Axial ligation and polypeptide matrix effects on the reduction potential of heme proteins probed on their cyanide adducts.

The enthalpic and entropic changes accompanying the reduction reaction of the six-coordinate cyanide adducts of cytochrome c, microperoxidase-11 and a few plant peroxidases were measured electrochemically. Once the compensating changes in reduction enthalpy and entropy due to solvent reorganization effects are factorized out, it is found that cyanide binding stabilizes enthalpically the ferriheme following the order: cyochrome c > peroxidase > microperoxidase-11. The effect is inversely correlated to the solvent accessibility of the heme. Comparison of the reduction thermodynamics for the cyanide adducts of cytochrome c and plant peroxidases with those for microperoxidase-11 and myoglobin, respectively, yielded an estimate of the consequences of protein encapsulation and of the anionic character of the proximal histidine on the reduction potential of the heme-cyanide group. Insertion of the heme-CN group into the folded peptide chain of cyt c induces an enthalpy-based decrease in E degrees ' of approximately 100 mV, consistent with the lower net charge of the oxidized as compared to the reduced iron center, whereas a full imidazolate character of the proximal histidine stabilizes enthalpically the ferriheme by approximately 400 mV. The latter value should be best considered as an upper limit since it also includes some solvation effects arising from the nature of the protein systems being compared.

The effects of the ergosteroid 7-oxo-dehydroepiandrosterone on mitochondrial membrane potential: possible relationship to thermogenesis.

Administered 3 beta-hydroxyandrost-5-ene-7,17-dione (7-oxo-DHEA) is more effective than 3 beta-hydroxyandrost-5-en-7-one (DHEA) as an inducer of liver mitochondrial sn-glycerol-3-phosphate dehydrogenase and cytosolic malic enzyme in rats. Like DHEA, the 7-oxo metabolite enhances liver catalase, fatty acylCoA oxidase, cytosolic sn-glycerol-3-phosphate dehydrogenase, mitochondrial substrate oxidation rate, and the reconstructed sn-glycerol 3-phosphate shuttle. The mitochondrial adenine nucleotide carrier is diminished by thyroidectomy and is restored to normal activity by administering 7-oxo-DHEA. The relationship between respiratory rate and proton motive force across the mitochondrial membrane was measured in the nonphosphorylating state. When treated with increasing concentrations of respiratory inhibitors liver mitochondria from rats treated with 7-oxo-DHEA or thyroid hormones show a more rapid decline of membrane potential than do normal liver mitochondria. Thus 7-oxo-DHEA induces an increased proton leak or slip as has been reported for the thyroid hormone by M.D. Brand [(1990) Biochem. Biophys. Acta 1018, 128-133]. This process may contribute to the enhanced thermogenesis caused by ergosteroids as well as by thyroid hormones.

Effect of cardiolipin on functional properties of isolated rat liver mitochondria.

The specific involvement of cardiolipin in modulating and/or controlling the activity of a number of mitochondrial carriers, enzymes and receptors is well documented; however, comparatively less understood is its role for the integrated functions of intact mitochondria. The aim of the present research was to get a better insight into this problem by investigating the effect of in vitro addition of cardiolipin on the properties of isolated liver mitochondria. The results obtained show that cardiolipin induces extensive structural and functional perturbations at the level of the inner mitochondrial membrane. In fact, addition of cardiolipin to intact mitochondria causes a significant increase of proton leak associated with a parallel increase of respiratory rate in State 4. Concomitantly, a slight uncoupling of phosphorylation associated with a moderate increase in ATPase activity is observed. Furthermore, the pore-mediated membrane permeability to calcium is drastically modified, an effect that can be reversed by addition of cyclosporin.

Effects of dehydroepiandrosterone and carnitine treatment on rat liver.

It is well established that DHEA treatment is associated in the rat to an increase in fatty acids metabolism. This condition would require levels of L-carnitine much higher than those physiologically present in the liver. The possibility thus exist that during DHEA treatment the concentration of L-carnitine may become a limiting factor for fatty acids oxidation and therefore responsible of some of the effects observed after administration of the hormone. The present experiments were designed to test this hypothesis. The results show that the increase in the levels of peroxisomal enzymes induced in hepatocytes by DHEA, is greatly reduced by parallel administration of L-carnitine. Furthermore, L-carnitine administration counteracts the effect of DHEA on mitochondrial structure. On the contrary, carnitine has no significant effect on the reduction in weight gain observed upon short- or long-term treatment with DHEA.

Comparative studies of effects of dehydroepiandrosterone on rat and chicken liver.

1. An attempt to identify the cause of decrease of gain in body weight during dehydroepiandrosterone (DHEA) treatment was made comparing the effects of hormone treatment on chickens and rats. 2. Chickens treated with DHEA for 7-10 days do not change their weight gain with respect to controls although their mitochondrial respiration and peroxisomal catalase (index of peroxisomal mass) were increased. 3. Liver cytosolic malic enzyme and sn-glycerol-3-phosphate dehydrogenase were depressed in chickens treated with DHEA in comparison with activities in untreated controls. DHEA treatment did not increase the activity of mitochondrial sn-glycerol 3-phosphate dehydrogenase. 4. In contrast to rat liver cytosolic sn-glycerol-3-phosphate dehydrogenase this enzyme in chicken liver was inactive with NADPH.

Concerning the mechanism of increased thermogenesis in rats treated with dehydroepiandrosterone.

Dehydroepiandrosterone (DHEA) treatment of rats decreases gain of body weight without affecting food intake; simultaneously, the activities of liver malic enzyme and cytosolic glycerol-3-P dehydrogenase are increased. In the present study experiments were conducted to test the possibility that DHEA enhances thermogenesis and decreases metabolic efficiency via transhydrogenation of cytosolic NADPH into mitochondrial FADH2 with a consequent loss of energy as heat. The following results provide evidence which supports the proposed hypothesis: (a) the activities of cytosolic enzymes involved in NADPH production (malic enzyme, cytosolic isocitrate dehydrogenase, and aconitase) are increased after DHEA treatment; (b) cytosolic glycerol-3-P dehydrogenase may use both NAD+ and NADP+ as coenzymes; (c) activities of both cytosolic and mitochondrial forms of glycerol-3-P dehydrogenase are increased by DHEA treatment; (d) cytosol obtained from DHEA-treated rats synthesizes more glycerol-3-P during incubation with fructose-1,6-P2 (used as source of dihydroxyacetone phosphate) and NADP+; the addition of citrate in vitro further increases this difference; (e) mitochondria prepared from DHEA-treated rats more rapidly consume glycerol-3-P added exogenously or formed endogenously in the cytosol in the presence of fructose-1,6-P2 and NADP+.

[Damage to calcium ion-loaded mitochondria by fatty acids and the protective effect of carnitine]. / Povrezhdenie nagruzhennykh ionami kal'tsiia mitokhondrii zhirnymi kislotami i zashchitnoe deistvie karnitina.

The effect of fatty acids and L-carnitine on Ca2+ retention in rat liver mitochondria have been studied. Ca(2+)-retention was estimated as a sum of consecutive Ca2+ additions which leaded to transient stimulation of respiration coupled with influx of Ca2+ L-carnitine increases the Ca(2+)-retention; such an effect requires ATP. The Ca(2+)-retention was increased in the presence of 50 microM ATP or ADP. In all cases carboxyatractylate prevented the increase in Ca(2+)-retention. Palmitate and FCCP added at concentrations producing similar stimulating effect on respiration inhibit Ca(2+)-retention to about the same degree. The effect of palmitate is strongly diminished by L-carnitine. Again, the L-carnitine effect requires ATP. The data obtained suggest that the protonophoric effect of fatty acid plays a crucial role in Ca(2+)-dependent damage of mitochondria.

Interaction of carnitine with mitochondrial cardiolipin.

The physiological role of L-carnitine is to determine the transport of acyl-CoA through the mitochondrial membrane. However, some observations may also suggest a direct effect of the molecule per se on the physical properties of the membrane, most probably at the level of the binding site. This possibility has been investigated by studying the influence of adriamycin, a drug that binds to cardiolipin, on the effect of carnitine on isolated rat liver mitochondria. It has been found that adriamycin almost abolishes the activating effect of carnitine on state 2 respiration. The effect and its inhibition is seen by using either the L-form of carnitine or the D-form or both. Cardiolipin removes the effect of adriamycin and restores the activation by carnitine. It is proposed that some effects of carnitine on mitochondrial properties may be the result of interaction of carnitine with cardiolipin at the membrane level.

Changes in liver structure and function after short-term and long-term treatment of rats with dehydroepiandrosterone.

The effects on the liver of feeding a diet containing 0.2% dehydroepiandrosterone were studied after short (7 d) and long (100 d) periods of treatment in rats. The short-term treatment caused hypertrophy of the hepatocytes that, at the ultrastructural level, seemed to be due to proliferation of peroxisomes and (to a minor extent) of mitochondria. The mitochondria seemed to have undergone transition from expanded to condensed configuration; accordingly, after isolation, their rate of coupled respiration was greater than that of control mitochondria. After long-term treatment, the structure of the hepatocytes reverted toward normal. In fact, at the ultrastructural level, the number and the size of peroxisomes was not significantly different from those of the controls, but degenerative phenomena were observed in the mitochondria. Attempts are made to explain the above ultrastructural and biochemical findings in view of the effects of dehydroepiandrosterone on the energy metabolism of liver.

The protecting effect of L-carnitine on Ca(2+)-loaded rat liver mitochondria.

It is shown that L-carnitine strongly increases the ability of rat liver mitochondria to respond to the train of Ca2+ additions by a transient stimulation of the State-4 respiration rate. Such an effect requires ATP and the L-carnitine efficiency strongly decreases when ATP is omitted. Oleate influences the mitochondria in a fashion opposite to that of L-carnitine. The oleate effect is strongly diminished by L-carnitine. Again, the L-carnitine effect requires ATP, and D-carnitine fails to substitute for L-carnitine. It is suggested that L-carnitine removes, in an ATP-dependent manner, endogenous or added fatty acids, which are involved in oxidative damage of Ca(2+)-loaded mitochondria.

Sources of intramitochondrial malate.

Liver mitochondria from rats treated with gluconeogenic hormones or subjected to vigorous exercise consume oxygen more rapidly than do mitochondria from control rats. These treatments result in elevated mitochondrial malate concentrations, which facilitate the entry of added substrate into the mitochondria. In this paper we describe experiments conducted to determine the source of the extra malate. Injections of glutamate plus alanine, two amino acids that are increased in blood after exercise and hormone treatment, caused liver mitochondrial malate to be increased. Injections of glucagon, cortisol, or both hormones elevated liver mitochondrial malate concentrations in both adrenalectomized and sham-operated rats.

Changes in mitochondrial activity caused by ammonium salts and the protective effect of carnitine.

Ammonium salts added to isolated rat liver mitochondria deviate alpha-ketoglutarate to glutamate synthesis, thus decreasing its availability as respiratory substrate. As a consequence a decrease of respiratory rate is observed which is paralleled by progressive mitochondrial swelling. It was demonstrated that L-carnitine may abolish this swelling thus improving structural and metabolic state of mitochondria.

The effectiveness of visual aids in teaching a microbiology course.

Uma das tecnicas de ensino, puramente convencional e outra, suplementada com audiovisuais, foram comparadas, visando melhorar a aprednizagem e reduzir as repetencias em um curso basico de microbiologia na UFV, Vicosa. O estudo foi efetuado no II semestre de 1980, envolvendo 282 estudantes, matriculados.Dois professores participaram no experimento. Tres classes experimentais e duas de controle assistiram a aula teorica de uma hora, durante 15 semanas. Durante a apresentacao teorica slides e transparencias ilustraram alguns aspectos do programa, no caso do grupo experimental. Tres pos-testes foram usados para avaliar os grupos. No inicio do experimento foi efetuado o teste de inteligencia G-36, para avaliar a homogeneidade do gruo. Nao foram observadas diferencas significativas (0,05), entre as medias das notas, obtidas pelos estudantes em cada pos-teste e a soma das notas dos pos-testes, entre os grupos experimental e de controle para ambos professores. Alguns fatores sao discutidos como responsaveis pelos resultados obtidos