Biomedical subjects
Publications and source records attributed to A Machado.
Intramitochondrial myelin-like structures were found in ventricular myocardial cells of the rat heart. These inclusions were found in both the first 2 postnatal weeks and in senescent stages. The origin of such myelin-like structures could be related to an age-dependent metabolic change, specifically to an inability to oxidize long-chain fatty acids described in both periods.
The use of the antineoplastic agent adriamycin is limited by its cardiotoxicity. The mechanism of cardiotoxicity has been investigated through the study of adriamycin effects on a number of heart enzymes. Adriamycin inhibited the activity of NADP-isocitrate dehydrogenase, malic enzyme, and 6-phosphogluconate dehydrogenase, three enzymes that have in common the presence of reactive SH groups involved in activity. Adriamycin action was prevented by the presence of proteins or dithioerythrol and mimicked by dithiobis dinitrobenzoate. It is suggested that adriamycin effects are due to interaction with enzyme SH groups by a product of adriamycin metabolism.
The location of peroxide-utilizing enzymes has been studied in rat brain. Glutathione peroxidase and glutathione reductase distributions indicate that both enzymes are located in the cytoplasm and in the matrix space of "synaptosomal" and "free" mitochondria. On the other hand, catalase distribution parallels that of NADH-cytochrome c reductase (rotenone-insensitive), and appears to be associated with the outer membrane of brain mitochondria. Whereas no gross age-dependent changes in various marker enzymes were found, a gradual but significant increase in glutathione peroxidase from the soluble fraction of free mitochondria was detected. The consequences of such increase are discussed with regard to the reducing potential of the cell.
The behavior of several enzymes was studied during rat heart development (4 days before birth to adult stage). Hexokinase has its highest activity during the fetal period; it decreases at birth and remains with low activity in the adult. The alpha-glycerophosphate dehydrogenase and alpha-glycerophosphate oxidase profiles are similar up to the 15th day of development. From there onwards, both profiles diverge, the cytoplasmic activity increasing 3-fold, while the mitochondrial activity remains unchanged. The developmental profiles of the malate dehydrogenases are almost parallel. The development of citrate synthase and succinate dehydrogenase results in a 2- to 4-fold increase in their activities. However, ATPase increases dramatically (20-fold) over the same period. With respect to the enzymes of the adenine nucleotide metabolism, adenylate kinase is fully expressed throughout all ages examined, showing no variation during development. AMP deaminase and creatine kinase increase during development, the cytoplasmic creatine kinase reaching a high level at birth whereas the increases of the mitochondrial enzymes take place gradually during development.
During growth on acetate, the pH of yeast cultures rises from 5.8 to around 7-8 in the stationary phase. This was found to result from acetic acid uptake and accompanying H+ loss. In addition, acetate-growing yeast were found to accumulate ammonia. The influence of pH on ammonia transport and accumulation was studied with the analogue [14C]methylamine with the following results. (a) Methylamine uptake kinetics from 0.1-50 mM were consistent with a single-component uptake system (NH+4 permease) at pH values more acidic than 6.5, and with a two-component system (NH+4 permease and NH3 diffusion) above pH 7.5. (b) Equilibrium accumulation of methylamine was found to increase with increasing pH. (c) Methylamine efflux from methylamine-loaded cells increased as the external pH decreased. It was concluded from measurements of the internal pH under various culture conditions that the accumulation of ammonia in acetate-growing alkaline cultures resulted from the sum of two processes: (1) an energy-driven NH+4 transport; and (2) NH3 diffusion dependent on the delta pH.
Citrate synthase (EC 4.1.3.7) from Tetrahymena pyriformis has been purified 185-fold. The molecular weight of the native enzyme was determined to be 120,000. The enzyme is labile at low ionic strength, but can be stabilized by KCl and glycerol. It is activated by KCl at low (below 60 mM) or high concentrations, and inhibited by divalent cations (Mn2+, Mg2+, Ca2+). The Michaelis constants are 0.1 mM for oxalacetate and 0.01 mM for acetyl-CoA. The kinetics with oxalacetate exhibit negative cooperativity, with a nH = 0.66. Among the metabolites tested, only ATP and GTP can inhibit the enzyme but Mg2+ relieves the ATP inhibition. Incubation with sulfhydryl reagents (DTNB) in the absence of its substrates results in a rapid inactivation of the enzyme. It is concluded that Tetrahymena citrate synthase is closer to the enzyme from Gram-positive bacteria than to those of eucaryotes.
The utilization by yeast of two carbon sources is carried out through the operation of the glyoxylic acid cycle. Kinetic acid from the isocitrate transforming enzymes suggest that the flow of isocitrate through the glyoxylic acid cycle depends upon the inhibition of the isocitrate decarboxylating enzymes. Both isocitrate dehydrogenases are inhibited by a mixture of glyoxylate + oxaloacetate, but for the reasons described in the text we consider that this inhibition is of no physiological significance. On the other hand, we have found that NADPH is a competitive inhibitor of NADP-isocitrate dehydrogenase with respect to NADP+, with a KI similar to its KM. It also produces an additive effect on the NADH-produced inhibition of NAD-isocitrate dehydrogenase. We propose NADPH as the compound that channels the utilization of isocitrate into the glyoxylic acid cycle. This is supported by the finding of an increased NADPH/NADP+ ratio in acetate grown yeast with respect to glucose grown cells.
The behavior of the principal NADPH-consuming pathways, fatty acid synthesis (-ATP-citrate lyase), transhydrogenase, glutamate dehydrogenase, glutathione reductase, NADPH-cytochrome c reductase and mitochondrial and cytoplasmic thioredoxin reductases were studied during the development of the heart, brain and liver of the rat. The liver is the tissue with highest NADPH-consuming activities. Transhydrogenase activity is highest in the heart; it undergoes two steep increases of activity, one at birth and another between 15 and 25 days. Liver cytoplasmic glutathione reductase activity increases mainly at birth. The activities in heart and brain are lower and the brain enzyme activity slightly increases throughout development. Liver NADPH-cytochrome c reductase activity greatly increases at weaning. The heart activity is the lowest, no changes taking place in the developmental period. Heart mitochondrial and brain cytoplasmic thioredoxin reductases undergo increases in activity at birth. The ratios of NADPH-producing/NADPH-consuming activities are constant in all tissues studied, and oscillate between congruent to 4 in fetuses to congruent to 2 in adults, the only exception being the adult heart ratio (congruent to 23). This otherwise constancy confirms that both processes are closely correlated.
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The levels and the distribution of monoamines within the rat circumvallate papilla have been studied. Noradrenaline was found in the connective tissue underlying the taste buds, whereas serotonin was located in the basal area of the gustatory epithelium but not inside the taste buds. Following denervation, noradrenaline levels decreased and serotonin levels increased. These results suggest that both neurotransmitters may have some mutual interaction in modulating transmission at the papilla.
The developmental changes in the activity of certain representative enzymes of adenine nucleotide metabolism, namely AMP deaminase, adenylate kinase and creatine kinase, have been studied in rat brain and liver. The principal points observed can be summarised as follows. (1) Fetal liver has a remarkably high AMP deaminase activity, which decreases immediately after birth. (2) The activities of both soluble and mitochondrial forms of brain creatine kinase are about 50-fold those of liver. The activities of these liver enzymes remain unchanged during the developmental period. In brain, however, these enzymes increase their activities about 3 and 5 times in the cytoplasmic and mitochondrial fractions, respectively. (3) The brain adenylate kinase activity from cytoplasmic fractions increased 4-fold during the 10- and 30-day period, whereas the enzyme from mitochondrial fractions remained unchanged.
Adriamycin is used for the treatment of leukemia and other neoplastic processes. Unfortunately the drug has a toxic effect on some tissues. Cardiotoxicity in particular limits the use of the drug. Several hypotheses have been given to explain adriamycin heart toxicity. The authors have studied the effect of the drug on the enzymes isocitrate dehydrogenase-NADP, malic enzyme, 6-P-gluconate dehydrogenase, malic dehydrogenase-NAD+, hexokinase, and phosphofructokinase. They observed that adriamycin inhibits the NADP-dependent enzymes, and that the sulfhydryl group of the enzymes may be involved in the inhibitory action of adriamycin.
The utilization of two-carbon sources is carried out through the operation of the glyoxylic acid cycle. The cycle involves several Krebs cycle steps as well as two additional specific enzymes, isocitrate lyase and malate synthase. Kinetic data from the isocitrate transforming enzymes suggest that the flow of isocitrate through the glyoxylic acid cycle depends upon the inhibition of the isocitrate decarboxylating enzymes. Three different mechanisms have been proposed at present that could account for this inhibition: a) Inhibition by concerted action of oxalacetate and glyoxylate; b) NADP-linked isocitrate dehydrogenase phosphorilation in E. coli, and c) competitive inhibition by NADPH with respect to NADP. In the present case the variations of the NADPH/NADP ratio would be channeling the utilization of isocitrate into the glyoxylic acid cycle.
The patterns of brain enzymes linked to energy metabolism have been determined in rats aged between 3 and 21 months and compared to those of the developing brain as an estimate of the senescent energy capacity of this organ. During aging, pyruvate kinase increases, pointing towards an enhancement of the glucose-dependence of this organ. However, NAD-isocitrate dehydrogenase declines, suggesting a reduction of Krebs cycle activity in the aged rat brain. An increase in cytoplasmic NAD-malate dehydrogenase found during aging could provide an alternative mechanism of NAD recovery.
This paper deals with the ultrastructure of two types of cytoplasmic inclusions, "myelin figures" and "lipid complexes", present in muscle cells of ventricular myocardium of the rat heart. The inclusions were numerous in both the first two postnatal weeks and in the senescent stages, being rarely observed in young adult animals. The myelin figures were entirely made up of myelin-like membrane whorls, whereas the lipid complexes exhibited discrete myeloid caps surrounding a central lipid core. The origin and functional significance of such myelin-like structures are discussed on the basis of ultrastructural and biochemical studies on myocardial physiology and age-related evolution.
The developmental and senescent patterns of a number of heart enzyme activities linked to energy metabolism have been studied in rats aged between 4 days and 21 months. A morphometric study of mitochondrial volume fractions and numbers has been also carried out. Developmental changes result in a rise of most mitochondrial enzymes (NADP+-isocitrate dehydrogenase, malic enzyme, succinate dehydrogenase, citrate synthase) and mitochondrial volume fractions. Exceptions are NAD+-isocitrate dehydrogenase, which declines from 4 days onwards, and NAD+-malate dehydrogenase, which declines and then rises over the same period. Senescent changes follow two different trends. While pyruvate kinase and those mitochondrial enzymes lying between citrate formation and isocitrate oxidation (citrate synthase, NADP+-and NAD+-isocitrate dehydrogenases) decline to some degree, mitochondrial succinate dehydrogenase and NAD+-malate dehydrogenase activities increase over the same period. This could point towards a partial impairment of Krebs cycle function, and a reduced energy-producing capacity in the aged rat heart.
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The variations in enzyme activities involved in the main pathways of liver energetic metabolism--glycolysis, Krebs cycle, gluconeogenesis and lipogenesis--have been studied in rats ranging between the age of 4 days and 21 months. The major changes observed are the following: (1) enzymes involved in glycolysis (pyruvate kinase) and lipogenesis (NADP-malic enzyme, ATP-citrate lyase) decrease in activity during ageing, and (2) gluconeogenic enzymes (phosphoenolpyruvate carboxykinase, glucose-6-phosphatase) are maintained or slightly increased over the same period. The results suggest that an increase in the capacity for gluconeogenesis with respect to that for lipogenesis takes place in the aged rat liver.