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Biomedical subjects

L Pereira-da-Silva

Publications and source records attributed to L Pereira-da-Silva.

At least 19 recordsLinked to original sources

Circulating leukocyte heat shock protein 70 (HSP70) and oxidative stress markers in rats after a bout of exhaustive exercise.

A novel method to measure oxidative stress resulting from exhaustive exercise in rats is presented. In this new procedure we evaluated the erythrocyte antioxidant enzymes, catalase (CAT) and glutathione reductase (GR), the plasma oxidative attack markers, reactive carbonyl derivatives (RCD) and thiobarbituric reactive substances (TBARS). Muscular tissue damage was evaluated by monitoring plasma creatine kinase (CK) and plasma taurine (Tau) concentrations. Also, we monitored total sulphydryl groups (TSG) and uric acid (UA), and the level of the 70 kDa heat shock protein (HSP70) in leukocytes as a marker of oxidative stress. In the study we found a correspondence between erythrocyte CAT and GR activities and leukocyte HSP70 levels, principally 3 h after the acute exercise, and this suggested an integrated mechanism of antioxidant defense. The increase in levels of plasma Tau was coincident with the increasing plasma levels of CK and TBARS, principally after two hours of exercise. Thus tissue damage occurred before the expression of any anti-oxidant system markers and the monitoring of Tau, CK or TBARS may be important for the estimation of oxidative stress during exhaustive exercise. Furthermore, the integrated analyses could be of value in a clinical setting to quantify the extent of oxidative stress risk and reduce the need to perform muscle biopsies as a tool of clinical evaluation.

Animals↗

[Neonatal bronchoscopy: a retrospective analysis of 67 cases and a review of their indications].

The availability of newer, more sophisticated and versatile bronchoscopes has expanded the spectrum and scope of the indications for bronchoscopy in the newborn infant both for diagnostic and therapeutic purposes. The aim of this study was to carry out a retrospective analysis of the bronchoscopies performed on newborn infants, and to review the indications of this procedure in this age group. Sixty-three patients were submitted to 67 bronchoscopies in a period of 13 years, allowing the diagnosis of 45 anomalies and malformations of the tracheo-bronchial tree, and the performance of 24 bronchoalveolar lavages. In six cases, endoscopic removal of secretions helped to resolve resistant atelectasia, while in another case, with esophageal atresia, intra-operative definition of the fistula tract was possible through catheterisation of the fistula with the bronchoscope. The flexible bronchoscope was preferred for diagnosis by direct visualisation, and the rigid bronchoscope for some diagnostic and therapeutic procedures. Stridor, unexplained cyanosis, hemoptysis, persistent or recurrent pulmonary images, difficulties in the intubation or extubation, and persistent disturbances in ventilation are among the main indications for bronchoscopy in the newborn infant. Bronchoscopy also allows the performance of subsidiary techniques, such as bronchoalveolar lavage, biopsy and laser therapy.

Bronchoscopy↗

Evaluation by blue native polyacrylamide electrophoresis colorimetric staining of the effects of physical exercise on the activities of mitochondrial complexes in rat muscle.

Blue native polyacrylamide electrophoresis (BN-PAGE) is a technique developed for the analysis of membrane complexes. Combined with histochemical staining, it permits the analysis and quantification of the activities of mitochondrial oxidative phosphorylation enzymes using whole muscle homogenates, without the need to isolate muscle mitochondria. Mitochondrial complex activities were measured by emerging gels in a solution containing all specific substrates for NADH dehydrogenase and cytochrome c oxidase enzymes (complexes I and IV, respectively) and the colored bands obtained were measured by optique densitometry. The objective of the present study was the application of BN-PAGE colorimetric staining for enzymatic characterization of mitochondrial complexes I and IV in rat muscles with different morphological and biochemical properties. We also investigated these activities at different times after acute exercise of rat soleus muscle. Although having fewer mitochondria than oxidative muscles, white gastrocnemius muscle presented a significantly higher activity (26.7 +/- 9.5) in terms of complex I/V ratio compared to the red gastrocnemius (3.8 +/- 0.65, P < 0.05) and soleus (9.8 +/- 0.9, P < 0.001) muscles. Furthermore, the complex IV/V ratio of white gastrocnemius muscle was always significantly higher when compared to the other muscles. Ninety-five minutes of exhaustive physical exercise induced a decrease in complex I/V and complex IV/V ratios after all resting times (0, 3 and 6 h) compared to control (P < 0.05), probably reflecting the oxidative damage due to increasing free radical production in mitochondria. These results demonstrate the possible and useful application of BN-PAGE-histochemical staining to physical exercise studies.

Animals↗

Osmolality of solutions, emulsions and drugs that may have a high osmolality: aspects of their use in neonatal care.

OBJECTIVE: Administration of some hypertonic substances to neonates has been associated with a variety of adverse effects. This study was conducted to determine the osmolality of intravenous drugs and solutions used in neonates receiving intensive care. METHODS: Osmolality was measured by freezing point depression. Vasoactive drugs, diuretics, anticonvulsants, antimicrobials, and glucose and electrolyte solutions were some of the substances analyzed. RESULTS: The osmolalities of 90 substances were measured; the respective intra-assay and interassay coefficients of variation were always less than 5%. A few drugs were found to be extremely hypertonic (> 8000 mOsm/kg), and most of them contain propylene glycol as vehicle (e.g. digoxin, phenytoin, diazepam and phenobarbital). Other drugs, at the same concentration, evidenced a significant discrepancy of osmolality depending on the trademark. CONCLUSIONS: The finding of some extremely hypertonic drugs highlights the need for further investigation in order to study their potential adverse effects in neonates, as well as to evaluate any advantage in diluting, infusing slowly or even avoiding such substances. Given the fact that there exists a discrepancy in osmolalities in some drugs at the same concentration depending on the trademark, the more isotonic solutions should be the preferred choice for intravenous administration.

Emulsions↗

Antioxidant effect of dipyridamole and its derivative RA-25 in mitochondria: correlation of activity and location in the membrane.

Dipyridamole (DIP), a coronary vasodilator, presents coactivator activity for a number of antitumor drugs as well as antioxidant activity in membrane systems. DIP and derivatives interact with membrane systems such as micelles, phospholipid monolayers and vesicles. The antioxidant effect of DIP and several derivatives upon iron-induced lipoperoxidation on mitochondria has been reported and a good correlation between the hydrophobicity and their protective effect was found (M.F. Nepomuceno et al., Free Radic. Biol. Med., 23 (1997) 1046-1054). In the present work an effort is made to better understand the role of DIP as inhibitor of Fe2+-induced lipid peroxidation in mitochondria. At low concentration, no significant effect on either state IV or state III respiration was found, discarding a possible direct interaction of DIP or RA-25 with the peripheral benzodiazepine receptor. The association constants for DIP and RA-25 in mitochondria were estimated, being 0.7 (mg/ml)-1 for DIP and 0.2 (mg/ml)-1 for RA-25. Oxygen consumption studies in the presence of FeSO4 showed that the antioxidant effect of DIP or RA-25 did not involved the initial step of Fe2+ oxidation. Our data strongly support the hypothesis that the antioxidant effect of both DIP and RA-25 is related to their partition in the lipid phase of the mitochondrial membrane and not to a specific interaction with membrane proteins. This protection may be due either to a direct inhibition of the propagation steps or a scavenger effect on the radicular species that would trigger the peroxidative process.

Animals↗

Upper arm measurements of healthy neonates comparing ultrasonography and anthropometric methods.

OBJECTIVE: To compare measurements of the upper arm cross-sectional areas (total arm area, arm muscle area, and arm fat area of healthy neonates) as calculated using anthropometry with the values obtained by ultrasonography. MATERIALS AND METHODS: This study was performed on 60 consecutively born healthy neonates: gestational age (mean+/-SD) 39.6+/-1.2 weeks, birth weight 3287.1+/-307.7 g, 27 males (45%) and 33 females (55%). Mid-arm circumference and tricipital skinfold thickness measurements were taken on the left upper mid-arm according to the conventional anthropometric method to calculate total arm area, arm muscle area and arm fat area. The ultrasound evaluation was performed at the same arm location using a Toshiba sonolayer SSA-250A, which allows the calculation of the total arm area, arm muscle area and arm fat area by the number of pixels enclosed in the plotted areas. STATISTICAL ANALYSIS: whenever appropriate, parametric and non-parametric tests were used in order to compare measurements of paired samples and of groups of samples. RESULTS: No significant differences between males and females were found in any evaluated measurements, estimated either by anthropometry or by ultrasound. Also the median of total arm area did not differ significantly with either method (P = 0.337). Although there is evidence of concordance of the total arm area measurements (r = 0.68, 95% CI: 0.55-0.77) the two methods of measurement differed for arm muscle area and arm fat area. The estimated median of measurements by ultrasound for arm muscle area were significantly lower than those estimated by the anthropometric method, which differed by as much as 111% (P < 0.001). The estimated median ultrasound measurement of the arm fat was higher than the anthropometric arm fat area by as much as 31% (P < 0.001). CONCLUSION: Compared with ultrasound measurements using skinfold measurements and mid-arm circumference without further correction may lead to overestimation of the cross-sectional area of muscle and underestimation of the cross-sectional fat area. The correlation between the two methods could be interpreted as an indication for further search of correction factors in the equations.

Adipose Tissue↗

Mitochondrial swelling and oxygen consumption during respiratory state 4 induced by phospholipase A2 isoforms isolated from the South American rattlesnake (Crotalus durissus terrificus) venom.

The non-covalent interaction between two molecular entities namely, phospholipase A2 and crotapotin, results in the main toxin, crotoxin, present in the venom of the South American rattlesnake Crotalus durissus terrificus. High performance liquid chromatography has enabled us the isolation of three phospholipase A2 isoforms (F1, F2 and F3), characterized through denaturing and non-denaturing polyacrylamide gel electrophoresis and also through the N-terminal amino acid sequence analysis. The effect of each purified phospholipase A2 isoform on isolated rat liver mitochondria was determined through mitochondrial swelling and O2 consumption during respiratory state 4. F1 showed a dose-dependent stimulation of O2 consumption while F2 and F3 caused stimulation only at low doses and inhibition at high amounts. These effects were completely suppressed by the presence of 0.1% bovine serum albumin or 0.5 mM EGTA in the incubation medium. Taking the mitochondrial swelling as an activity parameter, all of them presented the same behaviour at different intensities, leading to permeabilization of the mitochondrial membrane. In this case, addition of EGTA prevented it whereas bovine serum albumin was ineffective, indicating that the lipid microenvironment was affected. These results suggest that free fatty acids are directly responsible for the observed effects induced by phospholipase A2 isoforms on oxygen consumption experiments. The protection conferred by cyclosporin-A on swelling induced by the isoforms, when present in low concentrations, may suggest that cyclosporin-A binds to a mitochondrial membrane site protecting the membrane against the phospholipase A2 attack.

Amino Acid Sequence↗

Inhibitory effect of dipyridamole and its derivatives on lipid peroxidation in mitochondria.

Dipyridamole (DIP), 2,6-bis(diethanolamino)-4,8-dipiperidino-[5,4-d] pyrimidine, is a coronary vasodilator widely used in clinics. It has also been reported to have coactivator activity for a number of antitumour drugs and antioxidant activity in membrane systems. In recent years we have been studying the spectroscopic properties of this drug and several of its derivatives as well as their interaction with charged micelles and phospholipid monolayers. A strong interaction of DIP and DIP derivatives with these model membrane systems and a dependence of the strength of the interaction upon the chemical structure of the DIP derivative was observed. Here, the antioxidant effect of DIP and the derivatives, RA14, RA47, and RA25, was compared. We observed that although it strongly inhibits the iron-induced lipoperoxidation on mitochondria (IC50 = 1 microM), it shows no protection against an organic oxidant, cumene hydroperoxide. The order of hydrophobicity of the DIP derivatives, DIP > RA14 > RA47 > RA25, correlates very well with both the values of the association constants of these derivatives to micelles, their localization in the micelles, and phospholipid films and their antioxidant effect on mitochondria. So, a very good correlation of the structure of the drug in regarded to the nature of its substituents with the biological activity is observed. Essentially the same result was observed either measuring the lipid peroxidation or the membrane fluidity by ESR, suggesting that the effect of DIP and DIP derivatives is probably associated to their binding to the lipid bilayer and not to interaction with membrane proteins.

Animals↗

Inorganic pyrophosphate gives a membrane potential in yeast mitochondria, as measured with the permeant cation tetraphenylphosphonium.

Evidence for a membrane potential (delta psi) generating capacity of inorganic pyrophosphate (PPi) with inorganic pyrophosphatase in mitochondria from the yeast Saccharomyces cerevisiae is presented. Addition of PPi increases the accumulation of the permeant cation tetraphenylphosphonium (TTP+) in mitochondria, as measured with a TPP(+)-selective electrode or using [3H]TPP+. This accumulation is strongly inhibited by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) but not by oligomycin, whereas accumulation of TPP+ induced by ATP is strongly inhibited by both FCCP and oligomycin. The values of delta psi obtained upon addition of PPi were similar to those obtained when yeast mitochondria were energized by NADH or ATP. Energization of the wild type yeast mitochondria with ATP resulted in higher delta psi values than those achieved when PPi was hydrolyzed, whereas the effect of PPi was more pronounced in mitochondria from a pyrophosphatase (PPase) overproducing yeast strain. These results clearly indicate that PPi is significant as an energy donor and that the yeast mitochondrial PPase, the gene for which we have recently cloned and sequenced, may participate in energy transfer reactions.

Adenosine Triphosphate↗

Involvement of the ADP/ATP carrier in permeabilization processes of the inner mitochondrial membrane.

The effect of different agents on inner-mitochondrial-membrane permeabilization and lipoperoxidation induced by Ca2+ and the pyridine-nucleotide oxidant t-butylhydroperoxide or inorganic phosphate was investigated. Comparing the protection conferred by ADP, a substrate of the ADP/ATP carrier, dithiothreitol, a disulfide reductant and butylhydroxytoluene, a radical scavenger, it was found that ADP was always the most effective against mitochondrial damage, when present in the incubation medium from the beginning. Moreover, carboxyatractyloside, a specific inhibitor of the ADP/ATP carrier, abolished completely the protective effect of ADP on both the lipoperoxidation and mitochondrial swelling processes. Experiments where deenergized mitochondria were previously incubated with Ca2+ showed a decrease in the content of active ADP/ATP carrier, indicating a direct involvement of this protein in the formation of a non-specific Ca(2+)-dependent pore. Our results also eliminate the possibility of an attack of oxygen radicals on lipids or proteins of the mitochondrial membrane as the primary event triggering the permeability transition of the inner mitochondrial membrane.

Adenosine Diphosphate↗

Studies on the utilization of inorganic pyrophosphate by different metabolic systems in yeast mitochondria.

A mitochondrial pyrophosphatase (PPase) from yeast cells (Saccharomyces cerevisiae) was studied and characterized. The hydrolytic activity towards inorganic pyrophosphate (PPi) was inhibited by different SH-reagents and increased in the presence of uncouplers, indicating a possible involvement of this enzyme in energy-linked processes. This view was also supported by the observation that these mitochondria were able to hydrolyze PPi, generating an electrical membrane potential (delta psi) of the same magnitude as that obtained with ATP. Both ATP and PPi inhibited the pyruvate dehydrogenase complex and it was demonstrated that PPi can be used as substrate by mitochondrial kinases leading to the same pattern of protein phosphorylation as when ATP is used.

Diphosphates↗

Effect of cyclosporin A and trifluoperazine on rat liver mitochondria swelling and lipid peroxidation.

The effect of cyclosporin A (CsA) or trifluoperazine (TFP) on lipid peroxidation and mitochondrial swelling was determined using liver mitochondria incubated with 30 microM Ca2+ and 250 microM t-butylhydroperoxide or 5 mM inorganic phosphate (P(i)). Lipid peroxidation was not modified by either 1 microM CsA or 40 microM TFP. These compounds presented a distinct effect on mitochondrial permeability. Under oxidative conditions, CsA only showed a transient protective effect whereas TFP completely inhibited mitochondrial swelling. Conversely, CsA was very efficient when Ca2+ and P(i) were used, a condition under which TFP was unable to prevent the swelling. These data are consistent with our previous results (M.F. Nepomuceno, D.V. Macedo and L. Pereira-da-Silva (1991). Brazilian Journal of Medical and Biological Research, 24: 833-836) showing that lipid peroxidation is one among other different components of the permeabilization process. The data suggest that lipid peroxidation is independent of swelling, occurring later than swelling, presumably when the mitochondrial reductant systems are depleted. The differential effects of CsA and TFP suggest that these compounds can be used as specific probes in the elucidation of the two distinct mechanisms responsible for mitochondrial swelling.

Animals↗

Lipoperoxidation associated with mitochondrial membrane permeabilization in the presence of Ca2+.

Lipoperoxidation was investigated as a step for membrane protein thiol oxidation of rat liver mitochondria incubated in the presence of Ca2+ and t-butylhydroperoxide, by the determination of thiobarbituric acid reactive substances. Lipoperoxidation occurred only when the incubation medium contained 125 microM t-butylhydroperoxide (t-buOOH) in the presence of Ca2+ and phosphate (Pi). No lipoperoxidation was observed when acetate replaced Pi as permeant anion, or when the oxidant was omitted, even at high Ca2+ and Pi concentrations (up to 120 microM Ca2+ and 5 mM Pi), conditions under which the mitochondria are fully permeabilized. In both cases, ADP protected efficiently against permeabilization, indicating the possible involvement of the ADP/ATP carrier in the earlier stages of the process.

Adenosine Diphosphate↗

Membrane protein thiol cross-linking associated with the permeabilization of the inner mitochondrial membrane by Ca2+ plus prooxidants.

In a previous report (Macedo, D.V., Ferraz, V. L., Pereira-da-Silva, L., and Vercesi, A. E. (1988) in Integration of Mitochondrial Functions (Lemasters, J. J., et al., eds) pp. 535-542, Plenum Publishing Corp., New York), we proposed that the alterations in the inner mitochondrial membrane permeability caused by Ca2+ plus prooxidants could be the consequence of membrane protein sulfhydryl-disulfide transitions. In this study, we show that Ca2+ plus diamide, a thiol oxidant, significantly decrease the ability of beef heart submitochondrial particles to build up and sustain a membrane potential generated by succinate oxidation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins indicates that these effects on the membrane potential are associated with the production of protein aggregates due to thiol cross-linking. Evidence is also presented that these protein aggregates can be produced in mitoplasts previously loaded with Ca2+ and that this is potentiated by the presence of either diamide or t-butylhydroperoxide. Furthermore, dithiothreitol, a disulfide reductant, was found to be much more effective than NAD(P)+ reductants in reversing Ca2+ efflux induced by prooxidants. It is concluded that the perturbation of the inner mitochondrial membrane caused by Ca2+ plus prooxidants is associated with protein polymerization due to thiol cross-linking, resulting in the production of high molecular mass protein aggregates.

Animals↗

Comparison of the antiviral activity and toxicity of a protein magnesium ammonium phospholinoleate anhydride polymer with other antiviral drugs.

1. SB-73, a magnesium ammonium phospholinoleate anhydride aggregate, exhibited antiviral action in vitro in the concentration range of 50 to 100 micrograms/ml against herpes simplex type 1, stomatitis vesicular virus, adenovirus type 5, and in vivo in the dose range of 0.7 to 1.3 mg/kg against canine parvovirus and distemper virus. 2. The lethal dose (LD50) was 2.71 +/- 1.55 g/kg body weight in mice inoculated intraperitoneally. Oral ingestion of the aggregate up to 30 g/kg body weight by mice had no lethal effects during the 14 days of observation. 3. In in vitro cytotoxicity experiments with fibroblasts (V-79 Chinese hamster cell line), no toxic effects were observed with SB-73 concentrations (120 micrograms/ml) having antiviral activity. 4. In a cellular proliferation experiment using hamster V-79 cells, we observed 72% proliferation after treatment of the cells with a high concentration (500 micrograms/ml) of SB-73. 5. Compound SB-73 showed no genotoxicity for human lymphocytes at concentrations of 100 micrograms/ml. 6. When the cytotoxicity and genotoxicity of SB-73 were compared with those of acyclovir, idoxuridine and AZT at 500 micrograms/ml concentrations the compound was found to have effects similar to those of acyclovir.

Acyclovir↗