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Biomedical subjects

N Ramamurthy

Publications and source records attributed to N Ramamurthy.

At least 37 records · Page 2Linked to original sources
PubMed1992

Specificity of the anticollagenase action of tetracyclines: relevance to their anti-inflammatory potential.

The concentrations of doxycycline and 4-de-dimethylaminotetracycline required to inhibit 50% of collagenase activity were found to be 15 to 30 microM for human neutrophil and gingival crevicular fluid collagenases. Fibroblast collagenase was relatively resistant to inhibition by tetracyclines; the 50% inhibitory concentrations of doxycycline and 4-de-dimethylaminotetracycline were 280 and 510 microM, respectively.

Anti-Inflammatory Agents↗
PubMed1990

Minocycline-treatment of diabetic rats normalizes skin collagen production and mass: possible causative mechanisms.

Daily minocycline-treatment of streptozotocin-induced diabetic rats not only prevented a diabetes-caused atrophy of skin collagen mass (10-mos old rats), but also normalized skin collagen mass to match that of growing (ca. 1%/d) non-diabetic controls (4- and 5-mos old rats). The causative mechanism by which minocycline-treatment normalizes skin collagen mass must, in part, be related to a general anabolic effect on growth (body weight) because the effect on skin collagen mass correlates strongly to that on body weight. Consequently, a minocycline-stimulated increase of a systemic factor (such as insulin-like growth factor) is not unlikely. The anabolic effect of minocycline-treatment of diabetic rats is also expressed as a normalized cellular ribosome mass (an index of total protein synthetic capacity) and a normalized absolute rate of collagen production. (Calculation of an absolute rate was justified by an apparent maximum saturation of the prolyl-tRNA pool(s) of skin, maximum saturation obtained by the pool-flooding approach). The normalized skin ribosome amount does not, however, explain a selective effect of minocycline-treatment on collagen production as opposed to that for non-collagen protein, this selective effect measured as relative collagen production. To explain such selectivity, the inhibition of diabetes-induced excess skin collagenase activity seems unlikely. (This inference is based on results from a preliminary study indicating that recently [less than 2 h] synthesized collagen is not degraded by the excess collagenase in skin of diabetic rats). Thus, the principal collagen fraction acted on by pathologically excess collagenase might be collagen at a later stage (greater than 2 h after synthesis) in its life cycle. (Another possibility for the selective effect of minocycline on collagen production, as yet untested, is reduced intracellular procollagen degradation.) Overall, this is the first study aimed at discerning the mechanism(s) by which minocycline-treatment enhances the rate of collagen production in tissues of a diabetic rat. For future studies, the extent to which the positive effect on growth, ribosome mass, and rate of collagen production contributes to the change of collagen mass must, along with the known minocycline-inhibition of collagenase activity, be quantified. Such quantification is a prerequisite for evaluating the chemotherapeutic efficacy of minocycline-treatment on collagen-degradative diseases.

Animals↗
PubMed1990

Age for measles immunization seroconversion after measles vaccination at 6-8 months of age--a randomized controlled trial.

The objective of the study was to compare the effectiveness of measles vaccine by seroconversion in vaccinated children with non-vaccinated children of 6 to 8 months age group in a city slum community so as to study the feasibility of advancing the age of immunization. Live attenuated lyophilized Schwartz strain of measles vaccine was used. Hemagglutination inhibition (HI) antibody was estimated. Seroconversion was defined as either the conversion of negative to positive or a two fold rise in titre. One hundred and thirty two children completed the study. There was no difference in the age, sex and nutritional status between vaccinated and non-vaccinated groups (p greater than 0.7). The seroconversion rate in the vaccinated group was 65% and in the non-vaccinated group was 26%. The age, sex and nutritional status did not significantly affect the seroconversion. Our data suggest that immunization with measles vaccine may be effective as early as 6 months of age. Immunization at 6 months may be needed at least for children in densely populated areas like cities and towns.

Age Factors↗
PubMed1989

Gelatin-degrading type IV collagenase isolated from human small cell lung cancer.

The goal of this study has been to identify and characterize metalloproteinases from a highly metastatic human small cell lung cancer cell line. The cytosol isolated from NCI-H82 lung cancer cells propagated as solid tumors in nude mice contained a gelatinolytic enzyme that was subjected to ammonium sulfate precipitation, zinc chelate Sepharose column chromatography, anion exchange chromatography and gel permeation chromatography. This purification scheme resulted in a 280-fold enrichment of an active gelatin and type IV collagen-degrading enzyme. On gelatin zymography two bands of gelatinolytic activity were detected, corresponding to Mr of 75,000 and 63,000. Gelatinolytic activity was inhibited by metal chelators, tetracyclines, and serum. On immunoblotting using an affinity-purified polyclonal rabbit antibody to a peptide region of type IV collagenase, the tumor enzyme was identified as type IV collagenase. A second tumor metalloproteinase of Mr = 29,000, which degraded proteoglycan substrates, was also isolated.

Animals↗
PubMed1988

Purification of a gelatin-degrading type IV collagenase secreted by ras oncogene-transformed fibroblasts.

Connective tissue matrix-degrading metalloproteinases play an important role in cancer invasion. In this report we describe the isolation of a metalloproteinase exhibiting both type IV collagenolytic and gelatinolytic activities from the conditioned medium of NIH-3T3 fibroblasts transformed with DNA containing an activated c-Harvey-ras oncogene from T24 bladder cancer cells. This tumor proteinase was purified by anion exchange chromatography, zinc-chelate Sepharose chromatography, and gel permeation chromatography. The final product was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (relative molecular mass = 67,000). Gelatin zymography revealed two bands of gelatinolytic activity, corresponding to molecular weights of 67,000 and 62,000. Upon immunoblotting with the use of an affinity-purified polyclonal rabbit antibody to a peptide region of type IV collagenase that lacks homology with interstitial collagenase or stromelysin, the purified tumor enzyme was identified as type IV collagenase.

Cell Line↗
PubMed1988

Streptozotocin-induced diabetes and the rat periodontium: decreased relative collagen production.

This is the first study concerning the extent to which relative collagen production (RCP) in rat periodontal tissues is affected by diabetes. Determination of RCP, rather than individual production rates for collagen or for non-collagen protein, was deemed necessary because saturation of all proline pools in tissues of diabetics (and non-diabetic controls) was not achieved. Such non-saturation occurred despite the injection of a pool-expanding dose of proline (400-1150 mg/rat), non-saturation indicated by the lesser specific radioactivity (S.R.) of free-[3H]proline in tissues than that of the injected solution. RCP was decreased in five periodontal tissues (incisor and molar gingiva, incisor and molar periodontal ligament, antemolar palatal mucosa) and in skin. Diabetes-decreased RCP seems to result from decreased collagen synthesis and increased intracellular degradation, although some evidence is presented for increased extracellular degradation of recently secreted collagen.

Animals↗
PubMed1987

Purification and characterization of a connective-tissue-degrading metalloproteinase from the cytosol of metastatic melanoma cells.

A metalloproteinase with activity against type IV collagen, type I collagen and gelatin has been purified from the cytosol of a highly metastatic mouse melanoma by anion-exchange, zinc-chelated and lectin-affinity column chromatography. The purified enzyme has a molecular mass of approx. 59 kDa and on isoelectric focusing in two-dimensional gels produced three spots with apparent isoelectric points (pI) between 5.7 and 6.1. Enzymic activity with collagen, but not gelatin, substrates was latent, requiring activation by trypsin or organomercurials. Trypsin activation of this metalloproteinase was accompanied by a change in molecular mass, whereas autoactivation after 1 month's storage, was not. The degradation of types I and IV collagen by the melanoma enzyme yielded products of lower molecular masses than those yielded by mammalian collagenases, this characteristic thus differentiating this metalloproteinase from classical collagenases.

Animals↗
PubMed1987

Metastatic mouse melanoma cells release collagen-gelatin degrading metalloproteinases as components of shed membrane vesicles.

The purpose of this study has been to compare collagen-gelatin degrading enzymes isolated from cancer cell organelles and cytosol to the metalloproteinases released by cancer cells. To this end, metastatic mouse melanoma cell organelles were isolated by sucrose density gradient centrifugation and metalloproteinases were assayed using native and denatured [methyl-3H]collagen substrates. Solubilized proteinases were purified by ammonium sulfate precipitation, anion exchange, concanavalin A affinity and gel-filtration column chromatographic procedures and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The conclusions were as follows: malignant melanoma cells have a metalloproteinase (Mr = 59,000) which is shed from cells into conditioned medium as a component of intact membrane vesicles rather than as a soluble enzyme; storage of tumor-conditioned medium leads to the generation of autoactivated soluble metalloproteinases of lower molecular weight; purification of these metalloproteinase species yielded variant collagenases that have considerable gelatinolytic activity and a cleavage preference site for the Gly-Ile bond in a collagen-like synthetic octapeptide substrate which is typical for collagenase-type metalloproteinases. It is proposed that localization of potent proteinases to the surface of cancer cells facilitates the local breakdown of connective tissues during the invasive process.

Animals↗
PubMed1985

Dietary ascorbic acid normalizes diabetes-induced underhydroxylation of nascent type I collagen molecules.

Nascent collagen alpha chains (types I and III) isolated from diabetic rat skin were shown to be underhydroxylated, an underhydroxylation normalized (type I) or partially corrected (Type III) by dietary ascorbic acid. Increased hydroxylation occurred concomitantly with reduced intracellular procollagen degradation and increased production of nascent collagen molecules, both contributing to an increased total skin collagen mass. Overall, by correcting a defect (underhydroxylation) in a posttranslational event and by increasing collagen production, dietary ascorbic acid improved the collagen status of a diabetes-perturbed connective tissue.

Animals↗
PubMed1984

Skin collagen metabolism in the streptozotocin-induced diabetic rat: free hydroxyproline, the principal in vivo degradation product of newly synthesized collagen--probably procollagen.

We characterized the degradation products of recently synthesized collagen present in skins of control and diabetic rats. Specifically, the TCA-soluble fractions of homogenized skins from control and diabetic rats (killed 1 and 4 hours after [3H]-proline injection) were fractionated by molecular sieve chromatography, and eluted fractions were analyzed for hydroxyproline and [3H]-hydroxyproline. Free [3H]-hydroxyproline was the principal (greater than 95%), low molecular weight (greater than 2000 daltons), [3H]-hydroxyproline-containing material eluted from the molecular sieve column, this amount representing approximately 80% (controls) and approximately 87% (diabetics) of [3H]-hydroxyproline-containing material in TCA-soluble fractions of skin homogenates. These observations are similar to those from the intracellular degradation of cellular and secretory proteins in that the principal--almost exclusive--degradation product was the free amino acid. The free hydroxyproline had a greater specific radioactivity than that in any other [3H]-hydroxyproline-containing fraction (soluble and insoluble, see below); furthermore, the total radioactivity of free [3H]-hydroxyproline was greater at 1 hour than 3 hours later. These two properties (identity with free amino acid; time-dependent decrease in amounts) are consistent with [3H]-hydroxyproline arising from the intracellular degradation of procollagen. The [3H]-hydroxyproline-containing material eluting before free hydroxyproline (designated peptidyl [3H]-hydroxyproline) was similar to free [3H]-hydroxyproline in terms of specific radioactivity and the time-dependent decreases of specific and total radioactivities, these similarities indicating that the peptidyl [3H]-hydroxyproline are intermediates in the degradative pathway of procollagen to free amino acids. Results for control and diabetic rats were qualitatively similar, with regard to the inter-fraction ratios of specific radioactivities and their time-dependent changes. However, the degradative process, as assessed by the release of free and peptidyl [3H]-hydroxyproline, was dramatically enhanced by the diabetic state, extending our previous results based on analyses of uncharacterized degradation products.

Animals↗
PubMed1982

Skin collagen metabolism in the streptozotocin-induced diabetic rat. Enhanced catabolism of collagen formed both before and during the diabetic state.

Collagen catabolism has been measured in skins of streptozotocin-induced diabetic rats. For measuring catabolism of collagen synthesized de novo during the diabetic state, we measured the amounts of [3H]hydroxyproline-containing degradation products in skins of diabetic rats, killed 4 h after [3H]proline injection (protocol 1); degradation products were isolated in TCA-soluble fractions of skin homogenates. For measuring catabolism of collagen preexisting before the induction of the diabetic state, we measured the 21-day loss of [3H]hydroxyproline (and hydroxyproline) in entire skins of rats that were streptozotocin-treated after [3H]proline injection (protocol 2). A 2.5-fold increase in the relative amounts of [3H]hydroxyproline-containing degradation products was measured in the TCA-soluble fractions of skins from diabetic rats (protocol 1). These degradation products had a low molecular weight (as evident from their diffusibility), and they were derived from recently synthesized collagen, possibly procollagen (as evident from their high [3H]hydroxyproline specific activity). Furthermore, they were not derived from the degradation of [3H]hydroxyproline-labeled collagen present before induction of the diabetic state (protocol 2). Evidence for this conclusion is as follows: the amounts of [3H]hydroxyproline-containing degradation products in skins of diabetic rats were not greater than that in skins of control rats, despite a 50% resorption of collagen in skins of diabetic rats. Overall, the catabolism of collagen formed de novo during the diabetic state was distinguished from the catabolism of collagen formed before, and both catabolic processes were enhanced in rat skins of streptozotocin-induced diabetic rats.

Animals↗