Inhibition of H2O2 generation in rat liver mitochondria by radical quenchers and phenolic compounds

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.

Identification of amino acid residues essential for enzyme activity of sheep liver 5,10-methylenetetrahydrofolate reductase

Sheep liver 5,10-methylenetetrahydrofolate reductase was subjected to specific chemical modification with phenylglyoxal, diethyl pyrocarbonate and N-bromosuccinimide. The second-order rate constants for inactivation were calculated to be 54 M-1 X min-1, 103 M-1 X min-1 and 154 M-1 X min-1 respectively. This inactivation could be prevented by incubation with substrates or products, suggesting that the residues modified, namely arginine, histidine and tryptophan, are essential for enzyme activity.

A study on bullock carts. Part 2. Experimental study of forces in a bullock cart

A strain gauge load cell with separate bridges for measurement of the pull and the bending moment in the plane containing the net neck load and pull was developed and fixed in the longitudinal member of an experimental cart. A cart fitted first with pneumatic wheels and then with steel-rimmed wooden wheels was tested on three terrains—tar road, mud road and grassy terrain. Pull vs time and moment vs time records were obtained in each test and analysed. It is found that the bullocks pull the cart rather discontinuously at the low velocities at which these carts normally operate. On the tar road and the grassy terrain, the mean static coefficient of friction is significantly higher for the cart with steelrimmed wooden wheels. The dynamic frictional resistance of the terrain for the cart with steel-rimmed wooden wheels is lower than for the cart with pneumatic wheels so long as the wheels do not dig or sink into the terrain. The fluctuation in the neck load is lower in the cart fitted with pneumatic wheels. Also, the ground-induced low-amplitude high-frequency vibratory load content in the neck load is lower in the cart with pneumatic wheels.

Stimulation of liver tryptophan pyrrolase during heat exposure

Exposure of rats to heat (39 +/- 1 degree C) stimulated liver tryptophan pyrrolase 2-fold between 3 and 48 h. Plasma corticosterone increased 2-fold after 1 h of heat exposure and decreased to a low value of 50% by 16 h. The effect of heat exposure on the enzyme was obtained in adrenalectomized animals. Stimulation by cortisol and tryptophan of the enzyme was also obtained in heat exposure, and the effects seemed to be additive. The concentration of tryptophan in the liver remained unchanged, and that in the plasma decreased to about 50% at 8 h exposure to heat and reverted to normal by 46 h. Simultaneous administration of noradrenaline to heat-exposed rats had no effect, whereas that of thyroxine partly prevented the stimulation of the enzyme activity. Hypothyroid conditions obtained by thyroidectomy or treatment with propylthiouracil significantly stimulated the enzyme activity. Cycloheximide treatment of heat-exposed rats did not prevent the stimulation of the enzyme activity. The results indicate that the effect of heat exposure on liver tryptophan pyrrolase is obtained, due to the accompanying hypothyroid condition, by increasing the activity of the existing protein by a mechanism possibly different from those known at present.

Heat exposure and hypothyroid conditions decrease hydrogen peroxide generation in liver mitochondria

Exposure of rats to heat (39 +/- 1 degree C) decreased H2O2 generation in mitochondria of the liver, but not of the kidney or the heart. The effect was obtained with three substrates, succinate, glycerol 1-phosphate and choline, with a decrease to 50% in the first 2-3 days of exposure, and a further decrease on longer exposure. The dehydrogenase activity with only glycerol 1-phosphate decreased, which is indicative of the hypothyroid condition, whereas choline dehydrogenase activity remained unchanged and that of succinate dehydrogenase decreased on long exposure. The serum concentration of thyroxine decreased in heat-exposed rats. Thyroxine treatment of rats increased H2O2 generation. Hypothyroid conditions obtained by treatment with propylthiouracil or thyroidectomy caused a decrease in H2O2 generation and changes in dehydrogenase activities similar to those with heat exposure. Treatment of heat-exposed or thyroidectomized rats with thyroxine stimulated H2O2 generation by a mechanism apparently involving fresh protein synthesis. The results indicate that H2O2 generation in mitochondria of heat-exposed animals is determined by thyroid status.

Magnetic superconductors: Model theories and experimental properties of rare-earth compounds

Superconducting and magnetically long-range ordered states were believed to be mutually exclusive phenomena. The discovery of rare-earth compounds in recent years, which exhibit both superconductivity and magnetic ordering (ferromagnetic, antiferromagnetic or sinusoidal), has led to considerable theoretical and experimental work on such systems. In the present article, we give a review of various theoretical models and important experimental results. In the theoretical sections, we start with the Abrikosov-Gorkov pair breaking theory for dilute alloys and discuss its improvement in the work of Müller-Hartmann and Zittartz. Then, in the context of magnetic superconductors, various microscopic theories that have been advanced are presented. These predict re-entrant behaviour in some systems (ferromagnetic superconductors) and coexistence regions in others (particularly antiferromagnetic superconductors). Following this, phenomenological generalized Ginzburg-Landau theories for two kinds of orders (superconducting and magnetic) are presented. A section dealing with renormalization group analysis of phase diagrams in magnetic superconductors is given. In experimental sections, the properties of each rare-earth compounds (ternary as well as some tetranery) are reviewed. These involve susceptibility, heat capacity, resistivity, upper critical field, neutron scattering and magnetic resonance measurements. The anomalous behaviour of the upper critical field of antiferromagnetic superconductors near the Néel temperature is discussed both in theory sections and experimental section for various systems.

Heat-Capacity Measurements - Progress In Experimental-Techniques

The heat capacity of a substance is related to the structure and constitution of the material and its measurement is a standard technique of physical investigation. In this review, the classical methods are first analyzed briefly and their recent extensions are summarized. The merits and demerits of these methods are pointed out. The newer techniques such as the a.c. method, the relaxation method, the pulse methods, the laser flash calorimetry and other methods developed to extend the heat capacity measurements to newer classes of materials and to extreme conditions of sample geometry, pressure and temperature are comprehensively reviewed. Examples of recent work and details of the experimental systems are provided for each method. The introduction of automation in control systems for the monitoring of the experiments and for data processing is also discussed. Two hundred and eight references and 18 figures are used to illustrate the various techniques.

Identification of active-site residues of sheep liver serine hydroxymethyltransferase

Chemical modification of amino acid residues with phenylglyoxal, N-ethylmaleimide and diethyl pyrocarbonate indicated that at least one residue each of arginine, cysteine and histidine were essential for the activity of sheep liver serine hydroxymethyltransferase. The second-order rate constants for inactivation were calculated to be 0.016 mM-1 X min-1 for phenylglyoxal, 0.52 mM-1 X min-1 for N-ethylmaleimide and 0.06 mM-1 X min-1 for diethyl pyrocarbonate. Different rates of modification of these residues in the presence and in the absence of substrates and the cofactor pyridoxal 5'-phosphate as well as the spectra of the modified protein suggested that these residues might occur at the active site of the enzyme.

Functional changes in rat liver mitochondria on administration of 2-methyl-4-dimethylaminoazobenzene

Administration of 2-methyl-4-dimethylaminobenzene in the diet (0.1%, w/w) for 85-90 days doubled the content of mitochondria in the livers of rats. The azodye was covalently bound to liver proteins, and about 15% of the amount found in liver was associated with the mitochondrial fraction. Mitochondria isolated from the livers of azodye-fed animals showed drastically lowered ability to oxidize NAD+-linked substrates. The inhibited electron-transfer step was the reduction of ubiquinone. The organelles showed a large increase in succinate oxidase activity. The activity of cytochrome oxidase and the content of cytochrome aa3 were substantially higher in these organelles. Azodye-fed animals showed depressed serum cholesterol concentrations. The content of ubiquinone in liver also registered a small increase.

A comparative study of tbe regulation of cytochrome P-450 and glutathione transferase gene expression in rat liver

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.

Targeted delivery of hepatitis C virus-specific short hairpin RNA in mouse liver using Sendai virosomes

Internal ribosome entry site (IRES)-mediated translation of input viral RNA is the initial required step for the replication of the positive-stranded genome of hepatitis C virus (HCV). We have shown previously the importance of the GCAC sequence near the initiator AUG within the stem and loop IV (SLIV) region in mediating ribosome assembly on HCV RNA. Here, we demonstrate selective inhibition of HCV-IRES-mediated translation using short hairpin (sh)RNA targeting the same site within the HCV IRES. sh-SLIV showed significant inhibition of viral RNA replication in a human hepatocellular carcinoma (Huh7) cell line harbouring a HCV monocistronic replicon. More importantly, co-transfection of infectious HCV-H77s RNA and sh-SLIV in Huh7.5 cells successfully demonstrated a significant decrease in viral RNA in HCV cell culture. Additionally, we report, for the first time, the targeted delivery of sh-SLIV RNA into mice liver using Sendai virosomes and demonstrate selective inhibition of HCV-IRES-mediated translation. Results provide the proof of concept that Sendai virosomes could be used for the efficient delivery of shRNAs into liver tissue to block HCV replication.

Hormonal induction of riboflavin carrier protein in the chicken oviduct and liver: a comparison of kinetics and modulation

Estrogen (E) induction of riboflavin carrier protein (RCP) in the chicken oviduct and liver was investigated to compare and contrast the kinetics, hormonal specificity and modulation of its elaboration in the 2 steroid-responsive tissues. During primary stimulation, continued daily E administration to immature female chicks elicited, after an initial lag, rapid growth and RCP content of the oviduct; neither progesterone (P) nor testosterone (T) could substitute for E in this respect. Furthermore, P given along with E curtailed tissue growth and its RCP content, whereas E + T had a synergistic effect on tissue growth only. During secondary stimulation, E administration steeply enhanced both tissue weight and RCP content without any lag. Interestingly, P (but not T) could substitute for E in augmenting magnum RCP concentration to a comparable extent while a concomitant effect on tissue growth was less marked. In contrast, hepatic induction of RCP was absolutely E-specific during both primary and secondary stimulations. Secondary stimulation with either E or P of E-primed birds enhanced the rates of RCP synthesis in the oviduct relative to that of total protein, whereas in the liver only E was effective in this regard. The absolute rate of E-induced RCP synthesis in both the steroid-stimulated tissues was significantly higher than that of general protein elaboration.

Induction of carnitine acetyltransferase by clofibrate in rat liver

Administration of the anti-hypercholesterolaemic drug clofibrate to the rat increases the activity of carnitine acetyltransferase (acetyl-CoA-carnitine -acetyltransferase, EC 2.3.1.7) in liver and kidney. The drug-mediated increase in enzyme activity in hepatic mitochondria shows a time lag during which the activity increases in the microsomal and peroxisomal fractions. The enzyme induced in the particulate fractions is identical with one normally present in mitochondria. The increase in enzyme activity is prevented by inhibitors of RNA and general protein synthesis. Mitochondrial protein-synthetic machinery does not appear to be involved in the process. Immunoprecipitation shows increased concentration of the enzyme protein in hepatic mitochondria isolated from drug-treated animals. In these animals, the rate of synthesis of the enzyme is increased 7-fold.

Inhibition of monkey liver serine hydroxymethyltransferase by Cibacron Blue 3G-A

Cibacron Blue 3G-A inhibited monkey liver serine hydroxymethyltransferase competitively with respect to tetrahydrofolate and non-competitively with respect to L-serine. NADH, a positive heterotropic effector, failed to protect the enzymes against inhibition by the dye and was unable to desorb the enzyme from Blue Sepharose CL-6B gel matrix. The binding of the dye to the free enzyme was confirmed by changes in the dye absorption spectrum. The results indicate that the dye probably binds at the tetrahydrofolate-binding domain of the enzyme, rather than at the 'dinucleotide fold'.

Metabolism of geraniol and linalool in the rat and effects on liver and lung microsomal enzymes

1. Metabolites isolated from the urine of rats after oral administration of geraniol (I) were: geranic acid (II), 3-hydroxy-citronellic acid (III), 8-hydroxy-geraniol (IV), 8-carboxy-geraniol (V) and Hildebrandt acid (VI). 2. Metabolites isolated from urine of rats after oral administration of linalool (VII) were 8-hydroxy-linalool (VIII) and 8-carboxy-linalool (IX). 3. After three days of feeding rats with either geraniol or linalool, liver-microsomal cytochrome P-450 was increased. Both NADH- and NADPH-cytochrome c reductase activities were not significantly changed during the six days of treatment. 4. Oral administration of these two terpenoids did not affect any of the lung-microsomal parameters measured.