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Biomedical subjects

I Montfort

Publications and source records attributed to I Montfort.

34 records · Page 2Linked to original sources
PubMed1987

Collagenolytic activity in experimental cirrhosis of the liver.

Collagenolytic activity has been measured in insoluble sediments of normal and CCl4-induced cirrhotic rat liver. Two techniques were used: in one assay, the insoluble liver sediments were incubated with a radioactively labeled exogenous substrate; in the other assay, the endogenous collagen present in the insoluble liver sediments served as the substrate. Animals in various stages of development of CCl4-induced cirrhosis were used. Results suggest that in rat liver cirrhosis collagenolytic activity assayed with both techniques appears to increase when expressed as weight of collagen degraded in 24 hr. On the other hand, when the data are corrected for the variable amounts of collagen present in the insoluble liver sediments and expressed as weight of collagen degraded in 24 hr per unit weight of total collagen incubated, collagenolytic activity is shown to be significantly decreased below the normal level and to remain so for prolonged periods.

Animals↗
PubMed1987

Catalytic classes of proteinases of Entamoeba histolytica.

Endopeptidase inhibitors were used to determine the catalytic classes of proteinases present in extracts of Entamoeba histolytica (strain HM 1:IMSS) axenically grown in vitro. Cysteine proteinases account for most of the proteolytic activity; one or more proteinases with different catalytic mechanisms are also present but could not be unambiguously assigned to a particular catalytic class. Proteinases in amebic lysates were resolved by polyacrylamide gel electrophoresis with sodium dodecyl sulfate. The detergent was exchanged with Triton X-100 and the proteolytic activity in the gels was demonstrated by overlaying it on another gel containing the substrate. Four lysis zones were observed corresponding to molecular weights of 66,000, 56,000, 40,000 and 27,000. The first cannot be classified yet, but the last three showed properties consistent with those of cysteine proteinases. Finally, a novel technique is described which uses purified human alpha-2-macroglobulin to trap, purify and characterize proteases from amebic lysates. The results obtained with this technique confirm those of the overlay technique, since both methods reveal four distinct proteinases in the two different amebic preparations examined.

Animals↗
PubMed1987

Increase of biosynthesis and degradation of collagen in normal lungs induced by soluble factors obtained from experimental pulmonary silicosis.

We have studied the effects of soluble factors obtained from rat lungs with experimentally-induced pulmonary fibrosis of 2 months duration on the in vitro rates of biosynthesis and degradation of collagen in normal rat lung preparations. Factors soluble in phosphate-buffered saline were prepared from the minced lungs of normal controls and of silicotic animals. The in vitro rate of collagen biosynthesis of normal rat lung explants was measured as the rate of incorporation of radioactive proline into total and collagenous protein. The in vitro rate of collagen degradation in normal rat lung homogenates was measured as the rate of release of hydroxyproline-containing materials of less than 100,000 daltons to the supernatant. Our results suggest that in this experimental model of pulmonary fibrosis there are soluble factors that stimulate both collagen biosynthesis and collagen degradation in in vitro preparations of normal rat lung.

Animals↗
PubMed1985

A new model of diffuse interstitial pulmonary fibrosis in the rat.

We have produced experimental diffuse interstitial pulmonary fibrosis in rats with a combination of low and repeated doses of paraquat plus continuous exposure to normobaric 74% O2 in the breathing air for several weeks. Pulmonary fibrosis was evaluated histologically and biochemically, through the determination of total collagen content in the lung. Our procedure is characterized by low initial mortality, the development of extensive distortion of the pulmonary architecture, and the presence of severe and diffuse interstitial fibrosis. The model was compared with bleomycin-induced pulmonary fibrosis in the same rat strain, in which the process is focal and leaves most of the lung unaffected. We conclude that lung damage produced by the combination of low doses of paraquat plus normobaric 74% O2 concentration in the breathing air is an adequate experimental model of diffuse interstitial pulmonary fibrosis as it occurs in many of the human cases of this condition.

Animals↗
PubMed1985

The duration of the pulmonary paraquat toxicity--enhancement effect of O2 in the rat.

The duration of the pulmonary paraquat toxicity-enhancement effect of O2 has been examined in Wistar rats. In one experiment, various groups of normal animals were given a single dose (5 mg/kg body wt) of paraquat and after different periods were exposed to continuous breathing of normobaric 74% O2 in airtight chambers until dead or up to 10 days. In a reverse experiment, a large number of rats were first exposed for 6 days to continuous breathing of normobaric 74% O2 and were then separated into various groups which received a single dose of paraquat (5 mg/kg body wt) after various periods of breathing normal air, ranging from 0 to 96 hr. The extent of pulmonary damage in both experiments was evaluated by histologic examination and by biochemical determination of total collagen content of the lungs. It was found that the duration of the pulmonary damage induced by paraquat that is enhanced by continuous breathing of high O2 concentration lasts 24 to 48 hr. It was also observed that 12 to 24 hr after paraquat administration and continuous breathing of high O2 concentration pulmonary lesions are severe and extensive, and in animals surviving 6 or more days there was also incipient interstitial fibrosis. The reverse sequence of treatment (O2 + paraquat) resulted in no mortality and no pulmonary lesions. Additional controls treated with each of the pulmonary toxins alone also revealed no lung changes.

Animals↗
PubMed1983

Immunohistochemical identification of collagenase in carrageenin granuloma.

The collagenase present in experimental carrageenin granuloma in the guinea pig has been purified to homogeneity in acrylamide gel electrophoresis by a combination of ammonium sulfate salting out and affinity chromatography on Sepharose 4B--collagen-packed columns. The single protein band thus obtained was used as an antigen to obtain a monospecific antibody in heterologous conditions. Several immunodiffusion, immunoaffinity chromatography, and immunoinhibition tests of the antibody against the specific antigen and various possible serum and tissue contaminants suggested that the antibody was specifically directed against the enzyme protein collagenase. Indirect immunohistochemical staining of carrageenin granulomas, samples at different developmental phases with this specific anti-collagenase antibody, revealed that the specific antigenic protein (the enzyme collagenase) is universally present on the extracellular structures at both the collagen-deposition and the collagen-resorption stages. A hypothesis is proposed to account for these findings, namely, that the enzyme collagenase is bound to its substrate (collagen) under both normal and pathological conditions, and that the critical point of control of collagen degradation must be the activation of the collagen-bound enzyme.

Animals↗
PubMed1980

What controls collagen resorption in vivo?

The local control of collagen degradation in mammals in vivo is currently considered to depend primarily on variations on the level of activity of specific collagenases. Such variations are believed to depend on three factors: a) the rate of active collagenase synthesis and/or of activation of inactive enzyme precursors; b) the action of serum and/or tissue collagenase inhibitors; and c) different combinations of both mechanisms. We suggest that another element contributing to the regulation of collagen degradation in vivo is the susceptibility of the substrate. Support for this suggestion is derived from two sources: 1) experimental data, indicating that the rate of collagen degradation depends on the genetic type of substrate, on its state of aggregation (including degree of cross-linking), and on the nature and amount of other macromolecules associated with collagen in vivo. Other experimental findings supporting our hypothesis are the universal presence of collagen-bound collagenase, the apparent greater affinity of the enzyme for the more recently synthetized substrate molecules, and the increased amounts of intact collagen that may be solubilized from some tissues undergoing massive collagen degradation, 2) analogy with currently accepted views on intracellular protein catabolism, which cannot be rejected a priori as irrelevant to the problem.

Collagen↗
PubMed1980

Collagen-bound collagenase.

The presence of collagenase bound to collagen extracted and purified from several animal and human sources by a standard procedure has been confirmed by different methods. Polyacrylamide (10%) gel electrophoresis at pH 8.1 of intact or "spontaneously"degraded neutral salt soluble collagen results in the separation of two components: the upper one says at the origin and represents collagen or collagen ragments, whereas the lower protein component contains no collagen, often preserves specific collagenolytic activity, and migrates as a single band in SDS/polyacrylamide electrophoresis. With lower polyacrylamide gel concentration the electrophoretic separation of the two components is less clear. Removal of the lower protein component from collagen solutions by two different methods (TCA-ethanol purification cycles and pepsin digestion) results in concomitant loss of their "spontaneous" instability. Eluates of the lower protein component stimulate the heterologous production of a monospecific antibody capable of inhibiting the collagenolytic activity of homologous crude collagenase preparations. It is suggested that collagen-bound collagenase is not an artifact of the extraction procedure but rather a physiological reality, probably corresponding in the living animal to the enzyme closely associated with extracellular collagen fibers, revealed by immunohistochemical methods.

Animals↗
PubMed1980

The susceptibility of hepatic collagen to homologous collagenase in human and experimental cirrhosis of the liver.

The susceptibility of hepatic collagen to homologous collagenase in human and experimental CCl4 cirrhosis of the liver has been explored in vitro by exposure of cryostat liver sections to the corresponding enzymes for different time periods. The morphology and extent of collagen degradation was studied by the Picrosirius red/polarizing microscopy technique. The results of various experiments indicate that collagen present in cryostat sections of both human and rat normal and cirrhotic livers is resistant to trypsin digestion for periods of exposure of up to 48 hours but that heating the sections to 60 C for 1 hour renders the collagen susceptible to degradation by trypsin. Incubation of cryostat liver sections with bacterial collagenase revealed progressive degradation of collagen with a uniform pattern of changes in the original color and diameter of the fibers. Exposure of liver sections to homologous collagenases gave rise to the same pattern of changes observed with bacterial collagenase, although less extensive when equal incubation periods were compared. Nevertheless, sufficiently prolonged incubation of liver sections with their homologous collagenases eventually showed degradation of all collagen present in all normal and cirrhotic liver sections. These observations suggest that in the presence of non rate-limiting concentrations of homologous collagenase, the susceptibility of hepatic collagen to the corresponding degrading enzyme is probably not responsible for the irreversibility of the disease.

Animals↗
PubMed1978

Collagenase in experimental carbon tetrachloride cirrhosis of the liver.

The presence and distribution of collagenase in experimental CCl4 cirrhosis of the liver in rats has been studied by immunohistochemical techniques. A monospecific anti-rat uterus collagenase antibody was raised in rabbits and used for indirect immunofluorescence staining of liver sections obtained from rats in both the reversible and irreversible stages of CCl4-induced cirrhosis. Collagenase is present assoicated with connective tissue septums as long as cirrhosis is reversible, and it is not detectable in the irreversible stage. In animals sacrificed during the transition between the reversible and irreversible stages of cirrhosis, collagenase appeared bound to the outer surfaces of connective tissue septums and was absent from the deeper portions. These observation suggest that the irreversibility of experimental CCl4 cirrhosis of the liver is associated with a disturbance in the mechanisms of collagen degradation, which may be a deficiency in collagenase activity, a change in the susceptibility of the substrate, or a combination of both factors.

Animals↗
PubMed1975

The distribution of collagenase in normal rat tissues.

A rabbit monospecific anti-rat uterus collagenase antibody has been used to study the distribution of collagenase in normal rat tissues by immunohistochemical methods. Indirect staining was performed with fluorescein-conjugated goat anti-rabbit immunoglobulin G antibody. The organs studied were brain, lung, myocardium, liver, spleen, kidney, adrenal, testes, uterus, xiphoid cartilage, tail tendon, skeletal (triceps) muscle and skin. Collagenase is widely present throughout the connective tissue structures in all organs examined. The enzyme is apparently bound to collagen fibers, reticulum fibers and basement membranes. The results suggest that control of collagenase activity depends on factors other than the presence of the enzyme in tissues.

Animals↗
PubMed1975

The distribution of collagenase in the rat uterus during postpartum involution. An immunohistochemical study.

A monospecific rabbit anti-rat uterus collagenase antibody has been prepared and used to study the distribution of the enzyme in the rat uterus during postpartum involution. Cryostat sections of rat uteri from 24 to 240 hours postpartum were stained by the indirect immunofluorescent method. Nonpregnant rat uterus revelaed positive staining in basement membranes, in endometrial stroma, in perimuscular and in vascular connective tissue. During postpartum involution of the uterus two types of changes in uterine collagenase were observed: (1) variations in the distrubiton of the enzyme, which became selectively localized in the epithelial basement membrane and in the wall of small blood vessels, and (2) variations in the overall intensity of fluorescent staining, which decreased immediately after delivery and slowly increased back to nonpregnant levels in 5 days. The significance of these findings is discussed in relation to the mechanisms of control of collagenase activity in vivo.

Animals↗