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Biomedical subjects

R A Maciewicz

Publications and source records attributed to R A Maciewicz.

32 records · Page 2Linked to original sources
PubMed1991

Degradation of cartilage matrix components by the cysteine proteinases, cathepsins B and L.

Rat chondrosarcoma chondrocytes are able to secrete both the precursor and mature forms of cathepsins B and L. When these two cysteine proteinases were added to a native cartilaginous matrix near neutral pH they were found to release and degrade both the proteoglycan and collagen components. One cleavage site in the proteoglycan monomer was found to be near the amino terminal globular domain. Collagen types II, IX, and XI in the matrix were also released and partially degraded. Ultimately these proteinases act as matrix depolymerases as they attacked the regions which are involved in collagen crosslinking. We conclude that these enzymes may play a role in matrix destruction as seen in arthritis.

Animals↗
PubMed1990

Susceptibility of the cartilage collagens types II, IX and XI to degradation by the cysteine proteinases, cathepsins B and L.

We have investigated the susceptibility of both the helical and non-helical regions of isolated rat chondrosarcoma collagens, types II, IX and XI, to degradation by the cysteine proteinases, cathepsins B and L. Both enzymes degrade these collagens at temperatures from 20 to 37 degrees C and pH values from 3.5 to 7.0. Cleavage occurs only within the non-helical domains unless the helix is destabilized. Cathepsin L is more effective than cathepsin B on a molar basis and they appear to cleave at different sites. Since these cathepsins can degrade cartilage collagens at pH values near neutrality, they may contribute to the destruction of cartilage observed in arthritis.

Cathepsin B↗
PubMed1989

Immunodetection of cathepsins B and L present in and secreted from human pre-malignant and malignant colorectal tumour cell lines.

Pre-malignant and malignant human colorectal tumour epithelial cell lines both secreted precursor forms of the 2 cysteine proteinases, cathepsins B and L. The amount of proteinases secreted by these cell lines varied according to the cell density. Comparison at similar cell densities showed that the pre-malignant, adenoma-derived cell line (PC/AA) secreted as much, or more, of both cathepsin B and L precursors as did the malignant, carcinoma-derived cell line (PC/JW/FI). However, mature forms of cathepsins B and L were detected in the culture media of only the carcinoma-derived cell line, thus indicating that the invasive potential of a tumour may be related to its ability to process extracellularly the secreted precursor enzyme to a mature and consequently active enzyme, rather than to the amount of proteinase synthesized and/or secreted. Similar results were obtained using 2 other epithelium-derived tumour cell lines, HT/29 (carcinoma) and SP/AN (adenoma). Immunolocation studies showed that cathepsin B was lysosomal while cathepsin L appeared to have a distribution more consistent with a plasma membrane association. Purified human cathepsins B and L (mature form) were capable of solubilizing an isolated basement membrane matrix (bovine anterior lens capsule) in vitro, thus indicating that the secreted mature enzymes and the membrane-associated cathepsin L could potentially degrade basal laminae or sub-endothelial basement membranes in vivo.

Adenoma↗
PubMed1988

A comparison of four cathepsins (B, L, N and S) with collagenolytic activity from rabbit spleen.

We have separated four cathepsins (B, L, N and S) from rabbit spleen. They are all collagen-degrading cysteine proteinases, with Mr values of 25,250, 23,500, 34,000 and 30,000 for cathepsin B, L, N and S respectively. Cathepsins B, N and S have isoelectric points of 5.4, 6.2 and 6.8 respectively, whereas cathepsin L exhibited multiple charge forms in the range 5.0-5.7. A comparison of their specific activity against a variety of protein and synthetic substrates shows many differences. These differences can be visually illustrated through isoelectric focusing and detection of enzymic activity with protein and synthetic-substrate overlays. By using an enzyme-linked immunosorbent assay based on the binding to chicken cystatin and detection with polyclonal and monoclonal antibodies to native cathepsins B and L, no cross-reactivity of the four native enzymes was observed. Studies on the co-operative or synergistic effect in degrading collagen indicated that, of the different combinations tested, only the combination of cathepsin B and N exhibited enhanced collagenolysis.

Animals↗
PubMed1988

Transmission densitometry of stained nitrocellulose paper.

We report a simple solution to the problem of quantitative densitometry of stained nitrocellulose paper. By immersing the paper in a household lubricating oil of matching refractive index, the light-scattering properties of the paper are largely eliminated, allowing precise transmission densitometry in any flat bed densitometer. The method was evaluated on immunochemically stained Western blots of the proteinases cathepsins B and L. An approximately linear relationship was found between the integrated absorbance of the stained zone and the logarithm of the amount of protease applied to the polyacrylamide gel over the range of 150 to 350 ng of cathepsin B and 50 to 250 ng of cathepsin L.

Blotting, Western↗
PubMed1987

Collagenolytic cathepsins of rabbit spleen: a kinetic analysis of collagen degradation and inhibition by chicken cystatin.

We have investigated the steady state kinetics of the degradation of native fibrillar collagen at pH 3.4 by four collagenolytic cathepsins of rabbit spleen. For each enzyme, the dependence of initial velocity on collagen concentration was well described by the Michaelis-Menten mechanism. Km, expressed as the concentration of triple-helical chains, and kcat values were determined for cathepsins B, L, N and S. The ratio of Kcat to Km suggest that cathepsins L and N are far more effective at collagen solubilization than either cathepsins S or B. Ki values were determined for the inhibition of collagenolytic activity at pH 3.4 using cystatin, a naturally-occurring cysteine proteinase inhibitor. All four cysteine proteinases were inhibited by cystatin in this assay system, although it was found to be a tighter binding inhibitor of cathepsin L, than for cathepsins N and S (approximately 5-fold less), or cathepsin B (approximately 500-fold less).

Animals↗
PubMed1986

Monoclonal antibodies to rabbit liver cathepsin B.

Three stable hybridoma cell lines (AF8, BC11, CE2) have been produced that secrete antibodies specific for cathepsin B. These have been characterized by ELISA, SDS-PAGE immunostaining, immunoprecipitation and immunofluorescent staining. CE2 immunoprecipitated native cathepsin B with retention of enzymic activity, but failed to cross-react with the alkali-denatured enzyme. BC11 bound only to the denatured form of cathepsin B and AF8 cross-reacted with both native and denatured cathepsin B. However, unlike CE2-immunoprecipitated enzyme, activity could be detected only after dissociation of the antigen-AF8 antibody complex. No cross reaction was found with any lysosomal protein including the cysteine proteinases, cathepsins H and L.

Animals↗
PubMed1981

The loosely bound non-histone chromosomal proteins of rat prostate in androgen action.

Rat prostatic chromatin was fractionated by sequential salt extractions into 0.35 M NaCl-soluble (the loosely bound non-histone chromosomal proteins), 2 M NaCl-soluble and -insoluble (the residual proteins) fractions. Homologous and heterologous chromatins were reconstituted with these chromosomal fractions from castrated rats injected with testosterone for 2 h and from castrated control. Chromatin reconstituted with all hormone-treated fractions showed 30% more binding with polylysine and transcriptional template activity than did chromatin reconstituted with castrated fractions. Similar analyses of reconstituted heterologous chromatins indicated that the androgen-induced changes in chromatin were mainly determined by the androgenic state of the 0.35 M NaCl-soluble fraction. The 0.35 M NaCl-soluble fraction also contained 18% more total high mobility group protein than did the corresponding castrated fraction. Limited digestion of 5 alpha-[3H]dihydrotestosterone-bound nuclei with pancreatic DNAase 1 to 5% acid-soluble resulted in a rapid release of the nuclear-bound 3H-labelled androgen and protein, in contrast to similar digestion with microsomal nuclease. Since a large portion of the translocation androgen-receptor is bound to protein(s) in the 0.35 M NaCl-soluble fraction, these results suggest that the component(s) in the 0.35 M NaCl-soluble fraction to which androgen-receptor binds is associated with actively transcribing sequences on chromatin.

Androgens↗
PubMed1978

Effects of shearing on chromatin structure.

The effects of mechanical shearing on chromatin structure were investigated by using thermal denaturation and circular dichroism (CD) spectroscopy. Under ordinary conditions of mechanical shearing used for preparation of soluble chromatin, we observed only minor changes (less than 10%) of chromatin properties with respect to (a) absorption melting curves, (b) CD spectra, (c) CD melting curves and (d) histone transfer from chromatin to exogenous DNA. Such small pertubation of structural properties could be due to the generation of free ends when a large chromatin was cut into smaller fragments and by weakening the binding of histones to DNA near these free ends. In addition to mechanical shearing, sonication was used to shear some samples of chromatin. The effect of sonication on chromatin structure was investigated by the same physical methods used for mechanically sheared chromatin. The results indicate that sonication only slightly changes the chromatin properties with respect to CD spectra, similar to the results obtained by mechanical shearing, but sonication at high settings has a greater effect on the thermal denaturation property of chromatin as contrasted to our results from mechanically sheared chromatin.

Animals↗
PubMed1978

Protein dissociation from DNA in model systems and chromatin.

Salt induced dissociation of protamine, poly(L-lysine) and poly(L-arginine) from DNA was measured by relative light scattering at theta = 90 degrees and/or centrifugation. Dissociation of histones from DNA was studied using relative light scattering and intrinsic tyrosine fluorescence. Protamine was dissociated from DNA at 0.15 M MgCl2 (ionic strength mu = 0.45) or 0.53 M NaCl (mu = 0.53) based on light scattering data and at approximately 0.2 M MgCl2 (mu = 0.6) or 0.6 M NaCl based on centrifugation data. NaCl induced dissociation of poly(Lys) or poly(Arg) from natural DNAs measured by light scattering did not depend on the guanine plus cytosine content. To dissociate poly(Arg) from DNA higher ionic strength using NaCl, MgCl2, or CaCl2, similar ionic strength using NaClo4, and lower ionic strength using Na2SO4 was needed then to dissociated poly(Lys). Both the decrease in light scattering and the enhancement of tyrosine fluorescence of chromatin occurred between 0.5 and 1.5 M NaCl when histones were dissociated.

Anions↗
PubMed1977

Structural transition in chromatin induced by ions in solution.

Structural transition in chromatin was measured as a function of counter ions in solution (NaCl or MgCl(2)) and of histones bound on the DNA. The addition of counter ions to aqueous solutions of chromatin, partially dehistonized chromatin, and DNA caused a drastic reduction in viscosity and a significant increase in sedimentation coefficient. Transitions occurred primarily at about 2 x 10(-3) M NaCl and 1 x 10(-5) M MgCl(2) and are interpreted as a change in structure of chromatin induced by tight binding of cations (Na(+) or Mg(++)) to DNA, either free or bound by histones, and is an intrinsic property of DNA rather than of the type of histone bound. At a given ionic condition, removal of histone H1 from chromatin had only a minor effect on the hydrodynamic properties of chromatin while removal of other histones caused a drastic change in these properties. An increase in the sedimentation coefficient of DNA was observed also for protamine. DNA complexes wherein the bound protein contains only unordered coil rather than the alpha-helices found in histones.

Animals↗