Search PubMed⌕ Search

Biomedical subjects

M K Reddy

Publications and source records attributed to M K Reddy.

At least 91 records · Page 5Linked to original sources
PubMed1987

Peroxisome proliferator-binding protein: identification and partial characterization of nafenopin-, clofibric acid-, and ciprofibrate-binding proteins from rat liver.

Peroxisome proliferators (PP) induce a highly predictable pleiotropic response in rat and mouse liver that is characterized by hepatomegaly, increase in peroxisome number in hepatocytes, and induction of certain peroxisomal enzymes. The PP-binding protein (PPbP) was purified from rat liver cytosol by a two-step procedure involving affinity chromatography and ion-exchange chromatography. Three PP, nafenopin and its structural analogs clofibric acid and ciprofibrate, were used as affinity ligands and eluting agents. This procedure yields a major protein with an apparent Mr of 70,000 on NaDodSO4/PAGE in the presence of reducing agent and Mr 140,000 (Mr 140,000-160,000) on gel filtration and polyacrylamide gradient gel electrophoresis under nondenaturing conditions, indicating that the active protein is a dimer. This protein has an acidic pI of 4.2 under nondenaturing conditions, which rises to 5.6 under denaturing conditions. The isolation of the same Mr 70,000 protein with three different, but structurally related, agents as affinity ligands and the immunological identity of the isolated proteins constitute strong evidence that this protein is the PPbP capable of recognizing PP that are structurally related to clofibrate. The PPbP probably plays an important role in the regulation of PP-induced pleiotropic response.

Animals↗
PubMed1986

Transcription regulation of peroxisomal fatty acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase in rat liver by peroxisome proliferators.

The structurally diverse peroxisome proliferators ciprofibrate, clofibrate, and bis(2-ethylhexyl) phthalate [(EtHx)2 greater than Pht] increase the activities of hepatic catalase and peroxisomal fatty acid beta-oxidation enzymes in conjunction with profound proliferation of peroxisomes in hepatocytes. In order to delineate the level at which these enzymes are induced in the liver, the transcriptional activity of specific genes for fatty acyl-CoA oxidase (FAOxase) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (PBE), the first two enzymes of the peroxisomal beta-oxidation system, and for catalase were measured in isolated hepatocyte nuclei obtained from male rats following a single intragastric dose of ciprofibrate, clofibrate, or (EtHx)2 greater than Pht. All three peroxisome proliferators rapidly increased the rate of FAOxase and PBE gene transcription in liver, with near maximal rates (9-15 times control) reached by 1 hr and persisting until at least 16 hr after administration of the compound. FAOxase and PBE mRNA levels, measured by blot-hybridization analysis and FAOxase and PBE protein content, analyzed by immunoblotting, increased concurrently up to at least 16 hr following a single dose of peroxisome proliferator. The catalase mRNA level increased about 1.4-fold, but the transcription rate of the catalase gene was not significantly affected. The results show that the peroxisome proliferators clofibrate, ciprofibrate, and (EtHx)2 greater than Pht selectively increase the rate of transcription of peroxisomal fatty acid beta-oxidation enzyme genes. Whether the transcriptional effects are mediated by peroxisome proliferator-receptor complexes remains to be elucidated.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1986

Nephritogenicity of antibodies to proteoglycans of the glomerular basement membrane--I.

We investigated nephritogenic potential of antibodies to heparan sulfate-proteoglycan of glomerular basement membrane. Glomeruli were isolated, basement membranes were prepared, proteoglycans extracted, and purified core protein was obtained. We immunized rabbits with the core protein, IgG fraction prepared from the antisera and specificity of the antibody determined. A single immunoprecipitin line in agar diffusion plate and a single band (approximately 18,000 mol wt) on the immunoblot autoradiograms were visualized. The antibody showed precise reactivity with the glomerular basement membranes. The clearance studies indicated that approximately 75% of the radioiodinated antibody disappeared from circulation within 1 h and 1-2% bound to the kidney. For nephritogenicity experiments, the antibody was intravenously administered into rats and we examined their kidneys at 1 h to 24 d later. A linear immunofluorescence of glomerular basement membranes was observed with rabbit IgG at all times while that of C3 until the 10th day. Early morphologic changes included glomerular infiltration of polymorphonuclear leukocytes with focal exfoliation of endothelium. The leukocytic infiltration subsided by the third day and was followed by progressive thickening of basement membranes, focal mesangial cell proliferation, increase in mesangial matrix, and accumulation of monocytes. Focal knob-like thickening of glomerular basement membrane was observed from the 15th day onward. Regularly-spaced electrondense deposits were seen in the lamina rara interna and externa of glomerular basement membranes and persisted throughout the investigatory period. No significant proteinuria was observed at any stage of the experiment. These findings suggest that the antibodies to the basement membrane heparan sulfate-proteoglycan are nephrotoxic but possess weak nephritogenic potential.

Animals↗
PubMed1986

Comparison of hepatic peroxisome proliferative effect and its implication for hepatocarcinogenicity of phthalate esters, di(2-ethylhexyl) phthalate, and di(2-ethylhexyl) adipate with a hypolipidemic drug.

Peroxisome proliferation is inducible in hepatocytes of rodent and nonrodent species by structurally dissimilar hypolipidemic drugs and certain phthalate ester plasticizers. The induction of peroxisome proliferation appears to be a tissue specific response limited largely to the hepatocyte. Peroxisome proliferation is associated with increases in the activity of the H2O2-generating peroxisomal fatty acid beta-oxidation system and in the amount of peroxisome proliferation-associated 80,000 MW polypeptide (PPA-80). Chronic administration of these non-DNA damaging and nonmutagenic peroxisome proliferators to rats and mice results in the development of hepatocellular carcinomas. Comparative morphometric and biochemical data from rats treated with varying dose levels of ciprofibrate, a hypolipidemic drug, and di(2-ethylhexyl) phthalate, and di(2-ethylhexyl) adipate, the widely used plasticizers, indicate that the hepatocarcinogenic potency of these agents is correlatable with their ability to induce peroxisome proliferation, peroxisomal beta-oxidation and PPA-80. Available evidence strongly favors the role of peroxisome proliferation-associated oxidative stress in the induction of liver tumors by peroxisome proliferators.

Adipates↗
PubMed1986

Induction of peroxisome proliferation in hepatocytes transplanted into the anterior chamber of the eye. A model system for the evaluation of xenobiotic-induced effects.

The effect of two hypolipidemic peroxisome proliferators, ciprofibrate and di(2-ethylhexyl)phthalate (DEHP), on hepatocytes transplanted into the anterior chamber of the eye was examined. Young male F-344 rats transplanted with dissociated hepatocytes were fed either a control diet or a diet containing 0.025% ciprofibrate or 2% DEHP. After 4-5 weeks of treatment, all rats were sacrificed and the transplanted liver cells and portions of homotopic liver were processed for light and electron microscopy and for immunofluorescence microscopy. Morphometric analysis of transplanted hepatocytes showed a ninefold and fivefold increase in the volume density of peroxisomes in ciprofibrate and DEHP-fed rats, respectively. Indirect immunofluorescence studies revealed a marked induction of peroxisome-associated enzymes. From these data it is concluded that hepatic peroxisome proliferators cross the blood aqueous humor barrier and the transplanted hepatocytes in the anterior chamber of the eye retain their ability to recognize and respond to peroxisome proliferators.

Animals↗
PubMed1986

Duct-like morphogenesis of Longnecker pancreatic acinar carcinoma cells maintained in vitro on seminiferous tubular basement membranes.

We have investigated the behavior of dissociated cells of a moderately differentiated Longnecker transplantable pancreatic acinar carcinoma (Longnecker et al., Cancer Lett., 7: 197-202, 1979) maintained in vitro on acellular seminiferous tubular basement membranes of rat testis. The tumor cells, which grow as solid masses in vivo with little organization, undergo organogenesis in vitro into distinct duct-like structures with lumina in the presence of basement membrane scaffoldings. These duct-like structures were formed by flattened epithelial cells, which exhibited poorly differentiated acinar cell characteristics with few or no zymogen (secretory) granules. The cells lining the duct-like structures retained the pancreatic acinar cell specific antigen as determined by indirect immunofluorescence. DNA synthesis did not accompany duct-like organization; however, all of the cells lining these structures continued to incorporate [3H]leucine for up to 4-5 days of culture. They continued to synthesize and secrete amylase, a marker protein of pancreatic acinar cells, into the medium. These results demonstrate that neoplastic epithelial cells of Longnecker pancreatic tumor differentiate into duct-like structures when they come into contact with a basement membrane scaffolding and do not accumulate well-formed secretory granules. This is in marked contrast to the previously reported in vitro differentiation of cells derived from another transplantable rat pancreatic acinar cell carcinoma where the neoplastic cells were fully cytodifferentiated on seminiferous tubular basement membrane without forming duct-like structures but accumulated abundant well-developed zymogen granules (Watanabe et al., Cancer Res., 44: 5361-5366, 1984). Although the basal lamina promotes differentiation of cells of two different pancreatic carcinomas in vitro, we conclude that the in vitro expression of morphogenetic and cytodifferentiation patterns is dependent upon the intrinsic properties of cells of these two transplantable pancreatic tumors.

Animals↗
PubMed1985

Induction of fatty acid beta-oxidation and peroxisome proliferation in the liver of rhesus monkeys by DL-040, a new hypolipidemic agent.

Many structurally unrelated hypolipidemic agents and certain phthalate-ester plasticizers induce hepatomegaly and proliferation of peroxisomes in liver parenchymal cells of rodents, but there is relatively limited evidence regarding the ability of such compounds to induce peroxisome proliferation in the livers of nonrodent species including man. The present study was designed to determine if DL-040 (4-(((1,3-benzodioxol)-5-yl)methyl)amino-benzoic acid), a newly developed hypolipidemic agent, induces peroxisome proliferation in the liver of adult rhesus monkeys. Feeding of DL-040 (300 mg/kg body wt for 1 week; and 400 mg/kg body wt for 10 weeks) caused a significant increase in peroxisome population as determined by ultrastructural and morphometric analyses. The DL-040-induced peroxisome proliferation was accompanied by increases in the levels of catalase, carnitine acetyltransferase and the peroxisomal fatty acid beta-oxidation system. As expected, DL-040 caused a significant reduction of serum cholesterol and low density lipoprotein content. These data suggest that hepatic peroxisome proliferation is inducible in nonhuman primates at dose levels that exceed therapeutic levels.

Aminobenzoates↗
PubMed1984

Induction and origin of hepatocytes in rat pancreas.

2-[4(2,2- Dichlorocyclopropyl )phenoxy]2-methyl propionic acid (ciprofibrate), a peroxisome proliferator , induced hepatocytes in the pancreas of adult male F-344 rats when added to their diet at a dosage of 10 mg/kg body weight for 60-72 wk. These cells are morphologically indistinguishable from hepatic hepatocytes and were usually localized adjacent to islets of Langerhans with extensions into surrounding acinar tissue. A significant increase in the volume density of peroxisomes, together with immunochemically detectable amounts of two peroxisome-associated enzymes, was observed in pancreas with hepatocytes of rats maintained on ciprofibrate. Uricase-containing crystalloid nucleoids, specific for rat hepatocyte peroxisomes, were present in pancreatic hepatocytes. These structures facilitated the identification of cells with hybrid cytoplasmic features characteristic of pancreatic acinar and endocrine cells and hepatocytes. Such cells are presumed to represent a transitional state in which pancreas specific genes are being repressed while liver specific ones are simultaneously expressed. The presence of exocrine and/or endocrine secretory granules in transitional cells indicates that acinar/intermediate cells represent the precursor cell from which pancreatic hepatocytes are derived.

Animals↗
PubMed1984

Induction of hepatic peroxisome proliferation in nonrodent species, including primates.

It is well established that the administration to rodents of a variety of structurally diverse chemicals possessing hypotriglyceridemic properties results in hepatomegaly, the induction of hepatic peroxisome (microbody) proliferation, and the development of hepatocellular carcinomas. Studies have led to the hypothesis that persistent proliferation of peroxisomes serves as an endogenous initiator of neoplastic transformation in liver by increasing the intracellular production of H2O2 by the peroxisomal oxidase(s). The objective of the present study was to determine whether hepatic peroxisome proliferation can be induced in cats, chickens, pigeons, and two species of monkeys (rhesus and cynomolgus). The hypolipidemic drug ciprofibrate (2-[4-(2,2-dichloro-cylopropyl)phenoxyl]2-methylpropionic acid) induced peroxisome proliferation in the livers of cats (dose, greater than 40 mg/kg body weight for 4 weeks); chickens (dose greater than 25 mg/kg body weight for 4 weeks); pigeons (300 mg/kg body weight for 3 weeks), rhesus monkeys (50 to 200 mg/kg body weight for 7 weeks) and cynomolgus monkeys (400 mg/kg body weight for 4 weeks). In all five species examined in this study, a marked but variable increase in the activities of peroxisomal catalase, carnitine acetyltransferase, heat-labile enoyl-CoA hydratase, and the fatty acid beta-oxidation system was observed. These results suggest that peroxisome proliferation can be induced in the livers of several species and that it is a dose-dependent but not a species-specific phenomenon.

Animals↗
PubMed1983

Use of local electrostatic environments of cysteines to enhance formation of a desired species in a reversible disulfide exchange reaction.

The role of electrostatic factors has been evaluated for the reversible disulfide exchange reaction between N-acetylcysteine (A) and a peptide fragment (B) comprising residues 85-114 of Kunitz soybean trypsin inhibitor. In A, the sulfhydryl group has a negative carboxyl neighbor on the cysteine itself. In B, the only charged group within five residues of the single cysteine at position 86 is the positive N-terminal amino group on residue 85. The concentrations of the monomers A and B and of the disulfides AA, AB and BB have been determined as a function of time in kinetic experiments at pH 7, 23 degrees C and ionic strengths of 20 mM and 1 M. At both ionic strengths the sulfhydryl acid dissociation constants Ka have been determined for A and B, as well as the four rate constants for the disulfide exchange reaction. The electrostatic effects are small in magnitude but occur in expected directions. Local cysteine environments enhance formation of the mixed disulfide (AB), having a favorable configuration of adjacent unlike charges and generate decreases in the AA and BB disulfides joining regions of identical charge. These experiments represent an initial step towards use of intrinsic protein functional groups to direct formation of specific disulfides in a synthetic protein.

Acetylcysteine↗
PubMed1983

Induction of peroxisomal enzymes in livers of neonatal rats exposed to lactating mothers treated with hypolipidaemic drugs. Role of drug metabolite transfer in milk.

Lactating rats were administered by gavage 100 mg/kg body wt. twice a day of either nafenopin or Wy-14,643, two hypolipidaemic drugs with hepatic peroxisome proliferative property. Neonatal rats, after feeding from the drug-treated mothers for 8-14 days, showed sustained increases in both the proliferation of hepatic peroxisomes, as well as in levels of the peroxisome-associated enzymes catalase (3-fold), carnitine acetyltransferase (15-35-fold), peroxisomal enoyl-CoA hydratase (29-46-fold), and palmitoyl-CoA oxidation (12-14-fold). These increases in enzyme activities in suckling rats were similar to those seen in the livers of the drug-treated, lactating mothers after 14 days of treatment. After administering [3H]nafenopin or [3H]Wy-14,643 to lactating rats, significant levels of drug-derived radioactivity were observed in suckling rat gastric milk curds by 2-4 h with significant radioactivity seen in suckling rat livers by 4-6 h. T.l.c. analysis of organic extracts of milk samples from [3H]Wy-14,643 treated animals indicated no detectable levels of the parent drug, only more-polar metabolites. Wy-14,643 metabolites preparatively purified from a rat liver microsomal fraction incubation induced peroxisome proliferation when injected into a neonatal rat. Preparative high pressure liquid chromatography purification and mass spectral analysis has allowed preliminary assessment of the structures of the Wy-14,643 microsomal metabolites. It is concluded that one or more of the metabolite fractions of Wy-14,643 transferred in milk exert the biological ability to induce peroxisome proliferation and peroxisomal enzymes in neonatal livers.

Animals↗
PubMed1983

Stringent requirement for Ca2+ in the removal of Z-lines and alpha-actinin from isolated myofibrils by Ca2+-activated neutral proteinase.

Treatment of isolated myofibrils with Ca2+-activated neutral proteinase (CANP) results in specific removal of Z-line and of alpha-actinin. To investigate the ionic requirement for these processes, we measured Z-line removal by phase-contrast and interference microscopy and alpha-actinin removal by sodium dodecyl sulphate/polyacrylamide-gel electrophoretic analysis of myofibrillar proteins. The proteolytic digestion of native purified proteins was measured directly on polyacrylamide gels and by the fluorescamine technique. We found that the removal of Z-line and alpha-actinin as well as the release of proteolytic degradation products from isolated myofibrils by CANP occur only in the presence of Ca2+; Sr2+, Ba2+, Mn2+, Mg2+, Co2+ and Zn2+ are all ineffective. In contrast with this stringent requirement for Ca2+, the proteolytic activity of CANP measured with denatured casein, native and denatured haemoglobin, native actin and tropomyosin also occurs in the presence of other bivalent cations, in the following order: Ca2+ greater than Sr2+ greater than Ba2+. These data suggest that only Ca2+ can produce the conformational change in myofibrils that renders them susceptible to the action of CANP, whereas its proteolytic activity is stimulated by several bivalent ions.

Actinin↗
PubMed1983

Comparison of secretory protein and membrane composition of secretory granules isolated from normal and neoplastic pancreatic acinar cells of rats.

The diversity of cytodifferentiation in a transplantable rat pancreatic acinar carcinoma provides a biological model system for the study of regulatory molecular events that differ from those in normal acinar cells. Secretory (zymogen) granule proteins and granule membranes of neoplastic and normal pancreatic acinar cells were compared to determine the differences in gene expression between apparently well-differentiated secretory granule-containing neoplastic cells and normal cells. Nineteen proteins observed in two-dimensional polyacrylamide gels of normal secretory granule extracts were seen also in tumor granule extract profiles, with reduced but detectable amounts of lipase and four basic proteins. In addition, tumor granule extracts contained a new protein of Mr 24,000, designated p24, which was not detectable in normal extracts. Neoplastic granule membranes, while having phospholipid composition similar to that of normal membranes, lacked or contained a greatly reduced amount of a major glycoprotein of Mr 80,000 and three other proteins of Mrs 50,000, 37,000, and 36,000. The nature of p24 protein in secretory granule extracts and the significance of the reduction or absence of Mr 80,000 membrane glycoprotein in this tumor remain to be elucidated.

Animals↗
PubMed1983

Evaluation of selected hypolipidemic agents for the induction of peroxisomal enzymes and peroxisome proliferation in the rat liver.

There is a considerable interest in developing potent and safe hypolipidemic drugs for the prevention and management of coronary heart disease in man. In rodents, many of these hypolipidemic compounds induce hepatomegaly, proliferation of peroxisomes and a polypeptide with an approximate mol. wt. of 80000 in liver cells. In the present study, we have examined 10 hypolipidemic compounds for the induction of peroxisome proliferation associated 80000 mol. wt. polypeptide (polypeptide PPA-80), peroxisomal enzymes and peroxisome proliferation in rat liver, in view of the emerging evidence that hepatic peroxisome proliferators as a class are carcinogenic in rats and mice. All ten compounds, fenofibrate (isopropyl-[4-(p-chlorobenzoyl)2-phenoxy-2-methyl] propionate; LS 2265 (taurine derivative of fenofibrate); bezafibrate (2-(4-(2-[4-chlorobenzamido)ethyl] phenoxy)-methyl propionic acid; gemfibrozil (5-2[2,5-dimethylphenoxy]2-2-dimethylpentanoic acid); methyl clofenapate (methyl-2-[4-(p-chlorophenyl)phenoxy]-2-methyl propionate); DG 5685 (5-[4-phenoxybenzyl]trans-2-(3-pyridyl)1,3-dioxane); DH 6463 (5-[4-phenoxybenzyl] trans-2-(3-pyrimidinyl)-1,3-dioxane); tiadenol(bis[hydroxyethylthio]-7, 10-decane); ciprofibrate (2,-[4-(2,2-dichlorocyclopropyl)-phenoxy]2-methyl propionic acid) and RMI-14,514 ( [5-tetradecycloxy]-2-furancarboxylic acid), produced a marked but variable increase in the activities of peroxisomal enzymes catalase, carnitine acetyltransferase, heat-labile enoyl-CoA hydratase and the fatty acid beta-oxidation system and in the amount of polypeptide PPA-80 as demonstrated by SDS-polyacrylamide gel electrophoresis. The peptide map patterns of polypeptide PPA-80 in liver induced by these compounds were strikingly similar. The ultrastructural studies demonstrate that fenofibrate, ciprofibrate, LS 2265, DG 5685 and DH 6463 can induce proliferation of peroxisomes in liver cells of rats, and further confirm the previous reports of hepatic peroxisome proliferative activity of methyl clofenapate, tiadenol, bezafibrate, gemfibrozil and RMI-14514, as shown morphologically. Whether these structurally unrelated chemicals or their metabolite(s) directly activate the peroxisome specific genes to induce this multi-enzyme system or they exert their action on peroxisomes indirectly by causing fatty acid overload in hepatocytes remains to be elucidated. These chemicals offer a simple and reproducible means of stimulating peroxisomal enzymes in liver and should serve as useful tools, for evaluating the implications of hepatic peroxisome proliferation and in elucidating the mechanism of peroxisome proliferator-induced carcinogenesis.

Animals↗
PubMed1983

Immunocytochemical localization of fatty acid metabolizing heat-stable and heat-labile enoyl-coenzyme A (CoA) hydratases in liver and renal cortex.

Two enzymes, the heat-stable and the heat-labile enoyl-coenzyme A (CoA) hydratases, involved in the metabolism of fatty acids were localized in liver and renal cortex using specific antibodies, immunofluorescence, and the protein A-gold immunocytochemical technique. The qualitative and quantitative results have demonstrated that the heat-stable enoyl-CoA hydratase is a mitochondrial membrane-associated protein of hepatocytes and of epithelial cells in proximal and distal renal tubules. The hepatic sinusoidal cells, as well as the endothelial and epithelial cells of the glomeruli, fail to demonstrate any specific labeling. The heat-labile enoyl-CoA hydratase, on the other hand, was detected in the peroxisomal matrix of hepatocyte and proximal tubule epithelial cells. Its distribution was identical to that of catalase. The significance of the differential distribution of peroxisomal and mitochondrial enoyl-CoA hydratases is discussed in relation to their function.

Animals↗
PubMed1982

Response of chemically induced hepatocytelike cells in hamster pancreas to methyl clofenapate, a peroxisome proliferator.

Administration of N-nitrosobis (2-oxopropyl)amine during peak DNA synthesis of regenerating pancreas in hamsters has been shown to induce hepatocytelike cells in pancreas. We now present evidence to demonstrate that such cells respond to methyl clofenapate, a peroxisome proliferator. The response includes a marked proliferation of peroxisomes and enhanced activity of peroxisomal enzymes enoyl-CoA hydratase (8.5- to 13-fold), [1-14C]-palmitoyl-CoA oxidation (2.8- to 3.9-fold), catalase (1.6 to 3.4-fold), and carnitine acetyltransferase (greater than 2,000-fold). Cytochemical localization of catalase by the alkaline 3,3'-diaminobenzidine procedure and immunofluorescence localization of heat-labile enoyl-CoA hydratase showed that these peroxisome-associated enzymes are localized strictly in pancreatic hepatocytelike cells, while adjacent acinar, duct, and islet cells appeared consistently negative. Morphometric analyses of hepatocytelike cells showed a significant increase in the numerical density and an eightfold increase in the volume density of peroxisomes in methyl clofenapate treated animals. These results demonstrate that the hepatocytelike cells are responsible for the observed peroxisomal enzyme activity in pancreas of hamsters and suggest that the derepressed peroxisome specific genes in these cells respond to a peroxisome proliferator as do parenchymal cells in hamster liver.

Butyrates↗