Biomedical subjects
Publications and source records attributed to M C Pinto.
Accidental transmission of Chagas disease to man by blood transfusion is a serious problem in Latin America. This paper describes the testing of several naphthoquinones, some of which were active against blood trypomastigotes in vitro at 4 degrees C and might therefore warrant further study for preventing transmission of Chagas disease by blood transfusion.
We report in this paper the presence of fluorescence bands of tryptophan and tyrosine solutions centered above 550 nm. This long-wavelength fluorescence is of much lower intensity, (0.4-2.7)%, than the UV fluorescence of these aromatic aminoacids. The basic characteristic of these fluorescence bands are: (a) tyrosine: lambda em = 600 nm with two excitation peaks centered at 453 nm and 550 nm (b) tryptophan: lambda em = 675 nm with two excitation peaks centered at 455 and 560 nm. It has been found that irradiation of tyrosine solutions with a potent UV lamp promotes an important increase of absorption at 310 nm and above 400 nm.
The changes undergone by pure yeast glutathione reductase during redox interconversion have been studied. Both the active and inactive forms of the enzyme had similar molecular masses, suggesting that the inactivation is probably due to intramolecular modification(s). The glutathione reductase and transhydrogenase activities were similarly inactivated by NADPH and reactivated by GSH, while the diaphorase activity remained unaltered during redox interconversion of glutathione reductase. These results suggest that the inactivation site could be located far from the NADPH-binding site, although interfering with transhydrogenase activity, perhaps by conformational changes. The inactivation of glutathione reductase by 0.2 mM NADPH at pH 8 was paralleled by a gradual decrease in the absorbance at 530 nm and a simultaneous increase in the absorbance at 445 nm, while the reactivation promoted by GSH was initially associated with reversal of these spectral changes. The inactive enzyme spectrum retained some absorbance between 500 nm and 700 nm, showing a shoulder at 580-600 nm. Upon treatment of the enzyme with NADPH at pH 6.5 the spectrum remained unchanged, while no redox inactivation was observed under these conditions. It is suggested that the redox inactivation could be associated with the disappearance of the charge-transfer complex between the proximal thiolate and oxidized FAD in the two-electron-reduced enzyme. The inactive enzyme was reactivated by low GSSG concentrations, moderate dithiol concentrations, and high monothiol concentrations. These results and the spectral changes described above support the hypothesis attributing the redox interconversion to formation/disappearance of an erroneous disulfide between one of the half-cystines located at the GSSG-binding site and another cysteine nearby.
The redox interconversion of Escherichia coli glutathione reductase has been studied both in situ, with permeabilized cells treated with different reductants, and in vivo, with intact cells incubated with compounds known to alter their intracellular redox state. The enzyme from toluene-permeabilized cells was inactivated in situ by NADPH, NADH, dithionite, dithiothreitol, or GSH. The enzyme remained, however, fully active upon incubation with the oxidized forms of such compounds. The inactivation was time-, temperature-, and concentration-dependent; a 50% inactivation was promoted by just 2 microM NADPH, while 700 microM NADH was required for a similar effect. The enzyme from permeabilized cells was completely protected against redox inactivation by GSSG, and to a lesser extent by dithiothreitol, GSH, and NAD(P)+. The inactive enzyme was efficiently reactivated in situ by physiological GSSG concentrations. A significant reactivation was promoted also by GSH, although at concentrations two orders of magnitude below its physiological concentrations. The glutathione reductase from intact E. coli cells was inactivated in vivo by incubation with DL-malate, DL-isocitrate, or higher L-lactate concentrations. The enzyme was protected against redox inactivation and fully reactivated by diamide in a concentration-dependent fashion. Diamide reactivation was not dependent on the synthesis of new protein, thus suggesting that the effect was really a true reactivation and not due to de novo synthesis of active enzyme. The glutathione reductase activity increased significantly after incubation of intact cells with tert-butyl or cumene hydroperoxides, suggesting that the enzyme was partially inactive within such cells. In conclusion, the above results show that both in situ and in vivo the glutathione reductase of Escherichia coli is subjected to a redox interconversion mechanism probably controlled by the intracellular NADPH and GSSG concentrations.
The glutathione reductase from E. coli was rapidly inactivated following aerobic incubation of the pure and cell-free extract enzymes with NADPH, NADH and other reductants. The inactivation of the pure enzyme depended on the time and temperature of incubation (t 1/2 = 2 min at 37 degrees C), and was proportional to the [NADPH]/[enzyme] ratio, reaching 50% in the presence of 0.3 microM NADPH and 45 microM NADH respectively, at a subunit concentration of 20 nM. Higher pyridine nucleotide concentrations were required to inactivate the enzyme from cell-free extracts. Two apparent pKa, corresponding to pH 5.8 and 7.3, were determined for the redox inactivation. The enzyme remained inactive even after eliminating the excess NADPH by gel chromatography. E. coli glutathione reductase was protected by oxidized and reduced glutathione against redox inactivation with both pure and cell-free extract enzymes. Ferricyanide and dithiothreitol protected only the pure enzyme, while NADP+ exclusively protected the cell-free extract enzyme. The inactive glutathione reductase was reactivated by treatment with oxidized and reduced glutathione, ferricyanide, and dithiothreitol in a time-and temperature-dependent process. The oxidized form of glutathione was more efficient and specific than the reduced form in the protection and reactivation of the pure enzyme. The molecular weight of the redox-inactivated E. coli glutathione reductase was similar to that of the dimeric native enzyme, ruling out aggregation as a possible cause of inactivation. A tentative model is discussed for the redox inactivation, involving the formation of an 'erroneous' disulfide bridge at the glutathione-binding site.
Glutathione reductase from Saccharomyces cerevisiae was rapidly inactivated following aerobic incubation with NADPH, NADH, and several other reductants, in a time- and temperature-dependent process. The inactivation had already reached 50% when the NADPH concentration reached that of the glutathione reductase subunit. The inactivation was very marked at pH values below 5.5 and over 7, while only a slight activity decrease was noticed at pH values between these two values. After elimination of excess NADPH the enzyme remained inactive for at least 4 h. The enzyme was protected against redox inactivation by low concentrations of GSSG, ferricyanide, GSH, or dithiothreitol, and high concentrations of NAD(P)+; oxidized glutathione effectively protected the enzyme at concentrations even lower than GSH. The inactive enzyme was efficiently reactivated after incubation with GSSG, ferricyanide, GSH, or dithiothreitol, whether NADPH was present or not. The reactivation with GSH was rapid even at 0 degree C, whereas the optimum temperature for reactivation with GSSG was 30 degrees C. A tentative model for the redox interconversion, involving an erroneous intramolecular disulfide bridge, is put forward.
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This work describes the application of the N-demethylase activity of immobilized soybean lipoxygenase to the oxidative degradation of xenobiotics. Previously (1) we have shown that immobilized lipoxygenase produces the oxidative degradation of CPZ in the presence of hydrogen peroxide. As a continuation of this work, here we studied the N-demethylation of CPZ by the hydroperoxidase activity of covalent immobilized soybean lipoxygenase. The obtained results clearly reveal that the immobilized system produces the N-demethylation of CPZ in the presence of hydrogen peroxide, maintaining a high level of activity in comparison with free enzyme. Additionally, the immobilized lipoxygenase shows stability higher than that of free enzyme, making feasible its use in a bioreactor operating in continuous or discontinuous mode. The results obtained in this work, together with those obtained previously by us for the oxidation of CPZ, suggest that hydroperoxidase activity of immobilized lipoxygenase may constitute a valuable tool for oxidative xenobiotics degradation or for application to synthetic processes in which a N-demethylation reaction is involved.
In this study, lipoxygenase from potato tuber has been purified by a method involving hydrophobic chromatography and the purified enzyme immobilized by covalent coupling to oxirane acrylic beads. The immobilized lipoxygenase exhibited increased long-term stability without a significant modification of the kinetic parameters. The comparative study on the effects of inhibitors such as dithizone, NDGA, phenidone, and beta-mercaptoethanol on the free and immobilized enzyme highlighted the importance of the lipoxygenase--support interaction, concluding that the immobilization process could cause the protection of the iron atom in the enzyme. The enzymatic specificity was maintained for the immobilized lipoxygenase, and their stability increased as compared to the free enzyme, making if feasible to use the enzyme in a multistep reaction to produce large quantities of leukotriene A4 or other related compounds of interest in the chemical industry and medicine.
The glutathione reductase from Escherichia coli strain S33 was purified to homogeneity by a simple and fast procedure consisting of two affinity chromatography steps. After 40-80% ammonium sulfate fractionation, the enzyme was adsorbed to an N6-2'.5'-ADP-Sepharose affinity column from which it was specifically eluted by a 0-10 mM NADP+ linear gradient. The enzyme was finally purified to homogeneity after a second affinity chromatography step in a C8-ATPR-Sepharose column, from which it was eluted by means of the same NADP+ gradient. Starting from 182 g of E. coli cells, 6.9 mg of pure enzyme was obtained after a 2632-fold purification, with a total yield of 63%. The pure enzyme showed a specific activity of 361 U/mg, and its absorption spectrum was characteristic of a flavoprotein, with an A272/A450 of 7.84. The enzyme was a dimer with a molecular weight 109 000 and 40 A hydrodynamic radius. The optimum pH were 7.5 and 4.5 with NADPH and NADH, respectively, as reductants. Apparent K'm values of 16, 377, and 66 microM were determined at pH 7.5 for NADPH, NADH, and GSSG, respectively. Upon storage the enzyme was stable at pH values ranging from 7.5 to 9.5, being additionally stabilized by FAD, NADP+, dithiothreitol, or glycerol. The pure enzyme was quite heat stable, denaturing significantly only after 10 min at 70 degrees C. A marked activity loss was observed however, even at 0 degrees C, in the presence of 20 microM NADPH. The enzyme was inactivated by low concentrations of para-hydroximercuribenzoate; the sensitivity towards such mercurial was greatly enhanced after reduction of the enzyme by NADPH.
Accidental transmission of Chagas' disease to man by blood transfusion is a serious problem in Latin-America. This paper describes the testing of several synthetic, semi-synthetic, and natural compounds for their activity against blood trypomastigotes in vitro at 4 degrees C. The compounds embody several types of chemical structures: benzoquinone, naphthoquinone, anthracenequinone, phenanthrenequinone, imidazole, piperazine, quinoline, xanthene, and simple benzenic and naphthalenic derivatives. Some of them are for the first time tested against Trypanosoma cruzi. The toxic effect of these compounds on this parasite was done by two quite distinct sets of experiments. In one set, the compounds were added to infected blood as ethanolic solution. In this situation the most active one was a furan-1,2-naphthoquinone, in the same range as gentian violet, a new fact to be considered in the assessment of structure-activity relationships in this class of compounds. In other set, we tentatively evaluated the biological activity of water insoluble compounds by adding them in a pure form without solvent into infected blood. In this way some appear to be very active and it was postulated that the effectiveness of such compounds must result from interactions between them and specific blood components.
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BACKGROUND: We investigated the efficacy, safety, and stability of correction achieved with a silicone posterior chamber intraocular lens used to correct high myopia in phakic eyes. PATIENTS AND METHODS: A silicone posterior chamber plate-style intraocular lens (Chiron, Adatomed) was implanted in 38 consecutive phakic eyes with high myopia (-7.00 to -28.00 diopters (D) over a period of 21 months. Follow-up ranged from 3 to 24 months. RESULTS: Three months after surgery, refractions in 27 eyes (71%) ranged from -1.00 to +1.00 D and remained stable thereafter. Spectacle-corrected visual acuity improved at least two lines in 24 eyes (63%), and none was lost in any eye. Patient satisfaction was high (92%). The few complications that occurred were mostly related to imperfections in the surgical technique rather than lack of biocompatibility of the lens. No cataract or other vision-threatening complication was present at the end of the study. CONCLUSION: This silicone posterior chamber plate-style intraocular lens provides a reasonably predictable, safe, and stable means of correcting high myopia.
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