Faculty Bibliography

Several amino acid derived azolides (I) have been synthesized and investigated for their inhibitory activity toward human leukocyte elastase and porcine pancreatic elastase. The inhibitory activity was found to be dependent on the nature of the precursor amino acid ester. Thus, compounds derived from L-valine methyl ester 3, L-norvaline methyl ester 5, DL-norleucine methyl ester 9, and L-methionine methyl ester 10 were found to inhibit irreversibly both enzymes. Compound 10 was found to be a specific and selective inhibitor of human leukocyte elastase. In contrast to these, inhibitors derived from glycine methyl ester 1, D-valine methyl ester 4, and D-norvaline methyl ester 6 were found to be inactive. The results of the present study show that latent isocyanates derived from appropriate amino acids can serve as selective inhibitors of serine proteases and are of potential pharmacological value

This study examines the bronchial alveolar lavage (BAL) samples from a group of patients with acute bacterial pneumonia (n = 13) and makes a comparison with a control group (n = 5). The proteinase inhibitory capacity was examined and found to be composed primarily of alpha 1-proteinase inhibitor (PI, alpha 1-antitrypsin) and, to a lesser extent, bronchial mucosal inhibitor. Although the average PI concentration was elevated approximately 5-fold in the pneumonia group, its inhibitory function against elastase was decreased 15-fold when compared with that in the control group. The pneumonia group showed an increased concentration of immunologically identified elastin-derived peptides. Some of the BAL fluid from patients with pneumonia showed elastolytic activity against amorphous insoluble lung elastin. The majority of the elastase appears to be of neutrophil origin. Bronchial mucosal inhibitor is shown to be a component of both normal and pneumonia BAL fluids by both immunologic quantitation and by its resistance to perchloric acid inactivation. Compared with those from control subjects, BAL samples from patients with acute bacterial pneumonia showed a decreased proteinase inhibitor function and both increased elastolytic activity and elastin-derived peptide concentration

Several a-pyrones have been synthesized and investigated for their in vitro inhibitory activity using a-chymotrypsin (a-CT), porcine pancreatic elastase (PPE) and human leukocyte elastase (HLE). 4-Hydroxy-6-undecyl-2H-pyran-2-one 4, 4-Hydroxy-6-[(1-butyl)heptyl]-2H-pyran-2-one 5 and 4-Methoxy-6-[(1-butyl) heptyl]-2H-pyran-2-one 6 were found to be specific inhibitors of HLE. These compounds constitute a promising new class of HLE inhibitors

A quantitative assay of neutrophil degranulation was developed using flow cytometry. Dog neutrophils were purified to greater than 95% purity and viability by isopyknic density centrifugation in an isosmotic medium. These cells concentrated the fluorochrome acridine orange (AO) in their azurophilic granules, but not in specific granules. Also contained in the azurophilic granules are elastase, myeloperoxidase, and approximately 50% of the lysozyme activity. The fluorochrome was released concomitantly with elastase activity, as shown by flow cytometry, fluorescence microscopy, and biochemical assay in response to the ionophore A23187. By flow cytometry, unstimulated cells are distributed in a single broad peak of high fluorescence intensity. With increasing concentrations of A23187 (0.48-4.80 microM), a greater proportion of the cells shifted to a single peak of low fluorescence intensity. Few cells with intermediate fluorescence were observed. These analyses revealed that the neutrophils degranulated in a quantal, all-or-none response

Although there is good evidence for the presence of human neutrophil (PMN) collagenase, only moderate purification has been reported. The probable explanation for this fact is that most assays used to specifically measure collagenase activity are not reliable if high levels of several different proteases are also present in the assay mixture. The PMN granule is just such a concentrated mixture. Therefore, polyacrylamide gel electrophoresis was used to identify and quantitate the alpha 1 3/4 and alpha 2 3/4 cleavage products diagnostic for mammalian collagenase. White cells (85% PMN's) were lysed in 0.34 M sucrose and granules were obtained. The granules were lysed by sonication, and the lysate was chromatographed on a Sephadex G-200 column followed by a Trasylol-Sepharose 4B column. This procedure resulted in a 1350-fold purification and a yield of 75 micrograms of enzyme/unit of blood. The collagenase was inhibited by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid but not by sulfhydryl or serine protease inhibitors. The preparation was free of elastase, which has been shown to cleave type III collagen into alpha 1 3/4 and alpha 1 1/4 pieces. The pI of collagenase was shown to be 4.7 by isoelectric focusing, and the enzyme lost activity below a pH of 6.5 if collagen was absent. Antiserum was produced by 100-micrograms injections of the purified collagenase into rabbits. Titers were measured by the enzyme-linked immunosorbent assay. For determination of the specificity, collagenase and PMN extract were isoelectrically focused and blotted onto nitrocellulose. The antibody recognized only one band of protein in the PMN extract, which comigrated with the purified collagenase.

Lobar intrabronchial instillation of cadmium chloride (200 micrograms/ml) in saline causes a reproducible acute pulmonary inflammation in dogs. The influx of inflammatory neutrophils from the circulation into the alveolar spaces reaches a maximum approximately 16 hours after the cadmium chloride treatment in the treated lobe, while the controlateral lung appears normal. Morphometric quantitation of peroxidase-positive (azurophilic) granules in the inflammatory neutrophils shows a 74% loss of these granules, with little or no loss of the peroxidase-negative (specific) granules. These data are in good agreement with the measured loss of intracellular elastase, an enzyme known to be localized in the azurophilic granules. The results suggest that degranulation of azurophilic granules may occur selectively during this chemically induced acute inflammation

The major serum inhibitor of proteolytic activity, alpha-1-proteinase inhibitor (alpha-1-PI), (or alpha-1-antitrypsin) can be readily inactivated by oxidation [Carp, H. & Janoff, A. (1978) Am. Rev. Resp. Dis. 118, 617-621]. This inactivation appears to be due to the oxidation of a critical methionine(s) in alpha-1-PI that is required for the inhibition of elastase activity. An enzyme from Escherichia coli that reduces methionine sulfoxide residues in protein [Brot, N., Weissbach, L., Werth, J. & Weissbach, H. (1981) Proc. Natl. Acad. Sci. USA 78, 2155-2158] can restore the biological inhibitory activity of canine oxidized alpha-1-PI

The objective of this study was to develop an animal model representative of chronic human alpha-1-proteinase inhibitor deficiency. Eight dogs were treated with a mild oxidizing agent, chloramine T, with varying regimens for 3--27 wk. The capacity of the serum to inhibit both trypsin and elastase was examined and found to respond differently. Although immunologically determined levels of protease inhibitor did not change, the ability of serum to inhibit elastase in an in vitro assay decreased in direct response to chloramine T treatment. The trypsin inhibitory capacity was less affected. Emphysemalike alterations in lung morphology were observable when histologic sections were evaluated both subjectively and objectively by mean linear intercept measurements. The data suggest that this model parallels the emphysema associated with the genetic alpha-1-proteinase inhibitor deficiency in man

The protease hypothesis of emphysema development evolved from systems using intratracheal instillation or aerosols of heterologous enzymes, such as papain or porcine pancreatic elastase, which bear no relation to the animal species treated. Although these enzymes did produce experimental emphysema, their exogenous origin and superphysiological dosages limit their use in definitive model systems. The observation that dog leukocyte homogenates could induce canine emphysema led us to purify the causative agent from canine neutrophils. This report establishes that a single elastolytic enzyme from dog neutrophils is responsible for inducing experimental emphysema in the dog. Two purification methods were employed. The first used solvents of increasing ionic strength in a sequential extraction of acetone powders of purified dog neutrophils. The ability to initiate emphysema-like lesions was tested in every fraction of the purification and was localized in the extract with the highest true elastolytic activity. The second purification involved neutrophil intracytoplasmic organelle fractionation and established that only extracts of the lysosomal granules were capable of emphysema induction. Finally, the enzyme was purified to homogeneity from the granules using affinity chromatography and was shown to be a true elastase. Emphysema development was quantitated using mean linear intercept and was shown to be dependent on elastase concentration. There does not appear to be any other single enzyme in the canine neutrophil capable of inducing experimental emphysema