209 resultados para isolated perfused rat kidney
Resumo:
Thioacetamide, a hepatocarcinogen and an inhibitor of heme synthesis, blocks the phenobarbitone- mediated increase in the transcription of cytochrome P-450b+e messenger RNA in rat liver. This property is also shared by CoCl, and 3-amino-l,2,4-triazole, two other inhibitors of heme synthesis. Thus, it appears feasible that heme may serve as a positive regulator of cytochrome P-450b+e gene transcription. Thioacetamide enhances albumin messenger RNA concentration, whereas phenobarbitone decreases the same. However, these changes in albumin messenger RNA concentration are not accompanied by corresponding changes in the transcription rates. Therefore, drug-mediated changes in albumin messenger RNA concentration are due to posttranscriptional regulation. The property of thioacetamide to enhance the albumin messenger RNA concentration is not shared by CoC1, and 3-amino- 1,2,4-triazole. Therefore, heme does not appear to be a regulatory molecule mediating the reciprocal changes brought about in the concentrations of cytochrome P-450b+e and albumin messenger RNAs.
Resumo:
The biosynthesis of the cytoplasmic subunits of cytochrome oxidase from rat liver has been studied in vitro by translating liver poly (A)-containing RNA in the wheat germ cell-free system and immunoprecipitating the products with anti-cytochrome oxidase antibody. Analysis of the labelled immunoprecipitate on SDS-gels does not reveal the presence of a polyprotein precursor. On the other hand discrete products which are either slightly bigger or closely similar to the mature subunits present in purified cytochrome oxidase have been detected.
Resumo:
The antitumour antibiotic, adriamycin, inhibited oxidative phosphorylation in freshly prepared mitochondria from the heart, liver and kidney of the rat. It abolished respiratory control and stimulated ATPase activity. Sccinate oxidation by heart mitochondria was extremely sensitive to the drug when hexokinase was present in the reaction medium. The sensitive site has been identified to lie in the region between the succinate dehydrogenase flavoprotein and ubiquinone of the respiratory chain.
Resumo:
The enzyme carnitine acetyltransferase (acetyl-CoA:carnitine O-acetyltransferase, EC 2.3.1.7) has been purified to homogeneity from hepatic mitochondria of clofibrate-fed rats. It is a protein of molecular weight 56 000 composed of two non-identical subunits of molecular weight 34 000 and 25 000. The enzyme is inhibited by palmityl-CoA as well as acetyl carnitine. The inhibition by fatty acyl-CoA is competitive with respect to both the substrates, carnitine and acetyl-CoA. The inhibition by acetylcarnitine is reversed by carnitine but not by acetyl-CoA.
Resumo:
A cholesterol-esterifying enzyme which incorporates exogenous fatty acids into cholesterol esters in the presence of ATP and coenzyme A was demonstrated in 15-day-old rat brain. This enzyme was maximally active at pH 7.4 and distinct from the cholesterol-esterifying enzyme reported earlier (Eto and Suzuki, 1971), which has a pH optimum at 5.2 and does not require cofactors. Properties of the two enzymes have been compared. Both the enzymes showed negligible esterification with acetate and were maximally active with oleic acid. The pH 5.2 enzyme esterified desmosterol, lanosterol and cholesterol at about the same rate, while the pH 7.4 enzyme was only 50% as active ith lanosterol as it was with cholesterol and desmosterol. Phosphatidyl serine stimulated the pH 5.2 enzyme but not the pH 7.4 enzyme. Phosphatidyl choline and sodium taurocholate showed no effect on either of the enzymes. Both the enzymes were associated with particulate fractions, but the pH 7.4 enzyme was localized more in the microsomes. Purified myelin showed 2.6-fold and 1.5-fold higher specific activities of pH 5.2 and 7.4 enzymes respectively, when compared with homogenate. About 7-10% of total activity of both the enzymes was associated with purified myelin. Brain stem and spinal cord showed higher specific activity of pH 5.2 enzyme than cerebral cortex and cerebellum, while pH 7.4 enzyme specific activity was higher in cerebellum and brain stem than in cerebral cortex and spinal cord. Microsomal pH 7.4 activity showed progressive increase prior to the active period of myelination, reaching a maximum on the 15th day after birth and declined to 20% of the peak activity by 30 days. In contrast, pH 5.2 enzyme reached maximum activity about the 6th day after birth and remained at this level well into adulthood. In 15-day-old rat brain, pH 7.4 enzyme had five to six times higher specific activity than pH 5.2 enzyme, while in adults the activities were equal. The pH 7.4 enzyme showed a threefold higher specific activity than pH 5.2 enzyme in myelin from 15-day-old rats, but in adults the reverse was true. Key Words: Cholesterol esterifying enzymes-Developing rat brain-Myelination. Jagannatha H. M. and Sastry P. S. Cholesterol-esterifying enzymes in developing rat brain. J. Neurochem. 36, 1352- 1360 (1981).
Resumo:
The nucleic acid binding properties of the testis protein, TP, were studied with the help of physical techniques, namely, fluorescence quenching, UV difference absorption spectroscopy, and thermal melting. Results of quenching of tyrosine fluorescence of TP upon its binding to double-stranded and denatured rat liver nucleosome core DNA and poly(rA) suggest that the tyrosine residues of TP interact/intercalate with the bases of these nucleic acids. From the fluorescence quenching data, obtained at 50 mM NaCl concentration, the apparent association constants for binding of TP to native and denatured DNA and poly(rA) were calculated to be 4.4 X 10(3) M-1, 2.86 X 10(4) M-1, and 8.5 X 10(4) M-1, respectively. UV difference absorption spectra upon TP binding to poly(rA) and rat liver core DNA showed a TP-induced hyperchromicity at 260 nm which is suggestive of local melting of poly(rA) and DNA. The results from thermal melting studies of binding of TP to calf thymus DNA at 1 mM NaCl as well as 50 mM NaCl showed that although at 1 mM NaCl TP brings about a slight stabilization of the DNA against thermal melting, a destabilization of the DNA was observed at 50 mM NaCl. From these results it is concluded that TP, having a higher affinity for single-stranded nucleic acids, destabilizes double- stranded DNA, thus behaving like a DNA-melting protein.
Resumo:
Generation of H2O2 by rat liver mitochondria with choline, glycerol 1-phosphate and proline as substrates has been shown by using high-concentration phosphate buffer. Rates obtained under these conditions were higher and more consistent as compared with the earlier reports with high-concentration mannitol/sucrose/Tris buffer. Sulphate ions could replace phosphate indicating a requirement for a high concentration of oxygen-containing anions. H2O2 generation was dependent on the presence of native mitochondria and substrate. Maximal rates with various substrates were found to be the same as with succinate. Values of Km and Vmax for H2O2 generation were considerably less than those obtained for respective dehydrogenase activities, measured by dye reduction. Scavengers of O2-. and OH. inhibited generation of H2O2. ATP, ADP, thyronine derivatives and a number of phenolic compounds also showed very potent inhibitory effects of H2O2 generation, whereas phenyl compound had no effect. Phenolic compounds did not have any effect on mitochondrial superoxide dismutase and choline dehydrogenase activities as well as on O2-. generation by the xanthine-xanthine oxidase system. Inhibition by phenolic compounds may have potential for regulation of the intracellular concentration of H2O2, that is not considered to have a "second messenger' function.
Resumo:
The relative concentrations of the major histones, namely, H1, TH1, H2A, H2B, H3 and H4 are not significantly changed in the testes of the vitamin A-deficient rats, as compared to those in the normal controls. But the testis-specific protein, TP, which is synthesized at the mid-spermatid stage, is markedly reduced in the testes of the deficient rats. On supplementation of the deficient rats with retinyl acetate for 28 days, there was a 50% recovery in the relative concentration of the TP with respect to the total amounts of H1 and TH1.
Resumo:
A cDNA clone for cytochrome P-450e, a phenobarbitone-inducible species in rat liver, has been isolated and characterized. With the use of this cloned DNA, an attempt has been initiated to elucidate the factors regulating the cytochrome P-450 gene expression. Inhibitors of heme synthesis such as cobalt chloride and 3-amino-1,2,4-triazole block the induction of cytochrome P-450e by phenobarbitone at the level of transcription. This is evident from the decrease in the rate of synthesis of cytochrome P-450e, a decrease in the levels of specific translatable messenger RNA, a decrease in the specific cytoplasmic and nuclear messenger RNA contents, and nuclear transcription of cytochrome P-450e gene, as revealed by hybridization to the cloned probe, under these conditions. It is proposed that heme is a general regulator of cytochrome P-450 gene expression at the level of transcription, whereas the drug or its metabolite would impart the specificity needed for the induction of a particular species.
Resumo:
The ability of different LH-like hormones, such as hCG, PMSG/equine (e) CG, ovine (o) LH, eLH, and rat (r) LH, to bind to and stimulate steroidogenesis in two types of rat gonadal cells was studied under the same experimental conditions. In both Leydig and granulosa cells, the maximal steroidogenic responses elicited by optimal doses of different LHs present during a 2-h incubation were comparable. However, if the cells were exposed to the different LHs for a brief period and then subjected to interference with hormone action by removing the unbound hormone from the medium by washing or adding specific antisera, differences were observed in the amount of steroid produced during subsequent incubation in hormone-free medium. Thus, in the case of hCG, either of these procedures carried out at 15 or 30 min of incubation had little inhibitory effect on the amount of steroid produced at 2 h, the latter being similar to that produced by cells incubated in the continued presence of hCG for 2 h. With eCG and rLH, the effect was dramatic, in that there was a total inhibition of subsequent steroidogenic response. In cells exposed to eLH and oLH, inhibition of subsequent steroidogenesis due to either removal of the free-hormone or addition of specific antisera at 15 or 30 min was only partial. Although all of the antisera used were equally effective in inhibiting the steroidogenic response to respective gonadotropins when added along with hormones at the beginning of incubation, differences were observed in the degree of inhibition of this response when the same antisera were added at later times of incubation. Thus, when antisera were added 60 min after the hormone, the inhibition of steroidogenesis was total (100%) for eCG, partial (10–40%) for eLH and oLH, and totally lacking in cells treated with hCG. From this, it appears that hCG bound to the receptor probably becomes unavailable for binding to its antibody with time, while in the case of eCG and other LHs used, the antibody can still inhibit the biological activity of the hormone. Studies with 125I-labeled hormones further supported the conclusion that hCG differs from all other LHs in being most tightly bound and, hence, least dissociable, while eCG and rLH dissociate most readily; oLH and eLH can be placed in between these hormones in the extent of their dissociability. (Endocrinology 116: 597–603,1985)
Resumo:
A cDNA clone for the Ya subunit of glutathione transferase from rat liver was constructed in E.coli. The clone hybridized to Ya and Yc subunit messenger RNAs. On the basis of experiments involving cell-free translation and hybridization to the cloned probe, it was shown that prototype inducers of cytochrome P-450 such as phenobarbitone and 3-methylcholanthrene as well as inhibitors such as CoCl2 and 3-amino-l,2,4-triazole enhanced the glutathione transferase (Ya+Yc) messenger RNA contents in rat liver. A comparative study with the induction of cytochrome P-450 (b+e) by phenobarbitone revealed that the drug manifested a striking increase in the nuclear pre-messenger RNAs for the cytochrome at 12 hr, but did not significantly affect the same in the case of glutathione transferase (Ya+Yc). 3-Amino-l, 2,4-tnazole and CoCl- blocked the phenobarbitone mediated increase in cytochrome P-450 (b+e) nuclear pre-messenger RNAs. These compounds did not significantly affect the glutathione transferase (Ya+Yc) nuclear pre-messenger RNA levels. The polysomal, poly (A)- containing messenger RNAs for cytochrome P-450 (b+e) increased by 12–15 fold after phenobarbitone administration, reached a maximum around 16hr and then decreased sharply. In comparison, the increase in the case of glutathione transferase (Ya+Yc) mesenger RNAs was sluggish and steady and a value of 3–4 fold was reached around 24 hr. Run-off transcription rates for cytochrome P-450 (b+e) increased by nearly 15 fold in 4 hr after phenobarbitone administration, whereas the increase for glutathione transferase (Ya+Yc) was only 2.0 fold. At 12 hr after the drug administration, the glutathione transferase (Ya+Yc) transcription rates were near normal. Administration of 3-amino-l,2,4-triazole and CoCl2 blocked the phenobarbitone-mediated increase in the transcription of cytochrome P-450 (b+e) messenger RNAs. These compounds at best had only marginal effects on the transcription of glutathione transferase (Ya+Yc) messenger RNAs. The half-life of cytochrome P-450 (b+e) messenger RNA was estimated to be 3–4 hr, whereas that for glutathione transferase (Ya+Yc) was found to be 8-9 hr. Administration of phenobarbitone enhanced the half-life of glutathione transferase (Ya+Yc) messenger RNA by nearly two fold. It is suggested that while transcription activation may play a primary role in the induction of cytochrome P-450 (b+e), the induction of glutathione transferase (Ya+Yc) may essentially involve stabilization of the messenger RNAs.
Resumo:
Nucleolin is a major nucleolar phosphoprotein involved in various steps of ribosome biogenesis in eukaryotic cells. As nucleolin plays a significant role in ribosomal RNA transcription we were interested in examining in detail the expression of nucleolin across different stages of spermatogenesis and correlate with the transcription status of ribosomal DNA in germ cells.
Resumo:
A model has been developed to predict heat transfer rates and sizes of bubbles generated during nucleate pool boiling. This model assumes conduction and a natural convective heat transfer mechanism through the liquid layer under the bubble and transient conduction from the bulk liquid. The temperature of the bulk liquid in the vicinity of the bubble is obtained by assuming a turbulent natural convection process from the hot plate to the liquid bulk. The shape of the bubble is obtained by equilibrium analysis. The bubble departure condition is predicted by a force balance equation. Good agreement has been found between the bubble radii predicted by the present theory and the ones obtained experimentally.
Resumo:
Earlier workers have observed that in the leydig cell desensitization brings results in addition to down regulation of receptors, in leisons in the steroidogenlc pathway. In the present study immature rats having heavily leutinized ovaries were given 50 iu hCG and the desenasitized CL removed 48h later were used. At that time no change in the 5 3MSD activity and CAMP binding activity(a measure of CAMP dependent protein kinase) was observed.Followlng desensitization however,l)a significent increase in phosphodiestrase activity,ii)a 50% reduction in total mitochondrial cholesterol level, iii)a significant reduction in its ability to utilize cholesterol or hydrolyse its ester and iv)a significant lowering(by 66%)in cholesterol side chain clean age activity(by measuring pregnanalone formed) was observed. Pregnanalone production was restored to normalcy if exogenous cholesterol was added to the mitohondrial preparation. The results suggest that luteal desensitization is due in addition to down regulation of LH receptors, to a marked reduction in available cholesterol pool in the mitochondrial compartment. The increase in phosphodiestrase activity, though probably a secondary effect,might effectively contribute to the overall reduction in the steroid out-put by increasing the catabolism of CAMP.(Aided by grants from ICMR,New Delhi and WHO, Geneva).
Resumo:
Cell-free preparations of rat sciatic nerve were found to catalyze the reduction of fatty acid to alcohol in the presence of NADPH as reducing cofactor. The reductase was membrane-bound and associated primarily with the microsomal fraction. When fatty acid was the substrate, ATP, coenzyme A (CoA), and Mg2+ were required, indicating the formation of acyl CoA prior to reduction. When acyl CoA was used as substrate, the presence of albumin was required to inhibit acyl CoA hydro-lase activity. Fatty acid reductase activity was highest with palmitic and stearic acids, and somewhat lower with lauric and myristic acids. It was inhibited by sulfhydryl reagents, indicating the participation of thiol groups in the reduction. Only traces of long-chain aldehyde could be detected or trapped as semicarbazone. Fatty acid reductase activity in rat sciatic nerve was highest between the second and tenth days after birth and decreased substantially thereafter. Microsomal preparations of sciatic nerve from 10-day-old rats exhibited about four times higher fatty acid reductase activity than brain or spinal cord microsomes from the same animals. Wallerian degeneration and regeneration of adult rat sciatic nerve resulted in enhanced fatty acid reductase activity, which reached a maximum at about 12 days after crush injury.