells contained cathepsin G and elastase. Cathepsin G and elastase activities correlated with each other (r = 0.78, p < 0.05) suggesting PMN / primary granules as their likely source. There was a closer association between the cathepsin G or elastase and SF leukocyte count in ReA than in RA. In ReA and RA SF elevated cathepsin G and elastase activities are detected compared to activity levels in PB suggesting local production mainly from PMNs. The co-existence of highly cellular SF and cathepsin G and elastase activity in the documented presence of endogenous inhibitors in ReA SF together with the, known, usually self-remitting clinical course of ReA, suggest a brisk and even exaggerated local PMN serine proteinase release; sparing of joints does not seem to be due to lack or inhibition of PMN responses but rather to a successful down-regulation or cessation of the responses initially elicited.

Protective Effect of Elastase Inhibition Against Myocardial Dysfunction and Injury Induced by Ischemia and Reperfusion in Isolated Rat Hearts.

BACKGROUND: Elastase release has been incriminated in the genesis of reperfusion-induced myocardial dysfunction and injury, and elastase inhibitors have been reported to reduce myocardial dysfunction in dogs subjected to coronary artery occlusion and reperfusion. METHODS AND RESULTS: To examine if elastase inhibition will modify myocardial dysfunction and injured induced by ischemia and reperfusion in isolated hearts, hearts from male Sprague Dawley rats were subjected to 30 minutes of total ischemia followed by 30 minutes of reperfusion. Ischemia-reperfusion resulted in myocardial dysfunction (increase in coronary perfusion pressure and decrease in myocardial contraction), injury (measured as creatine kinase release), and lipid peroxidation (myocardial malondialdehyde). Perfusion of hearts with an elastase inhibitor, ICI200,880, protected against myocardial dysfunction, injury and lipid peroxidation following ischemia-reperfusion. As expected, perfusion with superoxide dismutase protected the hearts against hemodynamic deterioration following ischemia-reperfusion. In in vitro studies, there was no direct effect of ICI200,880 on superoxide anion generation. CONCLUSIONS: ICI200,880 seems to exert cardioprotective effects against ischemia-reperfusion-induced injury and myocardial dysfunction in isolated buffer-perfused hearts, most likely by an elastase-like protease inhibitory activity.

A protein complex from Choristoneura fumiferana gut-juice involved in the precipitation of δ-endotoxin from Bacillus thuringiensis subsp, sotto

A 75 kDa protein from spruce budworm ( Choristoneura fumiferana) gut-juice has been isolated and shown to cause a specific precipitation of the δ-endotoxin from Bacillus thuringiensis subsp. sotto. This 75 kDa protein, separated by either column chromatography or SDS-PAGE, caused precipitation of the sotto toxin in both agarose diffusion gels and the PAGE gels. The precipitation event leads to limited proteolysis of the toxin and loss of larval toxicity. SDSPAGE analysis of the precipitated toxin indicates that proteolysis of the toxin is not a prerequisite for precipitation. The protein responsible for precipitation, exhibits elastase-like activity and appears to be a complex which partially dissociates during boiling in SDS-PAGE sample buffer. Gut-juice from gypsy moth, forest tent caterpillar and white mark tussock moth also precipitated δ-endotoxin, but silkworm gut-juice gave a much weaker response. These results provide further evidence that, in the larval gut, differential processing of δ-endotoxin may play a role in the expression of activity towards various insect larvae.

Effects of the soybean (Kunitz) trypsin inhibitor on growth and digestive proteases of larvae of Spodoptera litura

Extracts from the digestive tract of final instar larvae of Spodoptera litura, reared on artificial diet, contained trypsin-like (BApNA-hydrolysing) activity with a pH optimum of 10.5, an elastase-like (SA 2PLpNA-hydrolysing) activity with a pH optimum of 10.5 and leucine aminopeptidase activity with a pH optimum of 8.5. No chymotrypsin amidase-like (BTpNA-hydrolysing) activity could be detected, but chymotrypsin esterase-like (BTEE-hydrolysing) activity was measured with a pH optimum of 8.5. Titration, in vitro of the soybean (Kunitz) trypsin inhibitor (SBTI) against each enzyme at its optimal pH revealed that the inhibitor was most effective at retarding the trypsin-like activity, only slightly effective against the elastase-like and chymotrypsin esterase-like activity, but completely ineffective against leucine aminopeptidase. Incorporation of SBTI into the diet of neonate larvae at 0.2% (w/v) and 0.5% (w/v) retarded growth rate when compared with larvae fed artifical diet only. The observed differences in growth rate occurred within the first three days, resulted in weight differences that were sustained over the whole period of the trial (12 days) and correlated with inhibitor concentration; the slowest growth rate and the lower weights were observed for larvae fed with the higher SBTI concentration (0.5%) in their diet. Assay of the digestive proteases in these larvae demonstrated that only the trypsin-like activity was stimulated significantly in larvae fed on diet containing SBTI.

A specific neutrophil elastase inhibitor (ONO-5046.Na) attenuates LPS-induced acute lung inflammation in the hamster.

We have examined the effect of ONO-5046.Na, a synthetic specific inhibitor of neutrophil elastase, on lipopolysaccharide (LPS)-induced acute lung inflammation. Syrian golden hamsters were injected intraperitoneally with either 300 mg.kg-1 of ONO-5046.Na or saline, 30 min before and 1 h after intratracheal administration of 0.1 mg.kg-1 LPS. Animals were sacrificed 2 and 24 h later and the wet-to-dry lung weight ratio (W/D) was determined. Bronchoalveolar lavage (BAL) was performed, and tissue sections were examined histologically. The effect of ONO-5046.Na on migration of isolated neutrophils was determined. W/D was not significantly different at 2 h, but was increased at 24 h in the LPS-treated animals. This increase was attenuated in the LPS-treated animals injected with ONO-5046.Na. Analysis of BAL fluid revealed that both at 2 and 24 h after LPS administration the total cell number and neutrophil number, albumin concentration, and elastase-like activity were significantly lower in the LPS-treated animals injected with ONO-5046.Na than in those given LPS alone. Histological examination of the lungs of the animals treated with LPS alone showed intra-alveolar haemorrhages and inflammatory cell infiltration 24 h after LPS administration, whereas the lungs of the LPS-treated ONO-5046.Na injected animals were only sparsely infiltrated by inflammatory cells, as indicated by the inflammation score.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgen Regulation of an Elastase-Like Protease Activity in the Seminal Vesicle1

The processing of secretory proteins in the guinea pig (GP) seminal vesicle epithelium (SVE) is altered by castration and restored by treatment of animals with androgens. To test the hypothesis that the changes in protein processing are due to changes in the activity of specific proteases, we examined the GPSVE for protease activities capable of cleaving a synthetic elastase substrate, succinyl-alanyl-alanyl-alanyl-p-nitroanilide (Suc(Ala)3pNA). We found that the GPSVE does contain a Suc(Ala)3pNA-cleaving activity that is sensitive to the serine protease inhibitor diisopropylfluorophosphate (DFP) and to the elastase inhibitor elastatinal. Furthermore, the amount of protease activity per milligram of SVE protein is reduced to about 50% of control levels by castration. The activity is completely restored within four days by treatment of castrated animals with androgens, but is not restored by treatment with estradiol, progesterone, or dexamethasone. Although the SVE enzyme did not yield a pattern of specific cleavage products when incubated with a secretory protein substrate in vitro, this enzyme activity was competitively inhibited by a peptide whose primary sequence included the cleavage site used by the processing machinery in vivo.

Protease activities in the larval midgut of Heliothis virescens: Evidence for trypsin and chymotrypsin-like enzymes

Protease activities in the midgut contents of larvae of the tobacco budworm, Heliothis virescens, were investigated. Two major activities were present: (i) a trypsin-like enzyme, hydrolysing the synthetic substrate N-benzoyl-arginine p-nitroanilide (BApNA), strongly inhibited by N-p-tosyl-lysine chloroketone (TLCK) and not inhibited by chymostatin at concentrations where this inhibitor is chymotrypsin-specific; (ii) an enzyme which hydrolysed synthetic chymotrypsin substrates containing more than one amino acid, but not N-benzoyl-tyrosine p-nitroanilide and was strongly inhibited by chymostatin. The latter activity was considered to represent a hitherto ill-characterised family of insect chymotrypsins, which differ from their mammalian counterparts in activity towards synthetic substrates. Both activities had strongly alkaline pH optima, in the pH range 10–11, but were shown to be due to distinct proteases. Low levels of elastase-like activity (hydrolysing succinyl (alanine) 3 p-nitroanilide) were also detected. The two major enzyme activities were shown to account for almost all the protease activity of gut contents towards protein substrates. The proteases showed different sensitivities to inhibition by plant protein protease inhibitors, which were effective in protecting exogenous proteins from digestion by gut extracts.

Influences of tobacco smoke and vitamin E depletion on the distal lung of weanling rats.

Tobacco smoke is associated with pulmonary emphysema via elastase-antielastase and oxidant-antioxidant imbalance. This study addressed the tobacco smoke-induced changes in the lungs of weanling rats with vitamin E depletion. Three-week-old Wistar rats fed on vitamin E-depleted or normal diet were intermittently exposed to tobacco smoke by Hamburg II machines for 4 weeks. Tobacco smoke significantly suppressed body weight increases, particularly in the vitamin E-depleted group. In the normal diet group, tobacco smoke induced emphysematous changes with significant increases in the mean linear intercept (Lm) and the destructive index (DI), which was supported by an increase in elastase-like activity and a decrease in elastase inhibitory capacity (EIC) in bronchoalveolar lavage (BAL) fluid. Vitamin E depletion alone altered neither Lm nor DI. In tobacco-exposed animals in addition to vitamin E depletion, elastase-like activity, EIC in BAL fluid and DI were comparable to that in tobacco-exposed animals on a normal diet. However, Lm was markedly decreased with thickened epithelium and shrunk alveolar space. These results suggest that vitamin E depletion, when linked to tobacco exposure, might induce impaired lung development in the weanling rats, which is different from the emphysematous changes.

Neutrophil-mediated degradation of segmented polyurethanes

The biostability of polyurethanes was evaluated using a human neutrophil cell culture. The polymers were synthesized with 14C radiolabelled components incorporated into the polyurethane chain and the amount of radiolabel released during exposure to cells and medium was used as a marker for material degradation. The effect of diisocyanate, soft segment and chain extender chemistry on the susceptibility of polymer degradation was examined. All polymers showed a release of material into the tissue culture medium which was unrelated to the cells. A significant cell-dependent release of radiolabel-containing material was found from one of the polymers (a polyester urea-urethane, TDI/ PCL/ED) which increased linearly up to 96 h. The polyether-containing polyurethanes showed no significant cell-mediated degradation under similar conditions as measured by radiolabel release. Scanning electron microscopy (SEM) showed that the cells adhered to the different polyurethanes. However, no effect of neutrophils on polymer structure could be detected by this technique. The cellular response to each polymer was evaluated by measuring release of elastase-like activity (ELA) into the tissue culture media. After 24 h TDI/PCL/ED showed the highest levels of ELA in the tissue culture medium. When TDI/PCL/ED was incubated with commercial elastase in vitro, a significant release of radiolabel was found which was comparable to the amount of radiolabelled material released from this polymer in contact with the neutrophils in culture. No significant amount of radiolabel was released from the corresponding polyether material (TDI/PTMO/ED) under similar conditions. This study has identified one enzymatic activity (elastase) as a possible contributor to the deterioration of biomedical implants in vivo in that the polymer which elicited the greatest response of ELA also showed release of radiolabel when incubated with elastase in vitro. The use of a live cell culture system to assess the susceptibility of polyurethanes to degradation has provided a significant step forward in understanding the mechanism of biodegradation.

Airways inflammation in subjects with chronic bronchitis who have never smoked.

Smoking is the single most common cause of chronic bronchitis but the disease can also occur in non-smokers. Alterations in the lung responsible for the disease, such as oxidant/antioxidant and protease/antiprotease imbalance, have been investigated in smokers. The aim of our study was to evaluate local cellular and soluble factors (albumin, immunoglobulins, proteases, alpha 1-antitrypsin, and transferrin) that may be involved in the development of chronic bronchitis in subjects who have never smoked. Sixteen clinically stable patients with chronic bronchitis who had never been smokers were studied and 17 healthy non-smokers served as controls. All subjects underwent bronchoalveolar lavage (BAL). Total and differential cell counts and concentrations of the main proteins (albumin, immunoglobulins, complement fractions, alpha 1-antitrypsin, and transferrin) were measured. Elastase-like activity was assessed in cells and supernatants. To estimate the oxidant burden the release of superoxide anion (O2-) from native cell populations was evaluated. Recovery of BAL fluid was reduced in older individuals in both the chronic bronchitis and control groups. There was no difference in total cell count, but neutrophil percentage count was higher in those with chronic bronchitis (median (range) 3.5 (1.6-14.2)) than in controls (1.3 (0.5-3.7)). These differences were most pronounced in the first recovery, representative of the bronchial lavage. There was no difference in bronchial epithelial cells. Total proteins and albumin levels were comparable and IgG, IgA, IgM, C3, C4, transferrin and alpha 1-antitrypsin values standardised to albumin did not show any significant differences. No differences in elastase-like levels in supernatants were detected. In cell lysates elastase-like activity x 10(7) cells (macrophages+neutrophils) was increased in patients with chronic bronchitis (0.25 (0.06-4.3) compared with controls 0.08 (0.03-0.9) micrograms PPEeq). The release of O2- both at baseline and after opsonised zymosan phagocytosis did not show any differences. Correlation analysis between FEV1 and BAL fluid data showed a negative correlation only with neutrophils/ml. Clinically stable non-smokers with chronic bronchitis show no alterations of local immune components, oxidant burden, and free elastase-like activity in BAL fluids, while the content of elastase-like activity in phagocytic cells is increased. As in smokers, bronchial neutrophilia is the most significant cellular modification which correlates with the degree of airflow obstruction.

Involvement of protein kinase C and an elastase-like enzyme in the processing of transforming growth factor-alpha in human colon carcinoma cell lines.

Human colon carcinoma cell lines secrete transforming growth factor-alpha (TGF-alpha). Previous work indicated that the apparent m.w. of the TGF-alpha secreted by these cells ranged between 5 and 25 kDa. The more differentiated GEO cell line secreted a higher percentage of high m.w. TGF-alpha than did the poorly differentiated HCT 116 cell line. In addition, the HCT 116 cells secreted 5-fold more TGF-alpha. Treatment of HCT 116 and GEO cells with a phorbol ester (TPA) resulted in a 4-fold increase in TGF-alpha in the conditioned media of both cell types. The TPA-induced release of TGF-alpha was blocked by an inhibitor of elastase-like enzymes. This suggested a role for protein kinase C (PKC) in TGF-alpha processing in colon carcinoma cells. Direct measurement of PKC activity indicated that the HCT 116 cells (which secrete more fully processed TGF-alpha) had 10-fold more PKC activity than GEO cells. The presence of an elastase-like activity in detergent extracts and the ability of an elastase inhibitor to block the TPA-induced secretion of TGF-alpha suggests that PKC and an elastase-like enzyme are involved in the processing and secretion of TGF-alpha by human colon carcinoma cell lines.

Matrix metalloproteinase and elastase activities in LPS-induced acute lung injury in guinea pigs.

Matrix metalloproteinases (MMPs) and elastase are proteolytic enzymes specifically directed against extracellular matrix (ECM) components. They are secreted by inflammatory cells and may consequently contribute to the lesions of the ECM observed during acute pulmonary edema. We therefore evaluated the MMP and elastase activities, which are secreted by cultured alveolar macrophages (AMACs) and polymorphonuclear neutrophils (PMNs) and present in the bronchoalveolar lavage (BAL) fluid in a guinea pig model of acute lung injury induced by intratracheal instillation of lipopolysaccharide (LPS). The control group was given 0.9% NaCl. 24 h after instillation, a BAL was performed, the BAL fluid was separated from the cells by centrifugation, and AMACs and PMNs were separately cultured for 24 h. In BAL fluid from LPS-treated guinea pigs, we found 1) an increase in free gelatinase activity, tested on [3H]gelatin (0.7 +/- 0.2 micrograms.200 microliters BAL fluid-1.48 h-1 vs. 0.2 +/- 0.1 in controls, P < 0.05), and 2) increased total gelatinase activities, as assessed by zymography. The molecular masses of the major gelatinase species found in BAL fluid by zymography were 92 and 68 kDa. The 92-kDa gelatinase was secreted by both AMACs and PMNs, as demonstrated by zymography of their respective culture media. When tested on [3H]elastin, the elastase activity of BAL fluid of LPS-treated animals exhibited no increase, but when tested on a synthetic peptidic substrate [N-succinyl-(L-alanine)3-p-nitro anilide (SLAPN)], increased elastase-like activity was observed (from 17 +/- 4 nmol of SLAPN.200 microliters BAL fluid-1.24 h-1 in control group to 34 +/- 8 in LPS group, P < 0.05). This increase was attributable to the activity of a metalloendopeptidase that was inhibited by the metal chelator EDTA but not by the specific tissue inhibitor of MMPs.

Diffuse aspiration bronchiolitis (DAB) produced in animals by repeated HCl microaspiration

We recently reported that diffuse aspiration bronchiolitis (DAB) was detected in 1% of autopsied lungs of aged cases of pneumonia. We hypothesized whether repeated HCl micro-aspiration (RHMA) is involved in DAB and established an animal model by administering HCl intratracheally to rats every two days for 2 weeks. Saline was given to control animals in the same fashion. Then, we performed bronchio-alveolar lavage (BAL) or excised lungs for histologic examination. There was no difference in BAL cell counts, TNF alpha-production, elastase-like activity or albumin levels between the HCl and control groups. Histologically, DAB-like findings were observed in the HCl-treated animals. These data suggest that RHMA might be involved in DAB, while neither TNF alpha-production nor elastase-like activity may play a significant role in inducing DAB.

Sulfur Mustard Increases Elastase-Like Activity in Homogenates of Hairless Guinea Pig Skin

AbstractThe pathologic mechanisms underlying sulfur mustard (HD)-induced skin vesication are as yet undefined. Enhanced proteolysis activity has been postulated as the cause of HD-induced dermal epidermal separation. Using a chromogenic assay, we previously reported that HD-exposed hairless guinea pig skin biopsies demonstrated increased proteolysis of the peptide substrate Tosyl-gly-pro-arg-p-nitroanilide, Chromozym TH (TH). Elastaise activity has been used as a biomarker of inflammation in skin. In this study we assayed human leukocyte elastase (HLE) and soluble extracts of biopsies from control and HD-exposed hairless guinea pig skin with both the TH and the HLE (N-methoxysuccinyl-ala-ala-pro-val-p-nitroanilide) substrates. HD-increased proteolysis of both substrates indicates that multiple enzymes may contribute to HD-induced pathology. The increased activities of an inflammation-associated enzyme such as elastase as early as 3-6 hr after HD exposure of hairless guinea pig skin indicate that inflammat...

Salivary collagenase, elastase‐and trypsin‐like proteases as biochemical markers of periodontal tissue destruction in adult and localized juvenile periodontitis

The profile of salivary proteases and their cellular origin, with special reference to polymorphonuclear leukocytes and bacteria, was studied in localized juvenile periodontitis and compared with adult periodontitis and healthy controls. General proteolytic activity in saliva as well as collagenase, elastase-like and trypsin-like activity was measured. In addition, the sensitivity of salivary collagenase of patients with localized juvenile periodontitis to doxycycline inhibition was studied. The saliva of localized juvenile periodontitis patients contained low amounts of collagenase compared with adult periodontitis saliva, and almost all salivary collagenase was found to exist in endogenously active form, as was found to be the case also in adult periodontitis patients and healthy controls. The salivary collagenase of localized juvenile periodontitis patients was relatively insensitive to 100 mumol/l doxycycline but was completely inhibited by 600 mumol/l doxycycline, reflecting rather matrix metalloproteinase-1 (fibroblast-type) than matrix metalloproteinase-8 (polymorphonuclear leukocyte) enzyme. The saliva of localized juvenile periodontitis patients also contained low amounts of elastase-like activity compared with the saliva of untreated adult periodontitis patients. Scaling and root planing caused a significant decrease in elastase-like activity in the saliva of adult periodontitis patients. General proteolytic and trypsin-like activities were also low in the saliva of localized juvenile periodontitis patients. Furthermore, the reducing agent beta-mercaptoethanol did not activate or inhibit the salivary trypsin-like activity of localized juvenile periodontitis or adult periodontitis patients, although the reductant readily activated partially purified Porphyromonas gingivalis trypsin-like protease in a characteristic manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Vitamin E attenuates induction of elastase-like activity by tumor necrosis factor-α, cholestan-3β,5α,6β-triol and linoleic acid in cultured endothelial cells

Disturbances in arterial wall elastin metabolism appear to be important factors in atherosclerosis development. To evaluate this hypothesis, elastase-like activity was determined in cultured endothelial cells and their surrounding media after exposure to tumor necrosis factor-α (TNF), cholestan-3β,5α,6β-triol (Triol) and linoleic acid (18:2). Significant increases in elastase-like activity both in the cells and in the media were observed when subconfluent endothelial cells were treated with 12 μM Triol, 500 U TNF/ml, or 90 μM 18:2, for 72 h in the presence of 5% calf serum. Even higher activities were measured when endothelial cells were seeded directly into media enriched with 18:2, TNF or Triol and treated for 72 h. Vitamin E supplementation (25 μM) attenuated elastase-like activity in cells and media, independent of treatment. These results suggest that elastase-like enzyme induction in endothelial cells may be involved in cellular pertubations induced by certain lipids and cytokines. Vitamin E may provide a protective function by preventing the induction of elastolytic enzymes. This may have implications in elastin metabolism and atherosclerosis.

Human cationic, pancreatic elastase in acute pancreatitis

Håkansson H.-O., Borgström A, & Ohlsson K. Human cationic, pancreatic elastase in acute pancreatitis. Scand J Clin Lab Invest 1993; 53: 17-24.Immunoreactive cationic pancreatic elastase (irPE) in serum and peritoneal exudate from 8 patients with severe acute pancreatitis was found mainly in complex with alpha-1-proteinaseinhibitor (α1PI). Only minor amounts of irPE were found in a molecular form corresponding to the free proenzyme. The presence of alpha-2-macroglobulin (α2M)-bound, elastase-like activity in the exudates indicates that active elastase was bound to this inhibitor. The results indicate that pancreatic cationic elastase is released to some extent as active enzyme in acute pancreatitis. However, no free elastolytic activity was present in the exudates.