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Biomedical subjects

R W Mason

Publications and source records attributed to R W Mason.

At least 19 recordsLinked to original sources
PubMed1992

Surface activation of pro-cathepsin L.

Pro-cathepsin L is an inactive zymogen that has been shown previously to undergo autolysis at pH 3.0 to give mature forms of the enzyme. We have now been able to demonstrate that this enzyme can undergo activation at pH 5.5 in the presence of negatively charged surfaces. Activation could also be measured at pH 6.0, but no activation occurred at pH 6.5 or higher. The initiation of activation depends upon the presence of a small percentage of active pro-enzyme, and this is then followed by a more rapid activation to give mature forms of the enzyme. No significant intermediate molecular forms of the enzyme were seen. The time taken for processing of the pro-enzyme to single-chain mature enzyme is comparable to that seen in biosynthetic pulse-chase experiments.

3T3 Cells↗
PubMed1992

Inhibition of cysteine proteinases in lysosomes and whole cells.

Inhibitors of cysteine proteinases have been used extensively to dissect the roles of these proteinases in cells. Surprisingly though, little work has been performed to demonstrate unequivocally that the inhibitors reach and inactivate their target proteinases in cell culture or in vivo. In the present study, the permeability of lysosomes and whole cells has been studied. Benzyloxycarbonyl (Z)-[125I]iodo-Tyr-Ala-diazomethane (CHN2), an inhibitor of cathepsins L and B, has been shown to label active forms of these enzymes in lysosomes and whole cells. The ability of other cysteine proteinase inhibitors to block this labelling has been used to indicate the permeation of these compounds. All the inhibitors were able to block labelling by Z-[125I]iodo-Tyr-Ala-CHN2 in lysosomal extracts. In intact lysosomes or cells, however, only N-[N-(L-3-trans-ethoxycarbonyloxirane-2-carbonyl)-L-leucyl]-3- methylbutylamine ('E-64d') Z-Tyr-Ala-CHN2, Z-Phe-Ala-CHN2 and Z-Phe-Phe-CHN2 were able to block labelling by Z-[125I]iodo-Tyr-Ala-CHN2. N-[N-(L-3-trans-Carboxyoxirane-2-carbonyl)-L-leucyl]amino-4-gua nidinobutane (E-64) and leupeptin were unable to block labelling by Z-[125I]iodo-Tyr-Ala-CHN2 in lysosomes or in cells. The ability to block labelling in lysosomes is an indication of the ability of the inhibitor to diffuse across membranes. Thus E-64 and leupeptin do not readily permeate membranes and therefore their uptake into cells probably only occurs via pinocytosis.

3T3 Cells↗
PubMed1992

Blindness associated with toxoplasmosis in canaries.

Seven of 30 canaries in an aviary in New Zealand developed ophthalmic problems. Clinically, 5 birds had unilateral and 2 birds had bilateral lesions characterized by conjunctivitis, crusty exudates on eyelids, and collapse of the eyeball. Microscopic lesions in 12 of 14 eyes examined included inflammation of the choroid and retina, with osseous replacement of the globe in some. Numerous Toxoplasma gondii tachyzoites were seen in the detached retina and vitreous humor of acutely affected birds. The diagnosis of toxoplasmosis was confirmed by immunohistochemical staining with T gondii antiserum. Affected birds had encephalitis, and T gondii was localized in the brains of these by immunohistochemical examination and by use of bioassays in mice. Toxoplasmosis should be considered in differential diagnosis of ophthalmitis in canaries.

Animals↗
PubMed1992

The specificity and elastinolytic activities of bovine cathepsins S and H.

Cathepsins S and H were purified from bovine spleen and their catalytic properties compared. The enzymes were shown to be similar by chromatographic properties and by the ability to hydrolyze Bz-Phe-Val-Arg-NHMec. They could however be distinguished by the fact that cathepsin S reacted with Z-[125I]Tyr-Ala-CHN2 and hydrolyzed Z-Phe-Arg-NHMec whereas cathepsin H did not. The substrate and inhibitor specificities of cathepsin H suggest that unlike cathepsins B, L, and S, it cannot accommodate peptides with aromatic side chains in P2. Cathepsins L and S can accommodate the aromatic side chain of tyrosine in P1 readily, whereas cathepsins H and B cannot. The specificities of each enzyme for synthetic substrates and inhibitors have enabled the construction of models of the architecture of the active sites of the mammalian cysteine proteinases which clearly show the differences between the four enzymes. A significant characteristic of cathepsin S is that it can hydrolyze insoluble elastin at both acidic and neutral pH; this distinguishes it from all of the other lysosomal proteinases.

Amino Acid Sequence↗
PubMed1991

Human cystatin C. role of the N-terminal segment in the inhibition of human cysteine proteinases and in its inactivation by leucocyte elastase.

Leucocyte elastase in catalytic amounts was observed to rapidly cleave the Val-10-Gly-11 bond of the human cysteine-proteinase inhibitor cystatin C at neutral pH. The resulting modified inhibitor had size and amino acid composition consistent with a cystatin C molecule devoid of the N-terminal Ser-1-Val-10 decapeptide. Leucocyte-elastase-modified cystatin C had more than 240-fold lower affinity than native cystatin C for papain. Removal of the N-terminal decapeptide of human cystatin C also decreased inhibition of human cathepsins B and L by three orders of magnitude, but decreased inhibition of cathepsin H by only 5-fold. A tripeptidyldiazomethane analogue of of the N-terminal portion of cystatin C was a good inhibitor of cathepsins B and L but a poor inhibitor of cathepsin H. It therefore appears that amino acid side chains of the N-terminal segment of cystatin C bind in the substrate-binding pockets of cathepsins B and L but not in those of cathepsin H. It is argued that the N-terminal cystatin C interaction with cathepsin B is physiologically important and hence that leucocyte elastase could have a function as a regulator of extracellular cysteine-proteinase inhibitory activity at sites of inflammation.

Amino Acid Sequence↗
PubMed1991

Cigarette smoking induces an elastolytic cysteine proteinase in macrophages distinct from cathepsin L.

Degradation of the interstitium of the lung by elastolytic enzymes is thought to be a critical component of the pathogenesis of emphysema. Alveolar macrophages are increased in numbers in cigarette smokers and contain the elastolytic cysteine proteinase cathepsin L. We sought to determine if cigarette smoking induces a change in cathepsin L levels in alveolar macrophages which would, in turn, alter the expression of elastolytic activity. Lysates of smokers' macrophages, assayed at pH 5.50, degraded more than seven times as much [3H]elastin as did lysates from nonsmokers' macrophages (44 +/- 20.8 vs. 6 +/- 1.6 micrograms.10(6) cells-1.24 h-1). Little or no activity was demonstrable at neutral pH. Immunoblots of macrophage lysates demonstrated that smokers' cells contain 3.7 +/- 1.1 times as much 25-kDa cathepsin L antigen as nonsmokers' cells. However, as judged by active site labeling, levels of active cathepsin L in smokers and nonsmokers are indistinguishable, suggesting that most of the 25-kDa antigen found in smokers' macrophages is inactive. Inhibitors of cathepsin L had little effect on lysate elastolytic activity, confirming that an enzyme other than cathepsin L is responsible for the increased elastolytic activity seen in smokers' macrophages. Further experiments demonstrated that this second enzyme(s) has a profile of inhibition indicating that it is a cysteine proteinase with optimal activity at pH 5.50. It is this second elastolytic cysteine proteinase(s) that is induced by exposure to cigarette smoke and is responsible for the sevenfold increase in elastolytic activity found in smokers' macrophage lysates.

Actins↗
PubMed1991

Regulation of cystatin C activity by serine proteinases.

The effect of four human serine proteinases on the human cysteine proteinase inhibitor, cystatin C, has been studied in vitro. Neutrophil elastase in catalytic amounts was observed to rapidly cleave cystatin C at neutral pH, thereby giving rise to a modified form of the inhibitor lacking the N-terminal Ser1-Val10 decapeptide. The two other leukocyte serine proteinases, cathepsin G and neutrophil proteinase 4, did not catalytically hydrolyse cystatin C bonds. Neither had the seminal plasma serine proteinase, prostate-specific antigen, any effect on cystatin C. The physiological implications of neutrophil elastase catalysed modification of cystatin C are discussed, and recent findings indicating that this reaction also occurs in vivo are reviewed.

Amino Acid Sequence↗
PubMed1991

Lethal toxoplasmosis in a little penguin (Eudyptula minor) from Tasmania.

A penguin (Eudyptula minor) from Tasmania, Australia, died following a 3-day illness. The liver and spleen parenchyma contained grossly visible foci of discoloration. Numerous tachyzoites of Toxoplasma gondii were seen associated with necrosis in sections of liver and spleen. The diagnosis was confirmed by immunohistochemical staining of parasites. This apparently is the first report of fatal toxoplasmosis in a penguin.

Animals↗
PubMed1990

Characterization of the active site of human multicatalytic proteinase.

The activity of multicatalytic proteinase against synthetic substrates and the kinetics of its inhibition by a range of class-specific inhibitors have been investigated. The enzyme was found to have a broader pH activity profile than previously noted, being active against succinyl-Ala-Ala-Phe-7-amino-4-methylcoumarin optimally at pH 4.5 and against benzyloxycarbonyl-Gly-Gly-Arg-7-amino-4-methylcoumarin optimally at pH 10.5. Neither activity was inhibited by the class-specific inhibitors 1,10-phenanthroline, EDTA, pepstatin, di-isopropyl fluorophosphate, peptidyl chloromethanes, peptidyl diazomethanes or L-3-carboxy-2,3-trans-epoxypropionyl-leucylamido-(4-guanidin o)butane (E-64), indicating that the enzyme is not a typical metallo-, aspartic, serine or cysteine proteinase. Inhibition by HgCl2, iodoacetamide and N-ethylmaleimide suggests that free thiols are necessary for the enzyme to maintain activity, but that these thiols are not particularly reactive as is the case for cysteine proteinases of the papain superfamily. The peptidyl aldehydes chymostatin and leupeptin were found to be reversible inhibitors of multicatalytic proteinase. Chymostatin inhibited activity against succinyl-Ala-Ala-Phe-7-amino-4-methylcoumarin at pH 4.5 (Ki 160 +/- 22 microM) whereas leupeptin (200 microM) was not inhibitory. Inhibition of activity against benzyloxycarbonyl-Gly-Gly-Arg-7-amino-4-methylcoumarin by these compounds was more complex, in that they behaved as slow tight-binding inhibitors. kon values were determined to be 12 +/- 2 M-1.s-1 and 1290 +/- 125 M-1.s-1 for chymostatin and leupeptin, respectively. The upper limit for Ki values for these two inhibitors was estimated as 5 +/- 1.5 microM and 25 +/- 5 nM, respectively. The different inhibition characteristics for each substrate were also apparent at an intermediate pH of 8.5, showing that the two activities are distinct. Dichloroisocoumarin, a mechanism-based inhibitor of serine proteinases, did inhibit activity against succinyl-Ala-Ala-Phe-7-amino-4-methylcoumarin with a rate constant of 250 M-1.s-1, suggesting that multicatalytic proteinase is an atypical serine proteinase.

Amino Acid Sequence↗
PubMed1990

Uptake of extracellular enzyme by a novel pathway is a major determinant of cathepsin L levels in human macrophages.

The phorbol myristate acetate (PMA)-differentiated myelomonocytic cell line, THP-1, and human alveolar macrophages contain the cysteine proteinase cathepsin L. This enzyme is synthesized as a 43-kD proenzyme and processed to the active 25-kD form. Differentiation of THP-1 cells in the presence of human serum resulted in an increase in the size of the vacuolar compartment and the accumulation of more 25-kD cathepsin L antigen, as compared with THP-1 cells differentiated in the presence of fetal calf serum. Cells cultured in both types of sera have equivalent levels of cathepsin L mRNA. Metabolic labeling experiments demonstrated equivalent rates of synthesis, processing to the active form, and persistence in both culture conditions. An extracellular source of enzyme was documented by immunoblotting human serum which demonstrated 25-kD cathepsin L antigen; furthermore, we demonstrated that both THP-1 cells, differentiated in human serum, and human alveolar macrophages take up the 43-kD proenzyme and process it to the 25-kD form. Thus, human serum contains a factor(s) that induces both a marked increase in the size of the vacuolar compartment in differentiated THP-1 cells and a novel pathway that is responsible for the uptake and processing of extracellular cathepsin L. The activity of this inducible pathway is a major determinant of levels of intracellular cathepsin L. Cathepsin L is a potent elastase and the regulation of its uptake and processing may play a role in the pathogenesis of disease processes characterized by the destruction of elastin, such as pulmonary emphysema.

Blotting, Western↗
PubMed1990

Studies on the pharmacokinetics and mutagenic potential of rhodamine B.

Rhodamine B is used as a marker dye in herbicide sprays. There is evidence that spray operators and others may absorb rhodamine B through the skin. This study was undertaken to investigate the in vivo mutagenicity of rhodamine B, to compare the in vitro mutagenicity of two commercial preparations of the dye with that of known mutagens including rhodamine 6G and to elucidate the pharmacokinetics of rhodamine B in the rabbit. Following the i.v. treatment of adult female New Zealand White rabbits with rhodamine B (1 mg/kg body wt), the plasma concentration of rhodamine B decreased rapidly and was accompanied by the appearance of at least four fluorescent polar metabolites. These metabolites, as well as a very small amount of rhodamine B, were also present in the urine which, when tested in the Ames assay was not significantly mutagenic against Salmonella typhimurium strains TA98 and TA100 either with or without metabolic activation. Urine from a human subject who had been contaminated with marker dye was also non-mutagenic. Both commercial preparations of rhodamine B were found to be weakly mutagenic, using the same assay system. It is concluded that while appropriate hygiene measures should be exercised by users of products containing this dye, the results do not support the hypothesis that rhodamine B is a genotoxic hazard in the mammalian organism.

Adult↗
PubMed1989

The use of benzyloxycarbonyl[125I]iodotyrosylalanyldiazomethane as a probe for active cysteine proteinases in human tissues.

The ability of benzyloxycarbonyl-(125I)Tyr-Ala-CHN2 to label cysteine proteinases in a variety of human tissues was investigated. The inhibitor bound only to cathepsin B in tissues homogenized at pH 5.0. When liver was autolysed at pH 4.0 for up to 4 h, the inhibitor also bound to a protein of Mr 25,000. This was identified immunologically and chromatographically as cathepsin L. Both cathepsins B and L were found primarily in kidney, liver and spleen. In spleen, an additional protein of Mr 25,000 was also labelled. This protein could not be precipitated by antibodies to any of cathepsins B, H and L. This protein has tentatively been identified as human cathepsin S by its tissue distribution, chromatographic properties and molecular size. This work clearly shows that peptidyldiazomethanes are specific probes for cysteine proteinases, and that benzyloxycarbonyl-(125I)Tyr-Ala-CHN2 binds to three such enzymes in human tissues.

Autolysis↗
PubMed1989

Glycosylation of procathepsin L does not account for species molecular-mass differences and is not required for proteolytic activity.

Cathepsin L is a major lysosomal cysteine proteinase in mouse and human cells. Despite similar predicted molecular masses, procathepsin L in these two species migrates on SDS/polyacrylamide gels with apparent molecular masses of 39 kDa and 42 kDa respectively. To determine if glycosylation differences account for this discrepancy, and to ascertain whether glycosylation is essential for enzymic activity, mouse and human procathepsins L were expressed at high concentrations in mouse NIH 3T3 cells or in human A431 cells after DNA-mediated transfection of cloned DNAs for these enzymes. In pulse-chase studies, human procathepsin L transfectants synthesized and secreted large amounts of enzymically active 42 kDa proenzyme and processed it into 34 kDa and 26 kDa intracellular peptides, a pattern of secretion and processing similar to that seen with endogenous or transfected mouse procathepsin L. Both translation of cloned procathepsin L cDNAs in vitro and Endoglycosidase H treatment of 39 kDa mouse and 42 kDa human procathepsin L resulted in non-glycosylated proteins 2 kDa lower in molecular mass than the untreated proteins for both species. This suggests that glycosylation differences are not responsible for the molecular-mass disparity between the two species. Moreover, Endoglycosidase H-treated mouse enzyme retained full proteolytic activity, indicating that glycosylation of cathepsin L is not essential for enzymic function.

Acetylglucosaminidase↗