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Biomedical subjects

A Janoff

Publications and source records attributed to A Janoff.

At least 37 records · Page 2Linked to original sources
PubMed1986

Inactivation of alveolar macrophage transglutaminase by oxidants in cigarette smoke.

The present study demonstrates the existence of tissue transglutaminase in rabbit alveolar macrophages and measures the effects of cigarette smoke extracts on the activity of enzyme partially purified from these cells. The effects of smoke on transglutaminase purified from guinea pig liver are also measured. A water soluble component of gas-phase cigarette smoke inhibits both enzymes in a dose-dependent manner, with a maximum inhibition of 65% occurring at concentrations of smoke as low as 1% in the case of the rabbit enzyme. The chemical oxidant N-chlorosuccinimide mimics the dose-dependent inhibitory effects of cigarette smoke on both enzyme systems. The thiol reducing agent, dithiothreitol, can prevent enzyme inactivation mediated by both smoke and N-chlorosuccinimide. Dithiothreitol is also capable of reversing inactivation mediated by cigarette smoke, but does not reverse inactivation caused by chemical oxidation with N-chlorosuccinimide. These data suggest that cigarette smoke inhibits transglutaminase activity via an oxidative mechanism that may selectively attack the active-site cysteine residue.

Animals↗
PubMed1986

Investigations into the biochemical mechanisms of pulmonary emphysema: effects of cigarette smoke on enzymes and anti-enzymes in the lung.

The mechanism(s) causing emphysema in the cigarette smoker are still poorly understood. However, circumstantial evidence is beginning to provide a tenuous link between smoking and the protease-antiprotease imbalance hypothesis. Independent effects on elastin synthesis may also be important. This triad of chemical and cellular events (leukocyte recruitment, inhibitor inactivation, depressed tissue repair) constitutes a reasonable first approximation from which to approach this complex question.

Animals↗
PubMed1986

A genetically engineered, mutant human alpha-1-proteinase inhibitor is more resistant than the normal inhibitor to oxidative inactivation by chemicals, enzymes, cells, and cigarette smoke.

Alpha-1-proteinase inhibitors (alpha 1PI) containing methionine (Met-358) or valine (Met----Val-358) at the reactive center were synthesized in and purified to homogeneity from recombinant yeast. The pure proteins were exposed to 1 of 4 different oxidizing systems: N-chlorosuccinimide (chemical oxidation), myeloperoxidase plus peroxide and halide (enzymatic oxidation), activated neutrophils (cellular oxidation), or gas-phase cigarette smoke. The effect of these treatments on the leukocyte elastase inhibitory function of both proteins was then assessed. After brief exposures, substantial inactivation of the normal inhibitor occurred, whereas the mutant inhibitor remained fully active. More prolonged exposures led to complete inactivation of the normal protein and partial inactivation of the mutant inhibitor. These results suggest that the reactive center methionyl residue in alpha 1PI is more rapidly affected by oxidants than are other oxidizable residues in the inhibitor; however, Met-358 is not the only residue in alpha 1PI whose modification can lead to the inactivation of the elastase inhibitory capacity of this protein.

Binding Sites↗
PubMed1986

The role of complement in cigarette smoke-induced chemotactic activity of lung fluids.

To help explain increased recruitment of leukocytes to smokers' lungs, we examined the possibility that the smoke-induced chemotactic activity in lung fluids may be partly complement-dependent. Acute inhalation exposure of rats to smoke from 2R1 Kentucky Reference cigarettes caused an increase in the leukocyte chemotactic activity of their lung secretions (collected by bronchoalveolar lavage 1 h after exposure). Prior depletion of circulating complement, by intravenous administration of purified cobra venom factor 24 h beforehand, prevented the smoke-induced rise in lavage fluid chemotactic activity. Crossed immunoelectrophoretic analysis of lavage C3 from smoke-exposed animals demonstrated a modified form of the molecule with increased electrophoretic (anodal) mobility. When 2 strains of mice, C5-deficient DBA/2 and C5-sufficient BALB/c, were acutely exposed to cigarette smoke, only the C5-sufficient animals (BALB/c) had increased neutrophil chemotactic activity in their lung fluids 1 h after exposure. These results show that the increased chemotactic activity in lung fluids of laboratory animals immediately after acute cigarette smoke exposure is complement-dependent.

Animals↗
PubMed1986

Tracing the leukocyte marker protein in lung fluids and lung-draining lymph nodes during endotoxemia in sheep.

Infusion of Escherichia coli endotoxin into sheep produces a form of acute lung injury that resembles the adult respiratory distress syndrome (ARDS). A large portion of the physiologic derangements produced by E. coli endotoxin in this model is thought to be granulocyte-dependent. We measured the level of L1, a granulocyte and monocyte marker protein, in various tissues and fluids after infusion of E. coli endotoxin into sheep. In an acute study, sheep received saline or 1.25 microgram/kg E. coli endotoxin dissolved in saline, or endotoxin after hydroxyurea-induced granulocytopenia. L1 was measured by radioimmunoassay in efferent lymph from the caudal mediastinal lymph node collected between 5 and 6 h after infusion. In addition, L1 was visualized in both lung-draining and extrapulmonary lymph nodes by indirect immunofluorescence. In a chronic study, sheep were prepared with lung lymph fistulas, and L1 was measured in draining pulmonary lymph, plasma, and bronchoalveolar lavage (BAL) fluid, serially, over a 24-h period after infusion. Mean L1 level in pulmonary lymph in the acute study was 6 times higher by absolute concentration, and 19 times higher when lymph flow rates were taken into account, in the sheep that received endotoxin than in saline-infused sheep or endotoxin-infused, granulocytopenic sheep. Fluorescence was greater in the outer cortical region adjacent to subcapsular, afferent sinuses of lung draining-lymph nodes of endotoxin-treated sheep than in the comparable nodes of saline-infused sheep and endotoxin-infused granulocytopenic sheep. In endotoxin-treated sheep, extrapulmonary lymph nodes were less reactive than lung-draining nodes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Subcellular localization and further characterization of a new elastase inhibitor from pneumococci.

Streptococcus pneumoniae contains an inhibitor of human neutrophil elastase. The agent does not inhibit other proteases, including neutrophil cathepsin G and pancreatic elastase. It is active in the presence of insoluble elastin as well as synthetic elastase substrates. The inhibitor is present in the pneumococcal cell membrane. [125I]elastase binding studies and inhibition experiments with intact bacterial autoplasts suggest that this agent has its elastase-binding site(s) exposed on the outside of the bacterial cell membrane. Native and randomized membrane vesicles also show equal inhibitory activity. Active inhibitor can be solubilized from pneumococcal membranes by treatment with a dipolar ionic detergent and can then be reconstituted, in active form, within artificial liposomes. Complex formation between the neutrophil elastase inhibitor and neutrophil elastase may involve noncovalent associations. Although elastase containing a covalently bound substrate analog no longer binds the pneumococcal inhibitor, the present study shows that complex formation is nevertheless independent of neutrophil elastase catalytic activity. Specific inhibitor activity and inhibitor release during bile salt-stimulated autolysis are greater in a nonnecrotizing pneumococcal strain (type I) than they are in a necrotizing strain (type III) or in Klebsiella pneumoniae. These results may help explain the frequent resolution of some pneumococcal pneumonias, despite the presence in the early pneumonic exudate of many neutrophils containing an elastolytic protease capable of injuring lung connective tissue.

Autolysis↗
PubMed1985

Elastase in tissue injury.

This article surveys elastinolytic proteinases in man, excluding enzymes of the pancreas and digestive tract. Special emphasis is placed on the elastase of polymorphonuclear neutrophils (PMN). The properties of this latter enzyme, its target molecules in plasma and connective tissues, and its endogenous regulators are briefly discussed. Persistent activity of the enzyme, even in the presence of its regulatory inhibitors, is explained. The chapter closes with a brief discussion of several pulmonary diseases in which elastase-mediated tissue injury is thought to play a role.

Blood Platelets↗
PubMed1985

A new elastase inhibitor from Streptococcus pneumoniae protects against acute lung injury induced by neutrophil granules.

A neutrophil elastase-inhibitor isolated from lysed pneumococcal cells, as well as trypsin-digest peptides derived from this factor, were tested for their ability to suppress acute lung injury in mice treated with human neutrophil granule extracts. Injury was assessed by measuring pulmonary sequestration of circulating 125I-labeled albumin, lung water, and lung hemoglobin. Both the native inhibitor and the tryptic-peptides gave good protection when preincubated with granule extract for brief periods before intrapulmonary instillation. Lesser, but still significant, protection was observed in the absence of preincubation. Protection was not simply due to addition of exogenous proteins to the granule extract because substitution of goat immunoglobulin for pneumococcal fraction was ineffective. These results suggest that pneumococcal elastase-inhibitors, recently described by us, may play a role in minimizing lung injury during pneumococcal pneumonia.

Acute Disease↗
PubMed1985

Elastases and emphysema. Current assessment of the protease-antiprotease hypothesis.

Many studies have been carried out in the past 10 yr dealing with the possible role of elastase in the pathogenesis of pulmonary emphysema. These include newer observations in animal models revealing augmentation of elastase-induced lesions by lathyrogens or by exposure to cigarette smoke. In general, the animal model experiments have focussed attention on repair-processes in the lung and shown that such processes may exert a major influence on the outcome of the initial proteolytic insult. Human studies exploring correlations between elastase levels in neutrophils or serum and development of disease have provided conflicting data; however, measurement of enzymes in pulmonary secretions have yielded more suggestive results. Assessments of lung elastase inhibitors in humans continue to support the importance of alpha-1-proteinase inhibitor in the protection of the lower respiratory tract, but newer information on locally produced, low molecular weight elastase inhibitors indicates that these, too, may play a significant role. Attempts have been made to link cigarette smoking to the development of emphysema at the chemical and cellular levels. These studies have focussed on: (1) the recruitment of elastase-producing leukocytes to smokers' lungs, (2) inactivation of lung elastase-inhibitors by tobacco products or by metabolites released from tobacco-stimulated lung cells, and (3) interference with elastin neosynthesis (repair) in the smoker. Additional information is also available concerning the biochemical properties of neutrophil and macrophage elastases, although it is still unclear which of these enzymes plays the predominant role in chronic lung injury associated with smoking. Perhaps the greatest advance in the emphysema field in recent years involves new discoveries concerning the structure and function of the alpha-1-proteinase (elastase) inhibitor. Applications of recombinant DNA technology and genetic engineering have made it possible to design modified inhibitors with striking new properties. These agents may enjoy significant clinical application in the not too distant future.

Animals↗
PubMed1985

Urinary excretion of desmosine (elastin cross-links) in subjects with PiZZ alpha-1-antitrypsin deficiency, a phenotype associated with hereditary predisposition to pulmonary emphysema.

To evaluate the concept that lung elastin degradation is accelerated in homozygous alpha-1-antitrypsin (AAT) deficient persons, we prepared acid hydrolysates of urine and used a radioimmunoassay for desmosine to measure urine concentrations of this elastin-specific cross-link in such persons and in control subjects. Excretion of desmosine in 17 homozygous AAT-deficient (PiZZ) patients with emphysema was compared with that in 27 patients with interstitial lung diseases (16 sarcoid, 5 idiopathic pulmonary fibrosis, 6 other interstitial lung diseases) and 26 healthy subjects. Both smokers and nonsmokers were present in all groups. Urinary desmosine concentration (microgram/100 mg creatinine) was 2.35 +/- 0.93 in the PiZZ patients, 2.49 +/- 1.01 in those with interstitial lung disease, and 2.05 +/- 0.54 in the healthy control subjects (p greater than 0.1, all comparisons). Because abnormal pulmonary elastolysis may be largely completed before symptoms of emphysema develop in AAT-deficient persons, we also tested 6 asymptomatic adults with homozygous AAT deficiency (PiZZ) and 5 PiZZ children. Urine desmosine (microgram/100 mg creatinine) was not significantly elevated in either group compared with that in the age-matched control subjects, although children (PiZZ and age-matched controls) showed higher excretions than did adults (6 asymptomatic PiZZ adults, 2.60 +/- 0.91; 5 PiZZ children, 3.27 +/- 0.62; 10 control children, 3.61 +/- 0.62). These data suggest that pathologic lung elastolysis in the PiZZ subject may constitute too small a fraction of total-body elastin turnover to be detected by this method.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1985

Cigarette smoke can activate the alternative pathway of complement in vitro by modifying the third component of complement.

Cigarette smoking is associated with significant increases in the number of pulmonary mononuclear phagocytes and neutrophils. A potent chemoattractant for these cells is C5a, a peptide generated during complement (C) activation. We, therefore, investigated the possibility that cigarette smoke could activate the complement system in vitro. Our results show that factor(s) (mol wt less than 1,000) present in an aqueous solution of whole, unfiltered cigarette smoke can deplete the hemolytic capacity of whole human serum in a dose-dependent manner. The particle-free, filtered gas phase of cigarette smoke is inactive. The smoke factor(s) do not activate serum C1, but do deplete serum C4 activity. Treatment of purified human C3 with whole smoke solution modifies the molecule such that its subsequent addition to serum (containing Mg/EGTA to block the classical pathway) results in consumption of hemolytic complement by activation of the alternative pathway. Smoke-modified C3 shows increased anodal migration in agarose electrophoresis, but this is not due to proteolytic cleavage of the molecule as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast to methylamine-treated C3, C3 treated with smoke is only partially susceptible to the action of the complement regulatory proteins Factors H and I. In addition, smoke-modified C3 has diminished binding to Factor H as compared with methylamine-treated C3. Finally, smoke-modified C3 incorporates [14C]methylamine which suggests that the thiolester bond may be intact. These data indicate that aqueous whole cigarette smoke solution can modify C3 and activate the alternative pathway of complement, perhaps by a previously unrecognized mechanism. Should this occur in vivo, complement activation might partly account for the extensive pulmonary leukocyte recruitment observed in smokers.

Complement Activation↗
PubMed1984

Inhibitors of human neutrophil elastase in extracts of Streptococcus pneumoniae.

Alveolar architecture is spared during most pneumococcal pneumonias, despite the presence in pneumonic exudate of many neutrophils containing a potent elastase. We explored the possibility that pneumococci might contain an inhibitor of this enzyme. We found that pneumococcal extracts prepared by sonication or by lysis with sodium deoxycholate contained 2 different inhibitors of human neutrophil elastase. Both inhibitors were specific for neutrophil elastase and did not affect pancreatic elastase or trypsin. Inhibitor I was partly purified by affinity chromatography and preparative acrylamide gel electrophoresis and shown to be a negatively charged, low molecular weight substance that inhibited competitively (Lineweaver-Burk analysis). Inhibition depended on ionic interaction with the cationic enzyme and could be blocked by 0.15 M NaCl. For this reason, the first agent seemed unlikely to play an important role in modulating neutrophil elastase activity in inflammatory exudates and was not studied further. The second agent (Inhibitor II) eluted in the high molecular weight fraction during Sephacryl S-300 chromatography. Gradient SDS-polyacrylamide gel electrophoresis of partly purified Inhibitor II revealed an apparent molecular weight of 140,000 daltons. This agent inhibited noncompetitively and remained active in the presence of 0.15 M NaCl. Prolonged incubation with TPCK-trypsin resulted in cleavage of Inhibitor II into smaller fragments, which could be further dissociated by reduction with dithiothreitol. Inactivation of neutrophil elastase with N-acetyl-alanyl-alanyl-prolyl-valyl-chloromethyl ketone prevented complex formation between this enzyme and Inhibitor II, suggesting that an unblocked binding pocket in neutrophil elastase is required for its complexation to the noncompetitive pneumococcal inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Biomechanical Phenomena↗
PubMed1984

Acute response to elastase in sheep lungs measured with Ga-67.

The early inflammatory changes in sheep's lungs were studied with Ga-67 citrate, injected i.v. immediately following intrabronchial instillation of different doses of elastase into the right diaphragmatic lobes of 15 sheep. The elastase-induced lesions in the first five sheep (two received 4,000 units; three got 6,000) were imaged up to seven times in an 8-day period to measure the temporal changes in the lesion and to select the appropriate imaging time; the other ten sheep (800-8,000 units) were imaged once at 52 hr. Localization of Ga-67, as seen on the posterior and right lateral projections, was confined to a well-circumscribed region in the right lung field. The lesion could be detected as early as 4 hr after elastase instillation. It decreased to 60% of its initial area at 4 hr, while the total Ga-67 activity in the sheep remained constant after 52-75 hr. Gallium-67 uptake in the lesion correlated positively with the dose of elastase (r = 0.88, p less than 0.001) and with the reduction in perfusion, as determined 4 wk after the elastase instillation (r = 0.66, p less than 0.05). Early Ga-67 uptake in inflammatory lung lesions could therefore be used as a reliable predictor of the size of the acute elastase-induced inflammatory reaction, as well as of the sequelae involving the regional vascular supply 4 wk later.

Animals↗
PubMed1983

Biochemical links between cigarette smoking and pulmonary emphysema.

A large body of circumstantial evidence has accumulated in the last 20 yr, suggesting that alveolar effacement in pulmonary emphysema is due to unrestrained proteolytic (elastolytic) activity in lung connective tissue. Even though this hypothesis still requires rigorous proof, newer information is rapidly developing that links the protease-pathogenesis model to the primary environmental risk factor associated with the disease, namely cigarette smoking. A triad of chemical and cellular effects produced by cigarette smoke have been suggested as factors contributing to altered elastin metabolism and eventual development of emphysema in smokers. The present article reviews some of these observations, seeks to place them within the overall framework of the protease model, and attempts to raise questions for future study.

Biomechanical Phenomena↗
PubMed1983

Cigarette smoke blocks cross-linking of elastin in vitro.

Water-soluble components of the gas phase of filtered cigarette smoke inhibit formation of covalent desmosine cross-links during conversion of tropoelastin to elastin in vitro. These same smoke components also suppress lysyl-oxidase-catalyzed oxidation of lysine epsilon-amino groups in tropoelastin (the chemical step preceding formation of all elastin cross-links, including desmosine) in a dose-dependent fashion. However, gas phase smoke does not block the oxidation of diaminopentane by lysyl oxidase. Thus, gas phase cigarette smoke may possess substrate-directed (rather than enzyme-directed) inhibitory components capable of interfering with elastin cross-linking in vitro. Similar effects occurring in smokers' lungs could impede elastin repair and contribute to the development of pulmonary emphysema.

Animals↗