Membrane modifications in nutritionally induced filamentous Escherichia coli B.

Nutritionally induced filamentous cell forms of Escherichia coli B were examined for their morphological and biochemical lesions. The filamentous forms showed no significant alteration in total DNA concentration, RNA synthesis, ability to form beta-galactosidase in response to isopropylthiogalactoside, or insensitivity to actinomycin D as compared to the normal cell form. The filamentous cells showed a marked decrease in the ability to incorporate N-acetylglucosamine-UL-(14)C into a phenol-soluble glycoprotein fraction relative to the normal cell form or relative to strain E-26 of E. coli grown in the filament-inducing medium. The filaments yielded an envelope-specific phenol-soluble protein fraction markedly reduced in or lacking three proteins as determined by acrylamide gel electrophoresis. Amino acid analysis, and chemical and enzymatic treatments of the envelope-specific phenol-soluble proteins showed striking differences between the fractions obtained from normal and filamentous cells. Electron microscope studies of divalent cation-induced aggregates of the envelope proteins showed different aggregation patterns dependent upon the cell form yielding the protein fraction.

Hexagonal pattern in cell walls of Escherichia coli B.

Cell walls, isolated from Escherichia coli B, as examined by electron microscopy and optical diffraction contain a hexagonal lattice structure, the (1,0) planes of which are separated by 140 +/- 8 angstroms. Unless the walls are briefly heated (10 minutes, 90 degrees C) early in the isolation, the hexagonal array cannot always be observed. Enzymatic digestion with pancreatin and amylase improves visualization of the lattice; subsequent treatment with pepsin and sodium dodecylsulfate removes the hexagonal pattern. Protein or lipoprotein globular units within the wall may thus be arranged in a hexagonal array uponthe mucopeptide layer.

Fibronectin degradation products containing the cytoadhesive tetrapeptide stimulate human neutrophil degranulation.

We investigated whether adhesive glycoproteins, such as fibronectin or fibrinogen, could function to provide a nidus for neutrophil degranulation. Elastase release in recalcified plasma was normal in afibrinogenemic plasma, but 73% less in plasma depleted of fibronectin. Proteolytic digests of fibronectin, but not intact fibronectin (50-1,000 micrograms/ml), induced a concentration-dependent release of neutrophil elastase and lactoferrin. MAbs N293, which recognized the mid-molecule of fibronectin, N294, which was directed toward the 11-kD cell adhesive fragment, and N295, generated against the amino terminal of the 11-kD fragment, inhibited the release of elastase by 7, 24, and 60%, respectively. The cytoadhesive tetrapeptide portion of fibronectin, Arg-Gly-Asp-Ser (250-1,000 micrograms/ml), released 1.94 +/- 0.10 micrograms/ml of elastase from 10(7) neutrophils, in contrast to the lack of release by the control hexapeptide, Arg-Gly-Tyr-Ser-Leu-Gly. Plasmin appeared to be the enzyme responsible for fibronectin cleavage, since neutrophil elastase release in plasma that had been depleted of plasminogen was decreased and reconstitution of plasminogen-deficient plasma with purified plasminogen corrected the abnormal release. Plasmin cleaved fibronectin to multiple degradation products, each less than 200 kD. This fibronectin digest released 1.05 microgram/ml of elastase from 10(7) neutrophils. We suggest that the activation of plasminogen leads to the formation of fibronectin degradation products capable of functioning as agonists for neutrophils.

Immunolocalization of elastase in human emphysematous lungs.

The current working hypothesis concerning the pathogenesis of human pulmonary emphysema proposes that neutrophils migrate through the alveolar interstitium and degranulate, releasing proteolytic enzymes into the interstitium. These enzymes, in particular elastase, can bind to and degrade interstitial elastin. This report describes an immunohistochemical, ultrastructural technique that utilizes polyclonal antibodies to localize neutrophil elastase in human lungs. Using both the immunoperoxidase and the immunogold methods on thin, embedded sections of surgically resected human emphysematous lung tissue, elastase was localized in neutrophils in the lung interstitium and extracellularly in association with interstitial elastic fibers in human lungs that showed local emphysema of varying severity. Quantitative morphometric data were obtained from the lungs of eight patients undergoing lobectomy for removal of pulmonary carcinomas. Patients had preoperative forced expiratory volume (FEV1)% levels ranging from 55 to 77. There was a correlation between a quantitative measure of the local distribution of neutrophil elastase in contact with alveolar interstitial elastin and the local presence of emphysematous change as determined by mean linear intercept of the various histologic sections. These data support the validity of the "protease-protease inhibitor balance hypothesis" as an explanation of the pathogenesis of human pulmonary emphysema.

A model of decreased functional alpha-1-proteinase inhibitor. Pulmonary pathology of dogs exposed to chloramine T.

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.

Glucosamine-labelled envelope proteins of Escherichia coli K-12. I. Electrophoretic studies and partial fractionation of phenol-soluble proteins.

Hydrophobic envelope proteins were extracted by phenol from a glucosamine- and leucine-requiring mutant of Escherichia coli K-12 (E-110). Three protein fractions labelled with D-[1-14C]glucosamine and L-[4,5-3H]leucine were obtained by electrophoretic separation. Envelopes were isolated from cells labelled with D-[1-14C]glucosamine--HCL and acid hydrolyzed. At least 68% of the radioactivity was recovered as glucosamine and glucose with no random distribution of label. Fingerprinting of pronase digests of glucosamine-labelled proteins showed four radioactive spots associated with peptides. The glycoproteins were pronase- and trypsin-sensitive and had apparent molecular weights of 11 000 (fast mobility), 35 000 (intermediate mobility) and 62 000 (slow mobility) as estimated by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. The two heavier fractions were labelled with meso-diamino[1,7-14C2]pimelic acid, while orth[32P]phosphate was not incorporated into any fraction. The glucosamine radioactivity of the fast fraction underwent rapid changes upon a chase with non-radioactive glucosamine. Using a Sephadex LH-20 column, the radioactive proteins were separated from the phenol and subsequently fractionated on a DEAE-cellulose column. The DEAE-cellulose fractions were distinct from each other in the number and composition of protein bands, when analyzed by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. Radioactive bands with intermediate and fast electrophoretic mobilities were found in separate DEAE-cellulose fractions.

Glucosamine-labelled envelope proteins of Escherichia coli K-12. II. Location in inner and outer membranes.

Outer and inner membranes were prepared from a culture of a glucosamine- and leucine-requiring mutant of Escherichia coli K-12, which was grown on L-[4,5-(3)H2]leucine and D-[1-(14)C]glucosamine and "chased" with unlabelled medium. Phenol-soluble glycoproteins were obtained from these membranes after phospholipid extraction. Sodium dodecyl sulfate-disc electrophoresis of the outer-membrane glycoproteins separated two components which were labelled with both isotopes and had a fast (F) and intermediate (I) migration. Dodecyl sulfate electrophoresis of the inner-membrane glycoproteins showed that only the F band incorporated both 14C and 3H labels. The 3H to 14C ratio in the F band from the outer membrane was completely different from the isotopic ratio in the F band from the inner membrane, indicating that these components were not identical despite their similar molecular weights. The F bands from both membranes lost their label during the chase with unlabelled medium, while the I band remained relatively stable. Chloroform-soluble 14C label extracted from the outer membrane decreased during the chase. In contrast, the chloroform-soluble 14C from the inner membrane, accumulated during the chase.

COPD and endobronchial polyposis associated with hypogammaglobulinemia. Are proteolytic enzymes involved?

The present report describes a patient who had severe obstructive lung disease in association with acquired hypogammaglobulinemia. Evidence obtained by bronchoalveolar lavage is presented that suggests that his lung disease resulted from both a marked increase in elastase load and a reduction in protease inhibitor function.

Effects of simulated extracorporeal circulation on human leukocyte elastase release, superoxide generation, and procoagulant activity.

Activated leukocytes are thought to contribute to respiratory dysfunction, alterations in microvascular permeability, disseminated intravascular coagulation, and thrombosis, all of which can complicate extracorporeal circulation. The purpose of this work was to determine the effects of extracorporeal circulation on leukocyte functions likely to mediate organ damage. White blood cell counts in the bubble circuits (n = 5) fell to 51% +/- 7% (mean +/- standard error of the mean; p less than 0.05) of initial levels within 2 hours of recirculation. In contrast, counts from both the spiral coil (n = 5) and hollow-fiber (n = 5) groups remained at 91% +/- 12% and 100%, respectively. Plasma levels of human neutrophil elastase rose from 0.28 +/- 0.06 micrograms/ml to 3.14 +/- 0.36 micrograms/ml (p less than 0.05) and 0.20 +/- 0.02 micrograms/ml to 1.61 +/- 0.35 micrograms/ml (p less than 0.05) in bubble and spiral coil circuits, respectively, but from only 0.20 +/- 0.03 micrograms/ml to 0.96 +/- 0.42 micrograms/ml in the hollow-fiber circuit despite 2 hours of recirculation. Consistently, in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine, a chemotactic peptide, cells from spiral coil and bubble circuits released and generated significantly less elastase and superoxide anion, respectively. In contrast, neutrophils from the hollow-fiber circuits demonstrated enhancement of N-formyl-L-methionyl-L-leucyl-L-phenylalanine-induced elastase release and superoxide generation. Finally, mixed leukocytes from all circuits expressed procoagulant activity reaching statistical significance in bubble circuits. In conclusion, extracorporeal circulation has pronounced effects on neutrophil elastase release, superoxide anion generation, and leukocyte procoagulant activity. Spiral coil and bubble oxygenators cause granule release and, subsequently, reduced sensitivity to soluble agonists. In contrast, hollow-fiber oxygenators "prime" cells, actually enhancing reactivity. Recirculation through all circuits induces leukocyte procoagulant activity that is likely to contribute to surface-induced thromboses and excessive bleeding.

Alpha 1-antitrypsin levels predict mortality from ethionine-induced pancreatitis in mice.

Experimental pancreatitis can be induced by an ethionine-containing, choline-deficient diet in mice. We investigated the role of circulating alpha 1-antitrypsin in this model using two strains of mice: ICR and C57BL-6. A 50% reduction in circulating alpha 1-antitrypsin occurred in all mice by day three of diet exposure. Total protein was reduced by only 9% and albumin was unchanged. Female mice of both strains had significantly lower alpha 1-antitrypsin levels than male mice prior to and after diet exposure. This was associated with significantly greater mortality in both female strains. Interstrain comparisons showed a significantly higher mortality in the C57BL-6 females (100%) compared to the ICR females (58%); this corresponded to significantly lower alpha 1-antitrypsin levels in C57BL-6 females. Regardless of sex or strain, alpha 1-antitrypsin levels prior to and after diet exposure were significantly higher in mice surviving than in mice dying. We conclude that circulating alpha 1-antitrypsin is a predictor of mortality from diet-induced pancreatitis.

The effect of the oxidizing agents chloramine-T and cigarette smoke on dog serum proteinase inhibitor(s).

Dog serum treated with the oxidant chloramine-T is rapidly and selectively depleted of its ability to inhibit porcine pancreatic elastase or dog neutrophil elastase. Trypsin inhibitory capacity of serum is not affected. Purified dog alpha-1-proteinase inhibitor (alpha-1-PI) is similarly oxidized with an apparent rate constant of 1.1 x 10(3) M-1 sec-1. Reversal of the oxidative inactivation using dithiothreitol was demonstrated. Cigarette smoke also directly affects the inhibitory capacity of both serum and pure alpha-1-PI. These studies form a basis for developing a model of functionally deficient alpha-1-PI by taking advantage of oxidative inactivation of normal proteinase inhibitor levels.