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Biomedical subjects

A Janoff

Publications and source records attributed to A Janoff.

At least 91 records · Page 5Linked to original sources
PubMed1976

The inhibition of human leucocyte elastase and chymotrypsin-like protease by elastatinal and chymostatin.

The ability of elastatinal and chymostatin, protease inhibitors of microbial origin, to inhibit human leucocyte proteases (EC 3.4.-) was studied. Elastatinal and chymostatin are capable of inhibiting the pancreatic enzymes elastase and chymotrypsin, respectively. It was found in these studies, with synthetic substrates, that elastatinal is a much weaker inhibitor of human leucocyte elastase than it is of porcine pancreatic elastase. Elastatinal caused no inhibition of the activity of human leucocyte chymotrypsin-like protease. Chymostatin was found to be a powerful inhibitor of human leucocyte chymotrypsin-like protease. Its affinity to the leucocyte protease was higher than its affinity to bovine pancreatic alpha-chymotrypsin. Chymsotatin had a weak inhibitory effect on the activity of human leucocyte elastase. Studies were also carried out on the ability of chymostatin to inhibit the release of 35SO2-4 from rabbit articular cartilage by human leucocyte chymotrypsin-like protease. Preincubation of the chymostatin with the protease before the latter was added to the 35SO2-4 -labeled cartilage caused inhibition of proteolysis as measured by 35SO2-4 release. Preincubation of chymostatin with 35SO2-4 -labeled cartilage prior to addition of the human chymotrypsin-like protease to the tissue also inhibited 35SO2-4 release. However, in the case of preincubation of cartilage with alpha1 -antitrypsin there was no such inhibition. It therefore appeared that chymostatin, unlike alpha1 -antitrypsin, was capable of penetrating the cartilage matrix and exerting its inhibitory effect upon the human leucocyte chymotrypsin-like protease that was subsequently added to the tissue.

Animals↗
PubMed1976

Degradation of cartilage proteoglycan by human leukocyte granule neutral proteases--a model of joint injury. I. Penetration of enzyme into rabbit articular cartilage and release of 35SO4-labeled material from the tissue.

The present work was undertaken to explore the effect of two purified neutral proteases derived from human peripheral blood polymorphonuclear leukocytes (PMN) on articular cartilage as a model of joint injury. Human leukocyte elastase and chymotrypsin-like enzyme, purified by affinity chromatography, released 32SO4 from labeled rabbit articular cartilage slices in vitro. Release of isotope was initially delayed, suggesting that either a lag in enzyme penetration occurs or that size of degradation fragments is a limiting factor in diffusion of label out of the tissue. The release of 35SO4 was inhibited by preincubation of elastase and chymotrypsin-like enzyme with human alpha 1-anti-trypsin, or with their specific chloromethyl ketone inactivators, and the action of elastase was also inhibited by a monospecific antiserum to PMN elastase, freed of major serum proteinase inhibitors. Immunohistochemical staining procedures revealed the presence of PMN elastase inside the matrix of cartilage slices after a 20-min exposure of tissue to either the pure enzyme or crude PMN granule extract. Serum alpha 1-antitrypsin failed to penetrate into the cartilage slices under identical in vitro conditions. In association with the results reported in the accompanying paper, these findings suggest a model of cartilage matrix degradation by PMN neutral proteases in which local protease-antiprotease imbalance, coupled with different rates of penetration of protease and antiprotease into target tissue, plays a key role in accounting for matrix damage.

Animals↗
PubMed1976

Degradation of cartilage proteoglycan by human leukocyte granule neutral proteases--a model of joint injury. II. Degradation of isolated bovine nasal cartilage proteoglycan.

Extracts of human peripheral blood polymorphonuclear leukocyte granules, and two purified proteases derived from such extracts, an elastase and a chymotrypsin-like enzyme, degrade isolated bovine nasal cartilage proteoglycan at neutral pH. Viscosity studies indicate that the leukocyte granule extracts lack hyaluronidase activity and that their degradative effect on proteoglycan at physiological pH is due entirely to proteolytic action. Sepharose 4B gel chromatography and SDS-polyacrylamide gel electrophoresis of proteoglycan fractions treated with leukocyte granule enzymes at pH 7.0 indicate that they degrade one of the proteoglycan link proteins, release a fragment from the hyaluronic acid-binding portion of the proteoglycan subunit core protein, and break down the remainder of the proteoglycan subunit molecule into peptide fragments with varying numbers of chondroitin sulfate chains. Immunodiffusion studies indicate that the antigenic determinants of the proteoglycan subunit core protein and the link proteins survive treatment with granule proteases. Similar degradation of human articular cartilage proteoglycan by granule neutral proteases can be presumed to occur, in view of the similarity of structure of human articular and bovine nasal cartilage proteoglycans. The release of granule enzymes in the course of neutrophil-mediated inflammation can thus result in the degradation of cartilage matrix proteoglycan, leading to cartilage destruction and joint injury.

Animals↗
PubMed1976

The role of lysosomal elastase in the digestion of Escherichia coli proteins by human polymorphonuclear leukocytes: experiments with living leukocytes.

Human polymorphonuclear leukocyte (PMN) elastase has been implicated in various pathological conditions. However, its physiological role remains undefined. One possible function of this enzyme may be digestion of bacterial proteins after phagocytosis. To test this hypothesis, we prepared Escherichia coli labeled with [3H]arginine and treated these bacteria with a lipid-soluble, active-site-directed chloromethyl ketone inactivator of pancreatic and granulocyte elastases (carbobenzoxy-L-glycyl-L-leucyl-L-alanine chloromethyl ketone, dissolved in methanol). Control bacteria were treated with methanol alone. When E. coli pretreated with the inactivator were incubated with solutions of porcine pancreatic elastase or with PMN granule extract, release of trichloroacetic acid-soluble radioactivity was significantly lower than in the control E. coli. Similar results were obtained when treated and control E. coli were fed to viable human PMN. In contrast, release of trichloroacetic acid-soluble radioactivity from E. coli containing [3H]thymidine was not affected by pretreatment of bacteria with elastase inactivator before feeding them to PMN, suggesting that phagocytosis of E. coli had not been inhibited by the chloromethyl ketone. When treated and control bacteria were fed to PMN, no significant difference was observed in the activity of lysosomal beta-glucuronidase recovered from post-granule supernatant fractions of homogenized leukocytes, suggesting that lysosomal degranulation had not been suppressed by the inactivator. However, elastase activity of the same fractions was depressed if the leukocytes had phagocytized chloromethyl ketone-treated E. coli, suggesting that inhibition of PMN elastase had occurred. We conclude that PMN elastase participates in digestion of E. coli proteins by human PMN.

Bacterial Proteins↗
PubMed1976

Variations in elastaselike esterase activities in human leucocytes during cell maturation.

Granules of human peripheral blood leucocytes contain four well-characterized elastase isozymes and one or two slow-moving elastaselike esterases (SE) which have not been as well characterized. SE are capable of hydrolyzing typical elastase synthetic sybstrates such as N-acetyl-dl-alanine-alpha-naphthyl ester (Ac-DL-Ala-1-ONap) and N-t-butyloxycarbonyl-L-alanine-p-nitrophenyl ester (Boc-Ala-ONp), but unlike the highly basic elastase isozymes, SE barely migrate into 13% acrylamide gels during cationic electrophoresis at pH 4.3. Hydrolysis of Ac-DL-Ala-1-ONap by SE requires the presence of Triton in the gel, and hydrolysis of Boc-Ala-ONp by the same enzyme(s) is also enhanced in the presence of the detergent. Triton is not required for these activities, in the case of the elastase isozymes. Diisopropylfluorophosphate (Dip-F) inactivates both SE and the elastase isozymes, whereas Ac-(Ala)2-Pro-AlaCH2Cl (a powerful inactivator of the leucocyte elastase isozymes at 10-4 M concentration) does not inactivate SE at the same concentration. Immunochemical studies revealed antigenic cross-reaction between the rapidly migrating leucocyte elastase isozymes and SE. Two preparations of leucocyte granules from nonleukemic bone marrow cells showed no activity of the rapidly migrating elastase isozymes, but did contain SE activity. SE may be a precursor or zymogen form of the elastase isozymes, present in immature cells and partly retained through later stages of development.

Aging↗
PubMed1975

A rapid method for purification of human granulocyte cationic neutral proteases: purification and characterization of human granulocyte chymotrypsin-like enzyme.

A chymotrypsin-like enzyme (EC 3.4.21.-) was purified from granules of human neutrophiles (polymorphonuclear leucocytes). The isolation procedure included differential salt extractions of the granules followed by affinity chromatography on 4-phenylbutylamine-Affi-Gel. This rapid purification method resulted in obtaining pure enzyme in relatively high yield in short time. The purified granulocyte chymotrypsin-like enzyme has a minimum Mr of 22 378, calculated from its amino acid composition. The Mr value obtained by sodium dodecyl sulphate gel electrophoresis was 20 000-23 000. The enzyme did not react with antibodies which are monospecific to granulocyte elastase. The granulocyte chymotrypsin-like enzyme was inactivated by Dip-F and by the chloromethyl ketone derivatives Z-PheCH2Cl and Z-(Gly)2-PheCH2Cl but not by Tos-PheCH2Cl. It therefore appears that the enzyme has serine and histidine side chains in its active site, like pancreatic chymotrypsin. The granulocyte enzyme substrate specificity is similar to that of pac-Tyr-Nan and Ac-Phe-1-ONap. It also has an intrinsic weak hydrolytic activity towards some classical elastase substrates such as Boc-Ala-ONp and Ac-DL-Ala-1-ONap. The granulocyte enzyme is inhibited by human serum and by human alpha1-antitrypsin. Its affinity for alpha1-antitrypsin is weaker than that of granulocyte elastase for the same inhibitor. The enzyme is stable at neutral pH at 37 degrees C, but unstable at pH 3.5 and at elevated temperature.

Amino Acids↗
PubMed1975

A rapid method of purification of human granulocyte cationic neutral proteases: purification and further characterization of human granulocyte elastase.

Human granulocyte elastase (EC 3.4.21.-) was isolated and purified (yield = 62%, purity = 91-100%) by a new short procedure using affinity chromatography using phenylbutylamine covalently linked to Affi-Gel. The granulocyte elastase was found to have a molecular weight of 34 400 by sodium dodecyl sulphate gel electrophoresis and the molecular weight obtained from the amino acid composition was 34 970. The composition of elastase purified from normal leucocytes showed some significant differences from that of enzyme purified by others from leukemic leucocytes. The granulocyte elastase hydrolysed typical pancreatic elastase substrates like Boc-Ala-ONp and Ac-(Ala)3-Nan. The enzyme was also found to have a weak enzymatic activity in hydrolysing acetyl-L-phenylalanine-alpha-naphthyl ester, a typical chymotrypsin substrate. A monospecific antiserum raised against the purified enzyme gave a single precipitin line with the pure enzyme and also with crude granular extract, both lines being identical.

Amino Acids↗
PubMed1975

In vitro transformation of BHK21 cells grown in the presence of calcium chromate.

Concentrations of 0.25 and 0.5 mug/ml calcium chromate (CaCrO4-2H2O)dissolved in Dulbecco's medium were found to alter the growth behavior of BHK21 cells in culture. Treated cells grew as shortened fibroblasts and in random orientation. The changes detected during the first two weeks of culture in the presence of the metal became more pronounced as the number of growth passages increased. In addition to the alterations noted above, chromate-treated cells grew into large clusters in Methocel (an alternative technique to the agar suspension system), while untreated cells underwent, at most, only one or two divisions in Methocel. These alterations in growth properties were irreversible and persisted after removal of the treated cells from chromate-containing medium, suggesting that a heritable change had occurred as opposed to a transient, chromate-dependent alteration of cell growth. This experimental observation suggests that chromate salts and perhaps salts of other metals can transform BHK21 cells in vitro or can select for spontaneously transformed cells.

Animals↗
PubMed1975

The course and nature of acinar cell death following pancreatic ligation in the guinea pig.

The course and nature of acinar cell death (ACD) following pancreatic ligation in the guinea pig was studied as a possible model for human disease. Ultrastructural studies after various periods of ligation suggested a biphasic pattern of ACD. Early phase ACD involved only a small portion of acinar cells and occurred within a few hours of ligation. It was preceded by swelling and vesiculation of the rough endoplasmic reticulum. Morphometric measurements disclosed celular swelling at this time, and NaCl equilibration studies demonstrated a change in cellular osmoregulation. Late phase ACD, characterized by cellular wasting and autophagic vacuole formation, became prominent several days after ligation. Marked increases in lysosomal enzyme activities were found in tissue homogenates at this time, and acid phosphatase electron histochemistry localized the majority of this increased activity to lysosomes and autophagic vacuoles within the acinar cells. The etiology and nature of both phases of ACD are discussed.

Acid Phosphatase↗