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Biomedical subjects

M S Murthy

Publications and source records attributed to M S Murthy.

At least 55 records · Page 3Linked to original sources
PubMed1987

Some differences in the properties of carnitine palmitoyltransferase activities of the mitochondrial outer and inner membranes.

Recent evidence has shown that the outer, overt, malonyl-CoA-inhibitable carnitine palmitoyltransferase (CPTo) activity resides in the mitochondrial outer membrane [Murthy & Pande (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 378-382]. A comparison of CPTo activity of rat liver mitochondria with the inner, initially latent, carnitine palmitoyltransferase (CPTi) of the mitochondrial inner membrane has revealed that the presence of digitonin and several other detergents inactivates CPTo activity. The CPTi activity, in contrast, was markedly stimulated by various detergents and phospholipid liposomes. These findings explain why in previous studies, which used digitonin or other detergents to expose, separate and purify the CPT activities, the inferences were drawn that (a) the ratio of latent to overt CPT was quite high, (b) both the CPT activities could be ascribed to one active protein recovered, and (c) the observed lack of malonyl-CoA inhibition indicated possible loss/separation of a putative malonyl-CoA-inhibition-conferring protein. Although both CPTo and CPTi were found to catalyse the forward and the backward reactions, CPTo showed greater capacity for the forward reaction and CPTi for the backward reaction. The easily solubilizable CPT, released on sonication of mitoplasts or of intact mitochondria under hypo-osmotic conditions, resembled CPTi in its properties. When octyl glucoside was used under appropriate conditions, 40-50% of the CPTo of outer membranes became solubilized, but it showed limited stability and decreased malonyl-CoA sensitivity. Malonyl-CoA-inhibitability of CPTo was decreased also on exposure of outer membranes to phospholipase C. When outer membranes that had been exposed to octyl glucoside or to phospholipase C were subjected to a reconstitution procedure using asolectin liposomes, the malonyl-CoA-inhibitability of CPTo was restored. A role of phospholipids in the malonyl-CoA sensitivity of CPTo is thus indicated.

Acyltransferases↗
PubMed1987

Enhanced therapeutic efficacy of cisplatin by combination with diethyldithiocarbamate and hyperthermia in a mouse model.

A spontaneously metastasizing solid tumor model derived by transplanting the TA3Ha murine mammary carcinoma into the s.c. tail tissue of mice was used to develop a treatment strategy for enhancing the therapeutic efficacy of cisplatin (CDDP). This strategy was based on the findings that diethyldithiocarbamate (DDTC) reduces the toxicity of CDDP, and that localized hyperthermia (HT) augments the antitumor efficacy of CDDP. DDTC (500 mg/kg) reduced the CDDP-induced nephrotoxicity and gastrointestinal toxicity as well as increased the CDDP LD10 from 8 to 20 mg/kg in strain A mice. When CDDP and DDTC were used in multiple treatment schedules at 5-day intervals, DDTC protected the hosts but not the tumors against the toxicity of CDDP. HT administered locally to the tumor 1 h after the injection of CDDP (8 mg/kg) in 1 ml Hanks' balanced salt solution increased the antitumor effect but not the host toxicity. While administration of 8 mg/kg CDDP alone or with HT three times at 5-day intervals caused 100% host mortality, this dose of CDDP could be used with no mortality by combining it with DDTC. A combination of 8 mg/kg CDDP with DDTC (750 mg/kg) and HT (43 degree C for 60 min), administered three times at 5-day intervals, retarded the local tumor growth significantly compared to the untreated, CDDP plus DDTC plus HT control groups of mice. The frequency of lung metastasis in these groups on day 30 of tumor inoculation were 0, 90, 90, and 80%, respectively. The mean survival days of the mice treated with CDDP plus DDTC plus HT was 61 +/- 6 compared to 34 +/- 5 in the controls. The results presented here demonstrate that by combining CDDP with DDTC, high doses of CDDP can be safely administered. When localized HT is combined with high dose CDDP and DDTC, the tumor growth retardation and the host survival prolongation are significantly better than those obtained with the highest tolerable dose of CDDP alone or CDDP plus HT.

Animals↗
PubMed1987

Factors influencing the growth and metastatic behavior of tumors.

The factors influencing the growth and metastatic behavior of experimental animal tumors are examined. 10(4) TA3Ha cells were injected intraperitoneally, intravenously, subcutaneously into the flank, and subcutaneously into the tail tissues of syngeneic strain A mice. The tumor takes from these injections were 50/50 (100%), 1/10 (10%), 10/10 (100%), and 7/12 (60%), respectively. The frequency of lung metastasis from these sites was 0, 100, 50, and 100%, respectively. At the time of host death, the flank and tail tumors were, respectively, 2.2 +/- 0.5 cm (geometric mean diameter) and 1.2 +/- 0.3 cm in the TA3Ha and 0.9 +/- 0.1 cm and 0.4 +/- 0.1 cm in the L1210 systems. TA3Ha tumors metastasized regularly to the lymph nodes but the L1210 tumors seldom metastasized to the lymph nodes. Tail implants of TA3Ha tumors behaved similarly in the athymic nude mice and strain A mice. TA3Ha cells inoculated into the Millipore chambers and maintained in mice for greater than 150 days were viable and able to form tumors. The results demonstrate that the anatomic location of the tumor affects the growth and metastatic behavior of the tumors, and that the tumors of different histologic origin metastasize differently even when grown in corresponding locations.

Adenocarcinoma↗
PubMed1987

Malonyl-CoA binding site and the overt carnitine palmitoyltransferase activity reside on the opposite sides of the outer mitochondrial membrane.

The overt carnitine palmitoyltransferase (palmitoyl-CoA:L-carnitine O-palmitoyltransferase, EC 2.3.1.21) activity of intact mitochondria from rat heart and liver was found to be resistant to the action of proteases such as Nagarse (subtilisin, EC 3.4.21.14). Nagarse under the same conditions, however, greatly decreased the malonyl-CoA inhibition of carnitine palmitoyltransferase activity, the high-affinity binding of malonyl-CoA to mitochondria, and the ability of malonyl-CoA to shift to the right the sigmoid activity curve of carnitine palmitoyltransferase observed with variations in palmitoyl-CoA concentration. No noticeable effect of Nagarse pretreatment was observed on the binding of octanoyl-CoA to mitochondria. Subfractionation of liver mitochondria using a combination of swelling, shrinking, and density gradient centrifugation yielded a membrane fraction in which the specific activities of the outer membrane marker enzymes were enriched greater than or equal to 16-fold together with a near-parallel enrichment of malonyl-CoA-inhibitable carnitine palmitoyltransferase activity. The percent recovery of this carnitine palmitoyltransferase in the outer membrane vesicles also matched that of the known outer membrane markers. The carnitine palmitoyltransferase activity of these out-side-out vesicles became susceptible to added Nagarse only on their cosonication. These findings show that whereas the malonyl-CoA binding site relevant to the inhibition of carnitine palmitoyltransferase is situated on the outer side of the outer membrane, the overt carnitine palmitoyltransferase activity resides on the inner side of the outer membrane.

Acyl Coenzyme A↗
PubMed1986

Differential effects of phosphatidylcholine and cardiolipin on carnitine palmitoyltransferase activity.

Rates of carnitine palmitoyltransferase-catalyzed conversion of palmitoylcarnitine to palmitoyl-CoA are markedly decreased with the progress of this reaction presumably owing to the build up of inhibitory palmitoyl-CoA in the enzyme vicinity. High, above micellar, concentrations of palmitoylcarnitine, phosphatidylcholine liposomes and high KCl concentrations increased the activity, apparently by facilitating the removal of palmitoyl-CoA from the enzyme surface. The presence of cardiolipin was found to be inhibitory. The enzyme activity followed in the direction of palmitoylcarnitine formation with low palmitoyl-CoA concentration as substrate, was inhibited by phosphatidylcholine, but stimulated by cardiolipin. Both of these lipids markedly stimulated the enzyme activity followed by the isotope exchange procedure which requires progression of both the forward and the backward reactions. The results indicate that one of the effects of phospholipids on carnitine palmitoyltransferase activity is exerted from the ability of these substances to bind the amphipathic reactants of this enzyme, particularly long-chain acyl-CoA. The possibility that the activity of the membrane-bound carnitine palmitoyltransferase may at times be affected by changes in the concentrations and composition of the various phospholipids in the enzyme's vicinity is raised by these findings.

Acyltransferases↗
PubMed1986

A carnitine/acylcarnitine translocase assay applicable to biopsied muscle specimens without requiring mitochondrial isolation.

A simple method for assaying the mitochondrial carnitine/acylcarnitine translocase of muscles that needs only few milligrams of fresh tissue is described. The procedure involves monitoring of the sulphobetaine (an inhibitor of the translocase)-sensitive acetylation of sub-saturating concentrations of carnitine in the medium, linked to the oxidation of [2-14C]pyruvate in the presence of malonate. Conditions affecting the reliability of the outlined procedure and the ancillary information to be collected, namely the activities of pyruvate oxidase system and carnitine acetyltransferase, for detecting possible deficiency of the translocase are described, together with data on the translocase activity in human skeletal muscle, in rat red and white skeletal muscles and in rat heart. The concepts outlined should allow development of assays of other mitochondrial transporters that also would require neither isolation of mitochondria nor availability of a large quantity of tissue, both of which are otherwise needed at present.

Acetylation↗
PubMed1985

Biodistribution and pharmacokinetics of vanadium following intraperitoneal administration of vanadocene dichloride to mice.

The biodistribution and pharmacokinetics of vanadium following i.p. administration of vanadocene dichloride (VDC), a representative of a new class of organometallic anticancer agents, is reported for Strain A mice. A convenient flameless atomic absorption spectroscopic assay is described and is used to determine kinetic profiles for vanadium in blood, kidney, liver, small intestine and brain tissue for times up to 24 h after administration. For a VDC dose of 80 mg/kg, vanadium concentration decreases rapidly from both the blood and small intestine, and the data can be fit to a phenomenological exponential function (blood: t1/2 = 118 +/- 43 min; small intestine: t1/2(alpha) = 18.10 +/- 0.14 min, t1/2(beta) = 341 +/- 45 min). In contrast, vanadium accumulates in both the kidney and liver up to a maximal concentration (1.12 +/- 0.06 mM and 0.56 +/- 0.06 mM after 12 and 8 h, respectively), and is then excreted with estimated half-lives of 7.9 +/- 0.7 and 12.1 +/- 0.1 h, respectively. No detectable levels of vanadium are found in the brain tissue over the temporal course of the experiment. These results are compared to previous mammalian studies with cis-dichlorodiammineplatinum(II) (CDDP) and related 'second generation' platinum derivatives; there are both qualitative similarities between the vanadium and platinum systems as well as important quantitative differences.

Animals↗
PubMed1985

Microcompartmentation of transported carnitine, acetylcarnitine and ADP occurs in the mitochondrial matrix. Implications for transport measurements and metabolism.

Monitoring of the exchange-diffusion of carnitine, acetylcarnitine and ADP by measuring the influx of radioactive substrates into mitochondria or their efflux, as commonly employed, underestimated their true transport. Higher transport rates were realized when the imports were monitored by analysing, in the entire incubation medium, formation of metabolites that could proceed only after the substrate import. A recycling of substrate present in an inner microenvironment near the translocase and in the external medium appeared to be responsible for these results. Microcompartmentation of carnitine was observable also at 30 degrees C. These findings strengthen the concept that a sharing of a microcompartment between transporters and enzymes metabolizing the entered substrates occurs and appears to offer a kinetic advantage for the reactions involved. The possibility that different segments of metabolism involving the same substrate may proceed at different loci within the matrix and thus be amenable to independent controls is also indicated by these findings.

Acetylcarnitine↗
PubMed1985

Combined effect of trans-diamminedichloroplatinum(II) and hyperthermia on murine and human tumor cells.

trans-Diamminedichloroplatinum(II), a paradigm of an inactive platinum compound, exhibited cytotoxic effect against HEP-2 human tumor cells, TA3Ha murine tumor cells, and freshly collected human ovarian carcinoma cells when combined with hyperthermia (43 degrees, 30 min). The heat treatment reduced the D0 of trans-platinum from 56 to 16.5 micrograms/ml in the HEP-2 system and from an undeterminable value at 37 degrees to 8.2 micrograms/ml in the TA3Ha system. Heat treatment before trans-platinum was more cytotoxic than that after trans-platinum in the TA3Ha system (P less than 0.001). TA3Ha cells treated in vitro with 40 micrograms/ml TDDP at 43 degrees failed to form tumors in mice upon subcutaneous implantation into the tails of mice. In contrast, these agents given singly did not alter the tumor-forming ability of TA3Ha cells. In vivo administration of trans-platinum after hyperthermia (43 degrees for 30 min) retarded the growth of TA3Ha tumors compared to either treatment alone. trans-Platinum did not form detectable DNA-interstrand cross-links in the HEP-2 cells treated at 37 degrees or 43 degrees. However, the DNA-protein cross-links were detectable under these conditions. The frequencies of DNA-protein cross-links were higher in the cells treated at 43 degrees than in those treated at 37 degrees, both immediately after and 12 h after the treatment with trans-platinum. Heat alone did not induce the formation of either DNA-interstrand or DNA-protein cross-links. Heat treatment did not appear to enhance the entry of trans-platinum into the cells.

Animals↗
PubMed1984

Mechanism of carnitine acylcarnitine translocase-catalyzed import of acylcarnitines into mitochondria.

Mitochondrial imports of acylcarnitine and carnitine have been measured by new methods based on the monitoring of deacylation of acylcarnitines and the acetylation of carnitine in the matrix, subsequent to their entry. These methods have shown higher import rates than those calculated from the uptake of radioactive carnitines into mitochondria as employed until presently. This new approach has permitted the import of long chain acylcarnitine to be followed unambiguously; the results have confirmed that the carnitine acylcarnitine translocase is indeed involved in this import which also proceeds by a mole to mole exchange-diffusion against internal carnitine. Depletion of matrix carnitine greatly decreased the substrate import rates based on their uptake assay but much less so when the deacylation and acylation techniques were employed to monitor imports. These results have revealed that there is a small pool of carnitine in the matrix which readily equilibrates with the medium carnitine through the translocase but which equilibrates with the larger matrix carnitine pool slowly. This finding has necessitated reinterpretation of several previous observations on the translocase that were based on the assumption of a single matrix carnitine pool and in which the translocase was assumed to constitute the rate-limiting step in the activity measurements.

Acetylcarnitine↗
PubMed1984

Sodium glycolate absorption in rat intestine.

Absorption of sodium [1-14C]glycolate by rat intestine was studied by using the tissue accumulation technique with everted intestinal rings. Saturation kinetics was observed for the absorption of glycolate in the jejunoileal region, with a Km of 6.25 mM for glycolate and a Vmax of 5.56 mumole/30 min/g wet wt. The absorption was linear up to a period of 25 min at 37 degrees C. Jejunum and ileum showed significantly higher absorption of glycolate as compared to colon. Sulfhydryl binding agents, viz., p-chloromercuribenzoate and iodoacetate, and respiration inhibitors, e.g., KCN and 2,4-dinitrophenol, had no significant effect on glycolate uptake. However, glyoxylate and lactate showed significant inhibition at 6 mM concentration of the inhibitor. Pyridoxine deficiency had no effect on glycolate uptake by the rat intestine.

2,4-Dinitrophenol↗
PubMed1984

Relationships between ethylation of hemoglobin, ethylation of DNA and administered amount of ethyl methanesulfonate in the mouse.

Ethylation of guanine-N-7 in the DNA of liver and kidney and of nucleophilic groups in hemoglobin has been studied as a measure of the in vivo dose in the mouse after i.p. administration of radiolabeled ethyl methanesulfonate (EMS). The degree of ethylation in both hemoglobin and DNA of the studied tissues was found to increase proportionally to the injected amount in the range 0.32-100 mumoles EMS/kg b.w. Above this range a somewhat higher than proportional degree of ethylation was found, indicating saturation of a system for detoxification of the compound, resulting in a decreased rate of elimination and consequently an increased dose in the tissues of this directly alkylating agent. The ratio between covalent binding to DNA and to hemoglobin, however, was approximately constant over the wide range of doses studied. For biological effects with a linear dose-response relationship, this demonstrates the validity of hemoglobin alkylation as an indicator of the risk.

Alkylation↗
PubMed1984

Effect of localized hyperthermia on TA3Ha tumor transplanted subcutaneously in the tails of mice.

Localized hyperthermia (43 degrees) in single or multiple fractions was applied to mouse mammary adenocarcinoma TA3Ha implanted into the s.c. tail tissue of strain A mice. The effects of heat on the growth of local tumors, on the pattern of metastasis, and on the survival periods of the hosts were studied. Hyperthermia was administered by heating the tumor-bearing tails in a water bath. Multiple 30-min hyperthermia treatments at 5- or 7-day intervals controlled local tumor growth better than did a single 30-min treatment or multiple 30-min treatments at 3-day intervals or at intervals longer than 7 days. Heat treatments that produced cytostatic effects on tumors, sparing the normal tissue, had no effect on either the survival of the hosts or the extent of metastasis to the lungs and the lumbar lymph nodes. However, local treatments reduced the frequency of renal lymph node metastasis, indicating that concurrent metastases in different sites may exhibit differential heat sensitivities.

Adenocarcinoma↗
PubMed1983

A study of the induction of gene conversion in yeast by sodium bisulphite under radical reaction conditions.

Sodium bisulphite (NaHSO3) at low concentrations is known to undergo radical reactions in the presence of Mn2+ ions, generating sulphite anion radicals (SO-3.). The kinetics of pH variation in aqueous solutions of NaHSO3 in the presence of 0.1 mM-MnSO4 suggests that the anion radicals are generated rapidly at low bisulphite concentration (10 mM) and quenched at a higher concentration (100 mM). The ability of NaHSO3 to induce gene conversion in the diploid yeast Saccharomyces cerevisiae BZ34 under conditions suitable for radical reactions has been investigated. The results show that NaHSO3 does not induce gene conversion under these conditions.

Dose-Response Relationship, Drug↗
PubMed1983

Effect of sodium glycolate and sodium pyruvate on oxalic acid biosynthesizing enzymes in rat liver and kidney.

Sodium glycolate feeding (50 mg/100 g body weight/day) to adult male rats for 7 days resulted in increased activities of glycolate oxidase in liver and lactate dehydrogenase in liver and kidney. However, the activity of glycolate dehydrogenase decreased both in liver and kidney. Treatment of sodium pyruvate (100 mg/100 g body weight/day) to the glycolate-fed rats resulted in lowered liver glycolate oxidase activity, and the glycolate dehydrogenase activity was further decreased as compared to glycolate-fed rats in both age groups. However, lactate dehydrogenase activity was not affected by pyruvate feeding in comparison to the glycolate-treated group. It is concluded that glycolate-induced oxalate biosynthesis in rats involves increased activity of liver glycolate oxidase, and pyruvate feeding inhibits glycolate oxidase, thereby decreasing oxalate biosynthesis.

Alcohol Oxidoreductases↗