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Biomedical subjects

A Janoff

Publications and source records attributed to A Janoff.

At least 55 records · Page 3Linked to original sources
PubMed1983

The role of oxidative processes in emphysema.

Elastase/elastase inhibitor imbalance in the lung has been implicated in the pathogenesis of pulmonary emphysema. In light of this, it may be significant that the activity of two major elastase inhibitors, alpha 1-proteinase inhibitor (alpha 1-antitrypsin, alpha 1Pi) and bronchial mucous proteinase inhibitor, can be decreased by oxidizing agents. The effect can be observed with ozone, substances present in cigarette smoke, and oxygen metabolites generated by lung macrophages as well as peroxidative systems released by other phagocytic cells. Thus alpha 1Pi recovered from lung washings of cigarette smokers has only half the predicted normal activity per mg inhibitor and contains 4 moles of methionine sulfoxide (oxidized methionine) per mole of inactive inhibitor. By contrast, alpha 1Pi purified from nonsmokers' lung washings is fully active and contains only native methionine. At the same time, lung washes from some smokers show significantly greater hydrolytic activity against a specific synthetic elastase substrate than do lung washes of nonsmokers. These findings suggest that some smokers may develop an acquired imbalance between elastase and elastase inhibitor in their lungs, favoring activity of the enzyme. In addition to the potential effect of cigarette smoking on lung elastase/elastase inhibitor balance, smoking also may interfere with elastin repair mechanisms. Specifically, acidic water-soluble gas phase components of cigarette smoke prevent synthesis of desmosine cross-links during elastinogenesis in vitro. This report will attempt to correlate the foregoing information on biochemical changes in the lung induced by cigarette smoking with the development of emphysema in the smoker.

Drug Synergism↗
PubMed1983

Human methionine sulfoxide-peptide reductase, an enzyme capable of reactivating oxidized alpha-1-proteinase inhibitor in vitro.

The present study demonstrates the presence of methionine sulfoxide [Met(O)] peptide reductase activity in human lung homogenates and in lysates of polymorphonuclear leukocytes (PMN) and alveolar type II cells. Enzyme activity was not detected in human bronchoalveolar lavage fluid or in pulmonary alveolar macrophage lysates. The Met(O)-peptide reductase derived from PMN is capable of reactivating alpha-1-proteinase inhibitor (alpha 1Pl) oxidized by treatment with chloramine-T or a myeloperoxidase oxidizing system. However, the PMN-derived enzyme does not reactivate alpha 1Pl inactivated by treatment in vitro with aqueous solutions of cigarette smoke plus peroxide. In addition, after the instillation of oxidized human alpha 1Pl into lungs of normal or ozone-tolerant rats, no reactivated alpha 1Pl could be found in the pulmonary lavage obtained from these animals. Finally, patients with chronic obstructive pulmonary disease appear to have normal levels of PMN Met(O)-peptide reductase.

Animals↗
PubMed1983

Levels of elastase activity in bronchoalveolar lavage fluids of healthy smokers and nonsmokers.

Elastase activity was measured in concentrated, cell-free bronchoalveolar lavage (BAL), using the synthetic substrate butyloxycarbonyl-L-alanyl-L-alanyl-L-prolyl-L-valyl-amino-methylcoumarin. The BAL fluids obtained from young, asymptomatic smokers with normal urine desmosine concentrations 1 h after they had smoked 2 cigarettes showed significant increases in elastase levels compared with those in nonsmoking control subjects [nanomoles substrate hydrolyzed (3 h) per milligram lavage albumin = mean 2.7 +/- 1.9 SD (11 smokers) versus 0.5 +/- 0.4 (11 nonsmokers), p less than 0.01]. Repeated BAL samples were obtained at later times from one smoker with a high initial enzyme value and from one nonsmoking control subject. Elastase activity varied over time, but both subjects consistently remained within their respective group ranges. Inhibition studies on pooled BAL from smokers showed that the elastase activity present had properties of both serine and metalloenzymes, suggesting that neutrophils and/or monocytes (serine enzyme) as well as macrophages (metalloenzyme) contributed to the observed activity. Lung lavage cells obtained from 2 of the smokers and 2 of the nonsmokers were stained with both a chromogenic substrate and by indirect immunofluorescence for the serine enzyme. Positively stained neutrophils were readily found in smokers' lavages, but no, or only rare, positive mononuclear cells could be identified. By contrast, peripheral blood mononuclear cells from all 4 subjects stained positively with either method. These results show that some asymptomatic smokers have significantly more elastase activity in their bronchopulmonary secretions than do nonsmokers (as measured with a low molecular weight synthetic substrate). Furthermore, the enzyme activity recovered in smokers' BAL appears to be derived mainly from neutrophils (serine enzyme) and macrophages (metalloenzyme), rather than from monocytes.

Adult↗
PubMed1983

Do neutrophils play a major role in elastin turnover of normal tissues?

Urinary desmosine excretion (a quantitative measure of endogenous elastin breakdown) was monitored in 2 patients with cyclic neutropenia. In the first one, measurements were carried out over 2 complete 25-day cycles (nonconsecutive), whereas in the second, measurements were limited to selected urine specimens obtained on days when circulating neutrophils were at undetectable levels. Desmosine excretion remained in the normal range in both patients during neutropenic episodes, suggesting that endogenous elastin breakdown was not dependent on neutrophil-mediated elastinolysis. Thus, in alpha 1-antitrypsin-sufficient subjects, any lung elastin turnover caused by neutrophils is probably intermittent and limited to times and foci marked by acute inflammatory changes in the lung.

Adult↗
PubMed1983

Evaluation of the urinary desmosine radioimmunoassay as a monitor of lung injury after endobronchial elastase instillation in sheep.

Thirty male sheep were treated with varying doses of endobronchial elastase. Urinary excretion of elastin peptides was then measured by desmosine radioimmunoassay and compared with pre-enzyme values. Mean linear intercepts were measured in treated and untreated lobes 4 wk later, and in addition, lung perfusion, ventilation, and volume were measured before enzyme treatment and 4 wk later using radionuclide-imaging techniques. Most of the elevation in urinary desmosine excretion occurred in the first 48 h after elastase administration. The increase in desmosine excretion was positively correlated with: enzyme dose (r = 0.74, p less than 0.01), increase in mean linear intercept (r = 0.61, p less than 0.05), decrease in lung perfusion (r = 0.77, p less than 0.01), and decrease in ventilation (r = 0.58, p less than 0.05). These results demonstrate that the urinary desmosine radioimmunoassay is a reliable index of pulmonary elastin breakdown and of several resultant anatomic and physiologic stigmata of pulmonary emphysema.

Amino Acids↗
PubMed1982

Potential mechanism of emphysema: alpha 1-proteinase inhibitor recovered from lungs of cigarette smokers contains oxidized methionine and has decreased elastase inhibitory capacity.

The elastase inhibitory capacity per mg of alpha 1-proteinase inhibitor (alpha 1 PI) was measured in the bronchoalveolar lavage (BAL) fluid from 26 healthy smokers and 24 nonsmokers. Activity was decreased by 40% in smokers' BAL fluid compared to nonsmokers. This effect was demonstrable by using human neutrophil elastase as well as porcine pancreatic elastase as test enzyme (elastase, EC 3.4.21.11) and was reproducible when selected individuals in each group underwent lavage on repeated occasions. In contrast, the functional activity of alpha 1-antichymotrypsin was not decreased in smokers' BAL fluid. Crossed antigen-antibody electrophoresis confirmed that inactivation of alpha 1 PI was responsible for the decrease in the elastase inhibitory capacity of smokers' BAL fluid. alpha 1 PI purified from smokers' BAL fluids contained methionine sulfoxide (4 mol/mol of inactive alpha 1 PI), whereas alpha 1 PI from nonsmokers' BAL fluid did not. Smokers' alpha 1 PI was indistinguishable from nonsmokers' alpha 1 PI on the basis of electrophoretic mobility, molecular weight, and immunoreactivity. Thus, oxidation of methionine residues in lung alpha 1 PI is associated with cigarette smoking. Because chemical oxidation of alpha 1 PI in vitro causes loss of its elastase inhibitory activity, the present observations suggest that methionine oxidation may also be the cause of decreased functional activity of lung alpha 1 PI in smokers. Oxidative inactivation of alpha 1 PI in the lungs of cigarette smokers may play a role in the development of pulmonary emphysema in this group.

Adult↗
PubMed1982

Evidence for in vivo internalization of human leukocyte elastase by alveolar macrophages.

Cell lysates from cultured human alveolar macrophages contain detectable amounts of an elastinolytic enzyme. Although particulate elastin was solubilized only after prolonged incubations, lysates readily hydrolyzed T-OC-alanyl-p-nitrophenol-ester. Hydrolysis of the latter substrate was inhibited by the leukocyte elastase site-specific inhibitor, N-ac-(ala)4-chloromethyl ketone. In addition, radioimmunoelectrophoresis of concentrated alveolar macrophage lysates, previously incubated with 3H diisopropyl-phosphofluoridate (DFP), revealed the presence of DFP binding material that comigrated with inactivated human leukocyte elastase. Human leukocyte elastase can cause lung lesions resembling pulmonary emphysema in experimental animals; therefore, the clearance of this enzyme by alveolar macrophages may represent a significant route for the removal of this potentially pathogenic enzyme from the lung.

Cells, Cultured↗
PubMed1982

Alpha 1-proteinase inhibitor is more sensitive to inactivation by cigarette smoke than is leukocyte elastase.

Aqueous solutions of gas phase cigarette smoke were incubated with pure human leukocyte elastase or with crude human leukocyte granule extract, and the effects on enzyme activity were determined using a synthetic amide substrate. Simultaneously, the same smoke solutions were incubated with 10% human serum under identical conditions, and the effects on serum inhibition of purified or crude leukocyte elastase were similarly measured. In addition, aqueous solutions of unfractionated cigarette smoke were incubated with leukocyte elastase or serum, and the abilities of the smoke-treated enzyme to digest elastin and of the smoke-treated serum to inhibit elastin digestion were determined. Both experimental protocols showed that serum elastase-inhibiting capacity (primarily caused by alpha 1-proteinase inhibitor) is more susceptible to inactivation by aqueous solutions of cigarette smoke than is leukocyte elastase, suggesting that elastase inhibition (rather than elastase activity) may be predominantly suppressed by cigarette smoke inhalation in vivo.

Blood Proteins↗
PubMed1982

Elastase-induced emphysema: retention of instilled proteinase in the rat.

Airway instillation of proteinases with the ability to degrade elastin has been used to produce disease in the rat analogous to human pulmonary emphysema. This study examined the retention, localization, and fate of endotracheally instilled elastase using 125Iodine labeled enzyme and immunoperoxidase histochemistry. Porcine pancreatic elastase labeled with 125I was detected in rat lungs through 96 h after instillation; over half of the label was still present after 7 h. Similar results were obtained when elastase was reacted with a specific, catalytic site inactivator prior to instillation. Trypsin and denatured elastase, however, were cleared much more rapidly from the lung (less than half of the label present after 30 min). When lungs were homogenized after instillation of active elastase, the soluble fraction contained elastase bound to rat alpha1-antitrypsin. In addition, a small amount of label (less than 10%) appeared bound to insoluble components for extended periods of time. Using immunoperoxidase histochemistry, it was found that exogenous elastase was rapidly contained with pulmonary alveolar macrophages, as well as associated with alveolar septums and other parenchymal structures. Similar results were obtained with elastase from both porcine pancreas and human neutrophils. These results suggest that exogenous elastase in the rat, and perhaps endogenous elastolytic enzymes in humans, may have several fates in the lungs: complex formation with endogenous inhibitors, containment within the macrophage, and/or association with connective tissue targets.

Animals↗
PubMed1981

The specificity of macrophage elastase on the insulin B-chain.

The specificity of macrophage elastase obtained from mouse peritoneal exudative macrophages was determined in the hydrolysis of the oxidized insulin B-chain. This elastase hydrolysed two bonds, namely Ala-Leu and Tyr-Leu. The rate of hydrolysis of the latter was two to three times greater than that of the former. The hexapeptide Glu-Ala-Leu-Tyr-Leu-Val, obtained by cleavage of the insulin B-chain, was not hydrolysed by macrophage elastase. When EDTA was present, proteolysis of the B-chain was not observed. The macrophage elastase is therefore different from the neutrophil elastase in specificity and mechanism.

Animals↗
PubMed1981

Urinary excretion of desmosine in patients with severe burns.

Urinary excretion of total desmosine was measured by a radioimmunoassay in severely burned adult males, as well as in normal adult males. Total urinary desmosine was significantly elevated in all the samples in the burned patients, who had injuries involving more than 19% of total body surface area. The values of 24-hr urinary desmosine for the burned patients ranged from 250--1,411 nmoles, as compared with 82--142 nmoles for normal controls. These were equivalent to 14--78 mg of elastin degraded for the burned patients and 5--8 mg for normal controls. Urinary desmosine values expressed as nmoles per g of creatinine were also higher than the corresponding normal values, ranging from 110--768 nmoles versus 63 +/- 6 nmoles for normal controls. Urinary excretion of total hydroxyproline in the burned patients was also higher than in normal controls, ranging from 56--471 mg per 24 hrs, or 36 to 413 mg per g of creatinine, vs. 31 +/- 6 mg per 24 hr, or 23 +/- 2 mg per g of creatinine, in burned patients and normal controls, respectively. These values of hydroxyproline were equivalent to 413--3,623 mg of collagen and 238 mg of collagen, respectively. In the burned patients, both urinary desmosine and hydroxyproline values were elevated from day 1 post-burn, and reached peak levels in days 2--12, declining thereafter but remaining higher than values for normal controls through day 60. The metabolism of elastin and collagen in skin of burned patients was probably highly accelerated for a long time, at least through day 60 post-burn.

Adult↗
PubMed1981

Secretion of alpha 1-proteinase inhibitor by cultured rat alveolar macrophages.

Alpha 1-proteinase inhibitor, metabolically labeled with 35S, was detected in concentrated culture medium of rat alveolar macrophages by immunoelectrophoresis autoradiography. The metabolically labeled macrophage alpha 1-proteinase inhibitor had the same electrophoretic mobility as did pure rat serum alpha 1-proteinase inhibitor. Moreover, monospecific antiserum to rat serum alpha 1-proteinase inhibitor precipitated a 35S-labeled protein from macrophage culture supernates, and this protein was shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis to have a molecular weight close to that of rat serum alpha 1-proteinase inhibitor. Cycloheximide (an inhibitor of protein synthesis) at a concentration of 0.50 micrograms/ml, inhibited secretion of labeled inhibitor. This, cultured rat alveolar macrophages secrete detectable concentrations of a proteinase inhibitor believed to play a major role in lung defense.

Animals↗