Ronald C. Roberts

Characterization of Dusts Collected from Swine Confinement Buildings

As part of a project to evaluate health hazards for workers in swine confinement buildings, the air in 21 different buildings was sampled with 37 mm cassette filters with and without cyclone preselectors and with cascade impactors. Filter results yielded a mean total aerosol of 6.3 mg/m3, a mean respirable aerosol of 0.5 mg/m3; the geometric mean diameter was 2.9 microns. Cascade impactor measurements revealed a mean total aerosol of 7.6 mg/m3, a respirable aerosol of 2.5 mg/m3 and a mass median diameter of 9.6 microns. The two major constituents in these aerosols were grain particles and dried fecal matter. The grain particles were larger than fecal particles and proportionately more abundant in finishing buildings where 50 kg X 100 kg animals are housed. Therefore the respirable fraction was less in finishing buildings than in farrowing and nursery buildings. Culturing of settled dusts yielded six different mold species, with the highest counts for Verticillium sp. (5 X 10(2) cfu/mg dry dust) grown at 37 degrees C. Thermophilic Actinomycetes and both gram negative and gram positive bacteria were isolated. Azocasein proteinase activity was found in most dust samples analyzed. This dust had a protein content of about 23% and a mean adsorbed ammonia content of 0.4%.

Human alpha-2-macroglobulin. Studies on the electrophoretic heterogeneity.

Purified alpha-2-macroglobulin may be resolved into as many as five electrophoretic bands on selected polyacrylamide gel systems. The microheterogeneity does not result from prior proteolytic attack but appears to correspond to different conformational states of the inhibitor. Trypsin binding capacity and the extent of subunit cleavage into 120,000 and 70,000 dalton fragments by mild alkaline treatment are related to the proportion of fast and slow electrophoretic forms. Study of proteinase binding after electrophoretic separation by special zymogram techniques confirms that the fastest electrophoretic form has very low binding capacity. No electrophoretic differences conld be observed in alpha-2-macroglobulin derived from cystic fibrosis plasma relative to control alpha-2-macroglobulin. Alpha-2-macroglobulin appears to exist as a simple, slow electrophoretic form in fresh plasma but converts into faster forms upon aging the plasma or during purification. Characterization of the electrophoretic microhetergeneity of alpha-2-macroglobulin preparations should be a prerequisite for the study of its proteinase binding properties.

Functional activities and nonenzymatic glycosylation of plasma proteinase inhibitors in diabetes.

The functional activity of three of the major plasma protease inhibitors, alpha 1-proteinase inhibitor, antithrombin III and alpha 2-antiplasmin, has been measured in a series of persons with diabetes mellitus and compared with healthy controls. The mean specific functional activity of both diabetic plasma antithrombin III (50.8 +/- 7.0 U/mg) and alpha 1-proteinase inhibitor (35.3 +/- 5.6 mU/mg) was found to be significantly lower than in healthy controls (57.9 +/- 6.0 U/mg) and (42.2 +/- 10.7 mU/mg). No difference was detected in alpha 2-antiplasmin activity or levels. The glycosylated protein fraction of 83% of diabetic plasmas showed the presence of less than 1% of the total alpha 1-proteinase inhibitor when isolated by phenylboronate affinity chromatography. Incubation of normal plasma with 250 nmol/l glucose (a level approximately 6 X higher than encountered in uncontrolled diabetes) resulted in a 17% and 6% decrease in antithrombin III and alpha 1-proteinase inhibitor activities. We conclude that the decrease in specific activity of alpha 1-proteinase inhibitor is not related to nonenzymatic glycosylation, but the decrease in antithrombin III specific activity may be related.

Comparison of the structure and aspects of the proteinase-binding properties of cystic fibrotic alpha-macroglobulin with normal alpha 2-macroglobulin.

Summary: Considerable attention has been focused recently on α2-macro-globulin (α2M), a major endopeptidase inhibitor in blood plasma, as a possible source of the primary defect in cystic fibrosis (CF). We report here studies designed to compare the structure of CF α2M with normal α2M to determine if there is a difference. The physicochemical properties of purified α2M as revealed by various electrophoretic techniques, covalent proteinase binding properties, and primary structural studies on a variety of partial hydrolyzates of CF α2M and normal α2M are compared. These studies were carried out on eight different individual isolates of CF α2M and three age-matched normal α2M preparations and α2M isolated from fetal cord blood. Three properties of CF α2M were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): (1) the existence of four identically-sized subunits in the native molecule (10), (2) the cleavage of this subunit into fragments of approximately 100,000 daltons upon interaction with proteinases (10), and (3) the cleavage of an alkaline/heat sensitive bond to produce 120,000 and 60,000 dalton fragments (11). Both CF and normal α2M were cleaved to the extent of 79–87%. CF α2M behaves identically with normal α2M with regard to all these properties. Salvesen and Barrett (24) have demonstrated that varying proportions of several [125I]-labeled proteinases form SDS-stable, non-reducible links to normal α2M. Two of the CF α2M preparations were studied to determine if similar covalent binding of proteinases occurred. The positions of the labeled and % of proteinase bound bands in SDS/reduced PAGE system were identical for normal α2M and CF α2M. These results indicate that CF α2M behaves normally with regard to covalent binding of proteinases. Qualitative comparison of the peptide fragments separated by SDS-PAGE or isoelectric focusing of CF and normal α2M produced by partial proteolysis with trypsin, chymotrypsin or Staphylococcus aureus V-8 proteinase did not reveal any differences unique to CF α2M. The cyanogen bromide fragmentation studies and the cysteine cleavage studies also indicated that no major change in the positions of methionyl residues or cysteinyl/cystinyl residues has occurred in CF α2M. The failure of all these different studies and those reported by others to demonstrate any differences between CF and normal α2M makes it highly unlikely that there is a primary defect in α2M in CF.Speculation: The abnormalities previously reported that are associated with α2-macroglobulin-proteinase complexes in cystic fibrosis do not arise from a primary defect in the structure of α2-macroglobulin.

Antibody-independent complement consumption by Micropolyspora faeni.

Micropolyspora faeni is an etiologic agent of hypersensitivity pneumonitis, a disease with an ill-defined mechanism of pathogenesis. Many reports have suggested an immunologic mechanism. A number of laboratories have demonstrated a nonantibody-mediated activation of the complement cascade. This study was undertaken to define the pathway of the complement consumption induced by M. faeni. We utilized an extract of M. faeni grown on synthetic media rather than the more common double dialysis extracts. Complement consumption by this extract was easily demonstrable in the absence of detectable antibody, but was inhibited by EDTA and MgEGTA. With the exception f C1, all of the early components of the classical pathway were markedly reduced following incubation of normal human serum with this extract. C1 was poorly consumed. In addition, factor B conversion to B was not inhibited by 10 mM MgEGTA suggests that the alternative pathway may also be affected. The generation of M. faeni-dependent chemotactic factor(s) from serum demonstrates that the complement cascade is being activated rather than inhibited.

Methionine oxidation and inactivation of α1-proteinase inhibitor by Cu2+ and glucose

The effect of glucose/Cu2+ incubation on (a) pure methionine oxidation, (b) the oxidation of active-site methionine in α1-proteinase inhibitor (α1PI) and (c) the resulting activity and structural changes of this inhibitor was investigated. While no methionine was oxidized during a 24 day, 37°C incubation with 0.01 M EDTA and 100 mM glucose, 64.2% oxidation occurred in 6 days when 0.01 mM Cu2+ was added to the 100 mM glucose. The first-order rate constant for oxidation in 10 mM glucose, 0.01 mM Cu2+ was 0.0218 day−1. Oxidation was inhibited by catalase, but accelerated by ascorbate ion. The active-site methionyl residue of α1PI was oxidized 71.3% after a 4 day incubation in 100 mM glucose, 0.01 mM Cu2+ (pH 7.45), 0.1 M phosphate buffer. The elastase and trypsin inhibiting activities were lowered to 3.1 and 1.5% of control samples during this incubation. The inclusion of 1 mM DETAPAC, a transition metal chelator, resulted in a 98+% retention of actiivty. Intrinsic fluorescence (350 nm excitation, 415 nm emission) of α1PI increased 576% over control for the sample incubated in 100 mM glucose, 0.01 mM Cu2+ and SDS-PAGE revealed protein fragment molecular weights of 44.4 and 39.8 kDa. These studies suggest that both methionine oxidation and free radical induced fragmentation contribute to loss of α1PI activity during glucose/Cu2+ incubations.

Characterization of synthetic medium antigens of Micropolyspora faeni and Thermoactinomyces candidus

A synthetic (Syn) medium was developed for growth of Micropolyspora faeni and Thermoactinomyces candidus, and optimum conditions for culture filtrate antigen production were found to be 3 days for T. candidus and 9 to 12 days for M. faeni. Appearance of proteolytic activity in the culture fluid supernatant coincided with a decrease in precipitating antigen content, protein content, and number of proteins on polyacrylamide gel electrophoresis (PAGE), probably as a result of proteolysis. The results suggest that protein content, number protein bands on PAGE, and proteolytic activity are predictive of precipitating antigen content. Antigens prepared in Syn medium were compared to those produced by the double-dialysis procedure and found to contain adequate amounts of antigenic material of sufficient quality to warrant clinical studies of their usefulness in the diagnosis of hypersensitivity pneumonitis.

Sedimentation equilibrium studies of human chymotrypsin II and bovine δ-chymotrypsin

Abstract Sedimentation equilibrium experiments indicate that neither human chymotrypsin II nor bovine δ-chymotrypsin molecules undergo association in the pH range 3–5 where dimerization occurs with α-chymotrypsin. The weight-average molecular weights of human chymotrypsin II and δ-chymotrypsin in a pH 4.4 0.1 ionic strength buffer are 26,200 and 26,400, respectively, using the measured partial specific volumes of 0.722 and 0.727 ml/g at 25 °C. Number-average molecular weight calculations also support the presence of monomeric species at this pH. In the pH range 6–7.6 where sedimentation velocity studies have shown that δ-chymotrypsin associates at concentrations above 3 mg/ml, no association was observed for either the human chymotrypsin II or bovine δ-chymotrypsin in the sedimentation equilibrium experiments where protein concentrations were below 1.2 mg/ml. These studies provide additional evidence that human chymotrypsin II is similar to bovine δ-chymotrypsin.