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A Janoff

Publications and source records attributed to A Janoff.

At least 73 records · Page 4Linked to original sources
PubMed1980

Partial purification and characterization of mouse peritoneal exudative macrophage elastase.

Mouse peritoneal exudate macrophage elastase can be significantly purified with 60% recovery of the starting activity by affinity chromatography against SDS-treated alpha-elastin covalently linked to agarose beads. The enzyme has an apparent Mr of 26 500 based on SDS-acrylamide gel electrophoresis. Molecular sieving chromatography on Sephadex gel gives a Mr for macrophage elastase of 21 000--28 000. The enzyme is not inhibited by chloromethyl ketone inactivators specific for pancreatic and leukocyte elastase nor by phenylmethylsulfonyl fluoride. Macrophage elastase also does not bind to tritiated diisopropylphosphorofluoridate. The enzyme is inhibited by EDTA and thus appears to be a metallo-protease. Macrophage elastase is resistant to human alpha 1-proteinase inhibitor and to human and mouse alpha 2-macroglobulin. In view of its lack of susceptibility to these endogenous serum proteinase inhibitors, macrophage elastase may play an important role in physiological and pathological remodeling of connective tissues.

Animals↗
PubMed1980

Desmosine radioimmunoassay for measuring elastin degradation in vivo.

Desmosine is a cross-link amino acid unique to elastin. Previous work has shown that during turnover in the body, desmosine is not reused, and that desmosine is not absorbed from the intestine. Instead, all desmosine released in the course of elastin metabolism is excreted in the urine attached to low molecular weight peptides. Therefore, measurement of desmosine in acid-hydrolysates of urine might be used to monitor elastin breakdown in several pathologic states, including pulmonary emphysema. In the present report, we have described a sensitive, highly specific radioimmunoassay capable of detecting as little as 200 pg of desmosine in acid-hydrolysates of urine. The assay was specific for desmosine; cross-reactivity with merodesmosine, isodesmosine, lysine, and mixed amino acids was 0.25%, 0.1%, less than 0.0003%, and 0%, respectively. Twenty-three normal, nonsmoking subjects had a mean 24-hr desmosine excretion of 47 +/- 15 microgram. In a group of smokers with evidence of chronic obstructive disease and/or lung infection, the values for desmosine excretion ranged from 40 to 400 microgram/24 h. Desmosine radioimmunoassay may find application in the study of diseases involving increased destruction of elastin in the body.

Adolescent↗
PubMed1980

Potential mediator of inflammation. Phagocyte-derived oxidants suppress the elastase-inhibitory capacity of alpha 1-proteinase inhibitor in vitro.

Human polymorphonuclear leukocytes, monocytes, or pulmonary alveolar macrophages, stimulated in vitro by phorbol myristate acetate (PMA), released reactive oxygen species able to suppress the elastase inhibitory capacity (EIC) of human serum. Immunoelectrophoresis using antibodies against alpha(1)-proteinase inhibitor (alpha(1)-Pi) and elastase showed that inactivation of alpha(1)-Pi was responsible for the decreased serum EIC. Treatment of phagocyte-inactivated serum with a reducing agent (dithiothreitol) resulted in significant recovery of EIC, suggesting that alpha(1)-Pi had been oxidatively inactivated. Serum EIC was partially protected by superoxide dismutase or catalase. Hydrogen peroxide alone had no effect on serum EIC. Thus, neither H(2)O(2) nor O(2) (-) alone, but a product of the two, may have oxidatively inactivated alpha(1)-Pi. In support of the foregoing, neutrophils or monocytes from a patient with chronic granulomatous disease failed to produce detectable levels of O(2) (-) after incubation with PMA. These cells also failed to suppress serum EIC. In the case of PMA-stimulated polymorphonuclear leukocytes or monocytes, extracellular myeloperoxidase may have also played a role in alpha(1)-Pi inactivation since serum EIC was partly protected by azide, cyanide, or the depletion of extracellular chloride. Indeed, in a cell-free system consisting of purified myeloperoxidase, a glucose oxidase-H(2)O(2)-generating system, and Cl(-), the EIC of human serum or purified alpha(1)-Pi could also be suppressed. Omission of any single reactant prevented this effect, as did NaN(3) or catalase, suggesting that enzymatically active myeloperoxidase and H(2)O(2) were necessary. Immunoelectrophoresis of myeloperoxidase-inactivated serum showed that, as before, inactivation of alpha(1)-Pi was responsible for the decreased EIC. Treating myeloperoxidase-inactivated serum with dithiothreitol led to significant recovery of EIC, again suggesting that oxidative inactivation of alpha(1)-Pi had occurred. Oxidative inactivation of alpha(1)-Pi in the microenvironment of inflammatory cells, at sites of acute or chronic inflammation, may allow proteases released from these cells to damage adjacent connective tissue components more readily.

Glucose Oxidase↗
PubMed1980

Inactivation of bronchial mucous proteinase inhibitor by cigarette smoke and phagocyte-derived oxidants.

Freshly prepared aqueous solutions of cigarette smoke suppressed the elastase inhibitory capacity (EIC) of the acid-stable proteinase inhibitor present in bronchial mucus (BMPi) and human seminal plasma (HUSI-I). Thin-layer gel-immunofiltration analysis of mixtures of smoke-treated BMPi and human leukocyte elastase showed decreased elastase: BMPi complexes, increased uncomplexed BMPi and increased free elastase. Phenolic antioxidants prevented the suppression of the EIC of BMPi or HUSI-I by cigarette smoke. In addition, treatment of BMPi or HUSI-I with chemical oxidants caused a similar suppression of EIC. Furthermore, treatment of BMPi or HUSI-I with the phagocyte-derived oxidizing system, myeloperoxidase + H2O2 + Cl-, suppressed EIC. Finally, the functional activity of BMPi was significantly reduced in tracheal aspirates of human smokers compared to that of nonsmokers. These results support the hypothesis that local inactivation of BMPi in the conducting airways of the lung by inhaled cigarette smoke or by phagocyte-derived oxidants may play a role in the pathogenesis of obstructive lung disease in smokers.

Bronchi↗
PubMed1979

Cigarette smoke inhalation decreases alpha 1-antitrypsin activity in rat lung.

Brief inhalation exposure of rats to three or six puffs of cigarette smoke significantly decreases elastase inhibitory capacity per milligram of alpha 1-antitrypsin in lung lavage fluid. This effect is not observed in ozone-tolerant rats and can be reversed by treating the lung lavage fluid from smoke-exposed rats with reducing agents. Samples of human serum obtained immediately after smoking also show decreased elastase inhibitory capacity per milligram of alpha 1-antitrypsin. Again, elastase inhibitory capacity can be restored by treatment with a reducing agent. Cigarette smoking may cause emphysema by inactivating alpha 1-antitrypsin through oxidation.

Animals↗
PubMed1979

In vitro suppression of serum elastase-inhibitory capacity by reactive oxygen species generated by phagocytosing polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMN) phagocytosing opsonized antigen-antibody complexes, produce dialyzable species of activated oxygen which are capable of partially suppressing the elastase-inhibiting capacity (EIC) of whole human serum or purified human alpha1-proteinase inhibitor. Serum EIC was partially protially protected by superoxide dismutase, catalase, or mannitol, suggesting that hydroxyl radical, formed by interaction of superoxide radical and hydrogen peroxide, might be responsible for this effect. NaN3 also partly protected EIC, implicating myeloperoxidase-mediated reactions as well. An artificial superoxide rradical-generating system, involving xanthine and xanthine-oxidase, could be substituted for phagocytosing PMN with resultant EIC suppression. These results are consistent with previous demonstrations of the release of potent oxidants by stimulated PMN, as well as earlier studies from our laboratory showing sensitivity of alpha1-proteinase inhibitor to inactivation by oxidants. Oxidative inactivation of proteinase inhibitors in the microenvironment of PMN accumulating at sites of inflammation may allow proteases released from these cells to more readily damage adjacent connective tissue structures.

Catalase↗
PubMed1979

Effects of cigarette smoke on elastase secretion by murine macrophages.

Mice were chronically exposed to cigarette smoke for various time periods up to 4 weeks. As a consequence of the exposure, there was an increase in the number of alveolar macrophages obtained from the lungs of these mice. Light microscopic examination of cultured cells revealed increased numbers of highly pleomorphic cells filled with pigmented residues of cigarette smoke. These cells were more mitotically active, with a five fold increase in the number of alveolar colony-forming cells compared to the controls. When macrophages derived from mice exposed to cigarette smoke were cultured at high density in the absence of serum, they secreted significantly greater amounts of elastase than did the same number of control macrophages. At concentrations as low as 0.50 micrograms/ml, cycloheximide reversibly inhibited elastase secretion from both the control and experimental cultures. The effects of cigarette smoke inhalation on elastase secretion by alveolar macrophages do not appear to be a direct effect of cigarette smoke on these cells. Exposure of normal mouse macrophages in vitro to pulses of aqueous extracts of cigarette smoke, while significantly increasing secretion of elastase by peritoneal exudative macrophages, did not augment that of resident or exudative alveolar macrophages. These results suggest that the increased elastase secretion observed with the use of cultured macrophages derived from mice exposed to cigarette smoke is the result of either indirect activation of resident macrophages or the recruitment of a highly activated exudative population into the lungs of the exposed animals.

Animals↗
PubMed1979

Lung injury induced by leukocytic proteases.

Human polymorphonuclear neutrophilic leukocytes (PMNs) contain large amounts of neutral proteases that can degrade elastin, collagen, proteoglycan, and basement membrane. The instillation of one of the purified enzymes (elastase) into dog lungs in vivo causes degradation of elastic fibers and other alveolar septal components and results in anatomic changes similar to those of human pulmonary emphysema. Cigarette smoking is a major risk factor associated with pulmonary emphysema in man. One mechanism for this association may be interference with the regulation of PMN elastase activity by alveolar antiproteases. This possibility is supported by the observation that the oxidizing activity of tobacco smoke inactivates alpha 1-proteinase inhibitor in vitro. Macrophages also secrete an elastolytic protease, albeit at low levels. The short-term exposure of cultured mouse macrophages to cigarette smoke augments the rate of elastase secretion by these cells. Mouse macrophage elastase is not inhibited by alpha 1-proteinase inhibitor or alpha 2-macroglobulin. This unusual property of macrophage elastase may facilitate its attack upon elastin over prolonged intervals despite very low levels of macrophage elastase production. A unified hypothesis of lung injury in pulmonary emphysema is presented, involving both PMN and macrophage elastases and the actions of cigarette smoke. (Am J Pathol 97:111--136, 1979).

Animals↗
PubMed1978

Possible mechanisms of emphysema in cigarette smokers. Release of elastase from human polymorphonuclear leukocytes by cigarette smoke condensate in vitro.

Human polymorphonuclear leukocytes, when incubated in vitro with cigarette smoke condensate, release beta-glucuronidase, acid phosphatase, and elastase and fail to exclude trypan blue. The released elastase is active on an elastin substrate even in the continuing presence of cigarette smoke condensate. When mixtures of human polymorphonuclear leukocytes and cigarette smoke condensate are instilled into rat lungs in vivo, the elastase is also released and can be traced to conneective tissue targets by immunohistochemical and enzyme-histochemical techniques.

Acid Phosphatase↗
PubMed1978

Possible mechanisms of emphysema in smokers. In vitro suppression of serum elastase-inhibitory capacity by fresh cigarette smoke and its prevention by antioxidants.

Freshly prepared, aqueous solutions of cigarette smoke suppressed the elastase-inhibitory capacity of human serum. Immunoelectrophoresis of mixtures of aqueous smoke solution, human serum, and pancreatic elastase showed decreased elastase/alpha1-proteinase inhibitor complexes and increased free, active protease. Phenolic antioxidants prevented the suppression of serum elastase-inhibition by cigarette smoke. By contrast, treatment of serum with model oxidants caused a similar suppression of elastase inhibition. These results suggested that emphysema in cigarette smokers might be due, in part, to the local suppression of alpha1-proteinase inhibitor in lung by oxidizing agents present in cigarette smoke.

Depression, Chemical↗
PubMed1977

Experimental emphysema induced with purified human neutrophil elastase: tissue localization of the instilled protease.

Human neutrophilic polymorphonuclear leukocyte (PMN) elastase was purified by affinity chromatography to greater than 95% homogeneity as judged by disc-gel electrophoresis. Dog lung elastin was prepared from alveolar-enriched tissue by prior extraction of soluble and collagenous lung proteins with 0.1 M NaOH at 98 degrees C. Digestion of the remaining insoluble residue by the purified PMN enzyme was monitored by Lowry assay of acid-soluble peptides released. The PMN enzyme possessed 60% of the digestive activity of crystallized porcine pancreatic elastase (weight:weight comparison) when tested in vitro against this substrate in phosphate-NaCl buffer at pH 7.5. Whole tissue studies were then performed in lungs of laboratory animals. One-ml samples containing purified PMN elastase were instilled into lavaged and saline-perfused isolated dog lung at the level of the sixth to seventh generation bronchus. Treatment with 384 mug of the PMN enzyme produced anatomic emphysema after a 90-min incubation at room temperature, which was comparable to that produced by 100 mug of porcine pancreatic elastase. Frozen sections of treated and control lungs were examined for the presence of PMN elastase by the indirect immunoperoxidase method using a monospecific rabbit antiserum against PMN elastase as the primary stain. Light microscopy revealed elastase bound to connective tissue in the treated lungs, in close proximity to aldehyde-fuchsin-counterstained elastic fibers. A similar experiment was tn of enzyme solutions containing 1;0 mg of elastase per ml produced discrete lesions within 90 min, as before. Light microscopic studies in conjunction with the indirect immunoperoxidase staining method again demonstrated elastase in association with connective tissue elements in the lesion area. In addition, part of the instilled protease could be demonstrated within alveolar macrophages. Electron microscopy combined with immunoperoxidase staining revealed direct attachment of th einstilled enzyme to elastic fibers within alveolar septa. In enzyme-treated tissue, some septa showed severe depletion of intercellular structures with the exception of colalgen, which was generally preserved. These results show that human leukocyte elastase penetrated dog alveolar septal connective tissue after airway instillation and that the enzyme attaches to elastic fibers, inducing histologic changes comparable to thos seen in human emphysema.

Animals↗
PubMed1977

Possible mechanisms of emphysema in smokers: cigarette smoke condensate suppresses protease inhibition in vitro.

To study the possible role of suppression of antiproteases by cigarette smoke in the pathogenesis of pulmonary emphysema in smokers, the following experiments were carried out. Elastin-agarose gels were impregnated with cigarette smoke condensate dissolved in dimethyl sulfoxide or with the solvent alone. This procedure affected neither local pH of the gel nor subsequent physical behavior (diffusion) of antiprotease. Elastases from various sources were then allowed to diffuse through the impregnated gels toward a counter-diffusing sample of antiprotease. The effectiveness of the antiprotease in blocking the enzyme was determined from the extent of elastolysis. The elastin substrates used included beef ligament elastin and dog lung elastin. The enzymes used were porcine pancreatic elastase and pure human leukocyte elastase. The antiproteases tested included human serum, pure human a1-antitrypsin, human bronchopulmonary lavage fluid, and a synthetic chloromethyl ketone inactivator of elastase. The results showed that whole, unfractionated cigarette smoke condensate suppressed all of the antiproteases tested, except for the chloromethyl ketone. These observations are discussed in terms of the protease-pathogenesis model of pulmonary emphysema.

Humans↗