Methylene blue (MB) is known to have trypanocidal activity. We tested the interactions of MB with... more Methylene blue (MB) is known to have trypanocidal activity. We tested the interactions of MB with a number of trypanosomatid-specific molecules of the antioxidant metabolism. At pH 7, trypanothione and other (di)thiols were oxidized to disulfides by the phenothiazine drug. MB inhibited Trypanosoma cruzi trypanothione reductase (TR) (K(i)=1.9 microM), and served as a significant subversive substrate of this enzyme (K(M)=30 microM, k(cat)=4.9s(-1)). With lipoamide dehydrogenase, the second thiol-generating flavoenzyme of T. cruzi, the catalytic efficiency for MB reduction was found to be almost 10(6)M(-1)s(-1). When the system MB-enzyme-molecular oxygen acts as a NAD(P)H-driven redox cycler, a reactive oxygen species, H(2)O(2) or superoxide, is produced in each cycle. Since MB is an affordable, available, and accessible drug it might be tested--alone or in drug combinations--against trypanosomatid-caused diseases of animal and man.
The mosquito, Anopheles gambiae, is an important vector of Plasmodium falciparum malaria. Full ge... more The mosquito, Anopheles gambiae, is an important vector of Plasmodium falciparum malaria. Full genome analysis revealed that, as in Drosophila melanogaster, the enzyme glutathione reductase is absent in A. gambiae and functionally substituted by the thioredoxin system. The key enzyme of this system is thioredoxin reductase-1, a homodimeric FAD-containing protein of 55.3 kDa per subunit, which catalyses the reaction NADPH + H+ + thioredoxin disulfide-->NADP+ + thioredoxin dithiol. The A. gambiae trxr gene is located on chromosome X as a single copy; it represents three splice variants coding for two cytosolic and one mitochondrial variant. The predominant isoform, A. gambiae thioredoxin reductase-1, was recombinantly expressed in Escherichia coli and functionally compared with the wild-type enzyme isolated in a final yield of 1.4 U.ml(-1) of packed insect cells. In redox titrations, the substrate A. gambiae thioredoxin-1 (Km=8.5 microm, kcat=15.4 s(-1) at pH 7.4 and 25 degrees C) was unable to oxidize NADPH-reduced A. gambiae thioredoxin reductase-1 to the fully oxidized state. This indicates that, in contrast to other disulfide reductases, A. gambiae thioredoxin reductase-1 oscillates during catalysis between the four-electron reduced state and a two-electron reduced state. The thioredoxin reductases of the malaria system were compared. A. gambiae thioredoxin reductase-1 shares 52% and 45% sequence identity with its orthologues from humans and P. falciparum, respectively. A major difference among the three enzymes is the structure of the C-terminal redox centre, reflected in the varying resistance of catalytic intermediates to autoxidation. The relevant sequences of this centre are Thr-Cys-Cys-SerOH in A. gambiae thioredoxin reductase, Gly-Cys-selenocysteine-GlyOH in human thioredoxin reductase, and Cys-X-X-X-X-Cys-GlyOH in the P. falciparum enzyme. These differences offer an interesting approach to the design of species-specific inhibitors. Notably, A. gambiae thioredoxin reductase-1 is not a selenoenzyme but instead contains a highly unusual redox-active Cys-Cys sequence.
The quorum sensor and signalling molecule pyocyanin (PYO) contributes significantly to the pathop... more The quorum sensor and signalling molecule pyocyanin (PYO) contributes significantly to the pathophysiology of Pseudomonas aeruginosa infections. Comparison to phenothiazine drugs suggests that the antimalarial compound methylene blue (MB) can be regarded as a sulfur analog of PYO. This working hypothesis would explain why the synthetic drug MB behaves as a compound shaped in biological evolution. Here we report on redox-associated biological and biochemical properties of PYO in direct comparison to its synthetic analog MB. We quantitatively describe the reactivity of both compounds toward cellular reductants, the reactivity of their reduced leuco-forms towards O2, and their interactions with FAD-containing disulfide reductases. Furthermore, the interaction of PYO with human glutathione reductase was studied in structural detail by x-ray crystallography, showing that a single PYO molecule binds to the intersubunit cavity of the enzyme. Like MB, also PYO was also found to be active against blood schizonts of the malaria parasite P. falciparum in vitro. Furthermore, both compounds were active against the disease transmitting gametocyte forms of the parasites, which was systematically studied in vitro. As shown for mice, PYO is too toxic to be used as a drug. It may, however, have antimalarial activity in numerous human patients with concomitant Pseudomonas infections. MB, in contrast to PYO, is well tolerated and represents a promising agent for MB-based combination therapies against malaria. Current and future clinical studies can be guided by the comparisons between MB and PYO reported here. Additionally, it is of interest to study if and to what extent the protection from malaria in patients with cystic fibrosis or with severe wound infections is based on PYO produced by Pseudomonas species.
Kwashiorkor is a severe edematous form of malnutrition with high prevalence and lethality in many... more Kwashiorkor is a severe edematous form of malnutrition with high prevalence and lethality in many African countries, and repeatedly has been reported to be associated with oxidative stress. The therapy of kwashiorkor is still ineffective. In this pilot study, we tested the hypothesis that oral application of thiol-containing antioxidants increases glutathione status and is beneficial for the clinical recovery of kwashiorkor patients. The longitudinal clinical intervention study was carried out at St Joseph's Hospital, Jirapa, Ghana. Children with severe kwashiorkor were randomly assigned to either a standard treatment (ST) receiving a therapeutic protocol based on the recommendations of the WHO or to one of three study groups receiving in addition 2 x 600 mg reduced glutathione or 2 x 50 mg alpha-lipoic acid or 2 x 100 mg N-acetylcysteine per day. Patients were followed up clinically and biochemically for 20 days and compared with 37 healthy controls. Both glutathione and alpha-lipoic acid supplementation had positive effects on survival. Also, the blood glutathione concentrations correlated positively with survival rates. Furthermore, the initial skin lesions, glutathione and total protein concentrations were found to be strong predictors of survival. The data strongly suggest that a therapy restoring the antioxidative capacity by applying cysteine equivalents in the form of glutathione and/or alpha-lipoic acid is beneficial for biochemical and clinical recovery of kwashiorkor patients.
Proceedings of the National Academy of Sciences, 2003
Selenium, an essential trace element for mammals, is incorporated into a selected class of seleno... more Selenium, an essential trace element for mammals, is incorporated into a selected class of selenoproteins as selenocysteine. All known isoenzymes of mammalian thioredoxin (Trx) reductases (TrxRs) employ selenium in the C-terminal redox center -Gly-Cys-Sec-Gly-COOH for reduction of Trx and other substrates, whereas the corresponding sequence in Drosophila melanogaster TrxR is -Ser-Cys-Cys-Ser-COOH. Surprisingly, the catalytic competence of these orthologous enzymes is similar, whereas direct Sec-to-Cys substitution of mammalian TrxR, or other selenoenzymes, yields almost inactive enzyme. TrxRs are therefore ideal for studying the biology of selenocysteine by comparative enzymology. Here we show that the serine residues flanking the C-terminal Cys residues of Drosophila TrxRs are responsible for activating the cysteines to match the catalytic efficiency of a selenocysteine-cysteine pair as in mammalian TrxR, obviating the need for selenium. This finding suggests that the occurrence of selenoenzymes, which implies that the organism is selenium-dependent, is not necessarily associated with improved enzyme efficiency. Our data suggest that the selective advantage of selenoenzymes is a broader range of substrates and a broader range of microenvironmental conditions in which enzyme activity is possible.
Proceedings of the National Academy of Sciences, 1997
Thioredoxin reductase, lipoamide dehydrogenase, and glutathione reductase are members of the pyri... more Thioredoxin reductase, lipoamide dehydrogenase, and glutathione reductase are members of the pyridine nucleotide-disulfide oxidoreductase family of dimeric flavoenzymes. The mechanisms and structures of lipoamide dehydrogenase and glutathione reductase are alike irrespective of the source (subunit M(r) approximately 55,000). Although the mechanism and structure of thioredoxin reductase from Escherichia coli are distinct (M(r) approximately 35,000), this enzyme must be placed in the same family because there are significant amino acid sequence similarities with the other two enzymes, the presence of a redox-active disulfide, and the substrate specificities. Thioredoxin reductase from higher eukaryotes on the other hand has a M(r) of approximately 55,000 [Luthman, M. & Holmgren, A. (1982) Biochemistry 21, 6628-6633; Gasdaska, P. Y., Gasdaska, J. R., Cochran, S. & Powis, G. (1995) FEBS Lett 373, 5-9; Gladyshev, V. N., Jeang, K. T. & Stadtman, T.C. (1996) Proc. Natl. Acad. Sci. USA 93, 6146-6151]. Thus, the evolution of this family is highly unusual. The mechanism of thioredoxin reductase from higher eukaryotes is not known. As reported here, thioredoxin reductase from human placenta reacts with only a single molecule of NADPH, which leads to a stable intermediate similar to that observed in titrations of lipoamide dehydrogenase or glutathione reductase. Titration of thioredoxin reductase from human placenta with dithionite takes place in two spectral phases: formation of a thiolate-flavin charge transfer complex followed by reduction of the flavin, just as with lipoamide dehydrogenase or glutathione reductase. The first phase requires more than one equivalent of dithionite. This suggests that the penultimate selenocysteine [Tamura, T. & Stadtman, T.C. (1996) Proc. Natl. Acad. Sci. USA 93, 1006-1011] is in redox communication with the active site disulfide/dithiol. Nitrosoureas of the carmustine type inhibit only the NADPH reduced form of human thioredoxin reductase. These compounds are widely used as cytostatic agents, so this enzyme should be studied as a target in cancer chemotherapy. In conclusion, three lines of evidence indicate that the mechanism of human thioredoxin reductase is like the mechanisms of lipoamide dehydrogenase and glutathione reductase and differs fundamentally from the mechanism of E. coli thioredoxin reductase.
The pathophysiology of kwashiorkor, a severe edematous manifestation of malnutrition, is still po... more The pathophysiology of kwashiorkor, a severe edematous manifestation of malnutrition, is still poorly understood. The syndrome is, however, known to be associated with alterations in redox metabolism. To further elucidate the role of oxidative stress in kwashiorkor, we carried out a longitudinal study on the major blood antioxidants at the St. Joseph's Hospital, Jirapa, Ghana. All kwashiorkor patients (K) were followed up for 20 d. In comparison with local healthy controls (C), the plasma total antioxidant status was reduced to less than 50% in the patients (C, 0.87 +/- 0.21 mM; K, 0.40 +/- 0.20 mM; p<0.001). Similarly, the major plasma antioxidant albumin (C, 40.9 +/- 2.5 g/L; K, 19.1 +/- 7.4 g/L; p < 0.001) and erythrocyte glutathione (C, 2.39 +/- 0.28 mM; K, 1.01 +/- 0.33; p < 0.001) were decreased, whereas the levels of bilirubin and uric acid were not significantly altered. Nitrite and nitrate were found to be increased by a factor of 2 in kwashiorkor (C, 120 +/- 46 microM; K, 235 +/- 107 microM; p < 0.001). Over the observation period, the trends of albumin and glutathione levels were related to clinical outcome. These concentrations rose in patients who recovered and fell in patients who did not. Our study strongly supports the hypothesis that oxidative and nitrosative stress play a role in the pathophysiology of edematous malnutrition. Prophylactic and therapeutic strategies should aim at the careful correction of the reduced antioxidant status of the patients.
The use of selenocysteine (Sec) as the 21st amino acid in the genetic code has been described in ... more The use of selenocysteine (Sec) as the 21st amino acid in the genetic code has been described in all three major domains of life. However, within eukaryotes, selenoproteins are only known in animals and algae. In this study, we characterized selenoproteomes and Sec insertion systems in protozoan Apicomplexa parasites. We found that among these organisms, Plasmodium and Toxoplasma utilized Sec, whereas Cryptosporidium did not. However, Plasmodium had no homologs of known selenoproteins. By searching computationally for evolutionarily conserved selenocysteine insertion sequence (SECIS) elements, which are RNA structures involved in Sec insertion, we identified four unique Plasmodium falciparum selenoprotein genes. These selenoproteins were incorrectly annotated in PlasmoDB, were conserved in other Plasmodia and had no detectable homologs in other species. We provide evidence that two Plasmodium SECIS elements supported Sec insertion into parasite and endogenous selenoproteins when they were expressed in mammalian cells, demonstrating that the Plasmodium SECIS elements are functional and indicating conservation of Sec insertion between Apicomplexa and animals. Dependence of the plasmodial parasites on selenium suggests possible strategies for antimalarial drug development.
Methylene blue (MB) is known to have trypanocidal activity. We tested the interactions of MB with... more Methylene blue (MB) is known to have trypanocidal activity. We tested the interactions of MB with a number of trypanosomatid-specific molecules of the antioxidant metabolism. At pH 7, trypanothione and other (di)thiols were oxidized to disulfides by the phenothiazine drug. MB inhibited Trypanosoma cruzi trypanothione reductase (TR) (K(i)=1.9 microM), and served as a significant subversive substrate of this enzyme (K(M)=30 microM, k(cat)=4.9s(-1)). With lipoamide dehydrogenase, the second thiol-generating flavoenzyme of T. cruzi, the catalytic efficiency for MB reduction was found to be almost 10(6)M(-1)s(-1). When the system MB-enzyme-molecular oxygen acts as a NAD(P)H-driven redox cycler, a reactive oxygen species, H(2)O(2) or superoxide, is produced in each cycle. Since MB is an affordable, available, and accessible drug it might be tested--alone or in drug combinations--against trypanosomatid-caused diseases of animal and man.
Human thioredoxin reductase is a pyridine nucleotide-disulfide oxidoreductase closely related to ... more Human thioredoxin reductase is a pyridine nucleotide-disulfide oxidoreductase closely related to glutathione reductase but differing from the latter in having a Cys-SeCys (selenocysteine) sequence as an additional redox center. Because selenoproteins cannot be expressed yet in heterologous systems, we optimized the purification of the protein from placenta with respect to final yield (1-2 mg from one placenta), specific activity (42 units/mg), and selenium content (0.94 +/- 0.03 mol/mol subunit). The steady state kinetics showed that the enzyme operates by a ping-pong mechanism; the value of kcat was 3330 +/- 882 min-1, and the Km values were 18 microM for NADPH and 25 microM for Escherichia coli thioredoxin. The activation energy of the reaction was found to be 53.2 kJ/mol, which allows comparisons of the steady state data with previous pre-steady state measurements. In its physiological, NADPH-reduced form, the enzyme is strongly inhibited by organic gold compounds that are widely used in the treatment of rheumatoid arthritis; for auranofin, the Ki was 4 nM when measured in the presence of 50 microM thioredoxin. At 1000-fold higher concentrations, that is at micromolar levels, the drugs also inhibited human glutathione reductase and the selenoenzyme glutathione peroxidase.
Methylene blue (MB) is known to have trypanocidal activity. We tested the interactions of MB with... more Methylene blue (MB) is known to have trypanocidal activity. We tested the interactions of MB with a number of trypanosomatid-specific molecules of the antioxidant metabolism. At pH 7, trypanothione and other (di)thiols were oxidized to disulfides by the phenothiazine drug. MB inhibited Trypanosoma cruzi trypanothione reductase (TR) (K(i)=1.9 microM), and served as a significant subversive substrate of this enzyme (K(M)=30 microM, k(cat)=4.9s(-1)). With lipoamide dehydrogenase, the second thiol-generating flavoenzyme of T. cruzi, the catalytic efficiency for MB reduction was found to be almost 10(6)M(-1)s(-1). When the system MB-enzyme-molecular oxygen acts as a NAD(P)H-driven redox cycler, a reactive oxygen species, H(2)O(2) or superoxide, is produced in each cycle. Since MB is an affordable, available, and accessible drug it might be tested--alone or in drug combinations--against trypanosomatid-caused diseases of animal and man.
The mosquito, Anopheles gambiae, is an important vector of Plasmodium falciparum malaria. Full ge... more The mosquito, Anopheles gambiae, is an important vector of Plasmodium falciparum malaria. Full genome analysis revealed that, as in Drosophila melanogaster, the enzyme glutathione reductase is absent in A. gambiae and functionally substituted by the thioredoxin system. The key enzyme of this system is thioredoxin reductase-1, a homodimeric FAD-containing protein of 55.3 kDa per subunit, which catalyses the reaction NADPH + H+ + thioredoxin disulfide-->NADP+ + thioredoxin dithiol. The A. gambiae trxr gene is located on chromosome X as a single copy; it represents three splice variants coding for two cytosolic and one mitochondrial variant. The predominant isoform, A. gambiae thioredoxin reductase-1, was recombinantly expressed in Escherichia coli and functionally compared with the wild-type enzyme isolated in a final yield of 1.4 U.ml(-1) of packed insect cells. In redox titrations, the substrate A. gambiae thioredoxin-1 (Km=8.5 microm, kcat=15.4 s(-1) at pH 7.4 and 25 degrees C) was unable to oxidize NADPH-reduced A. gambiae thioredoxin reductase-1 to the fully oxidized state. This indicates that, in contrast to other disulfide reductases, A. gambiae thioredoxin reductase-1 oscillates during catalysis between the four-electron reduced state and a two-electron reduced state. The thioredoxin reductases of the malaria system were compared. A. gambiae thioredoxin reductase-1 shares 52% and 45% sequence identity with its orthologues from humans and P. falciparum, respectively. A major difference among the three enzymes is the structure of the C-terminal redox centre, reflected in the varying resistance of catalytic intermediates to autoxidation. The relevant sequences of this centre are Thr-Cys-Cys-SerOH in A. gambiae thioredoxin reductase, Gly-Cys-selenocysteine-GlyOH in human thioredoxin reductase, and Cys-X-X-X-X-Cys-GlyOH in the P. falciparum enzyme. These differences offer an interesting approach to the design of species-specific inhibitors. Notably, A. gambiae thioredoxin reductase-1 is not a selenoenzyme but instead contains a highly unusual redox-active Cys-Cys sequence.
The quorum sensor and signalling molecule pyocyanin (PYO) contributes significantly to the pathop... more The quorum sensor and signalling molecule pyocyanin (PYO) contributes significantly to the pathophysiology of Pseudomonas aeruginosa infections. Comparison to phenothiazine drugs suggests that the antimalarial compound methylene blue (MB) can be regarded as a sulfur analog of PYO. This working hypothesis would explain why the synthetic drug MB behaves as a compound shaped in biological evolution. Here we report on redox-associated biological and biochemical properties of PYO in direct comparison to its synthetic analog MB. We quantitatively describe the reactivity of both compounds toward cellular reductants, the reactivity of their reduced leuco-forms towards O2, and their interactions with FAD-containing disulfide reductases. Furthermore, the interaction of PYO with human glutathione reductase was studied in structural detail by x-ray crystallography, showing that a single PYO molecule binds to the intersubunit cavity of the enzyme. Like MB, also PYO was also found to be active against blood schizonts of the malaria parasite P. falciparum in vitro. Furthermore, both compounds were active against the disease transmitting gametocyte forms of the parasites, which was systematically studied in vitro. As shown for mice, PYO is too toxic to be used as a drug. It may, however, have antimalarial activity in numerous human patients with concomitant Pseudomonas infections. MB, in contrast to PYO, is well tolerated and represents a promising agent for MB-based combination therapies against malaria. Current and future clinical studies can be guided by the comparisons between MB and PYO reported here. Additionally, it is of interest to study if and to what extent the protection from malaria in patients with cystic fibrosis or with severe wound infections is based on PYO produced by Pseudomonas species.
Kwashiorkor is a severe edematous form of malnutrition with high prevalence and lethality in many... more Kwashiorkor is a severe edematous form of malnutrition with high prevalence and lethality in many African countries, and repeatedly has been reported to be associated with oxidative stress. The therapy of kwashiorkor is still ineffective. In this pilot study, we tested the hypothesis that oral application of thiol-containing antioxidants increases glutathione status and is beneficial for the clinical recovery of kwashiorkor patients. The longitudinal clinical intervention study was carried out at St Joseph's Hospital, Jirapa, Ghana. Children with severe kwashiorkor were randomly assigned to either a standard treatment (ST) receiving a therapeutic protocol based on the recommendations of the WHO or to one of three study groups receiving in addition 2 x 600 mg reduced glutathione or 2 x 50 mg alpha-lipoic acid or 2 x 100 mg N-acetylcysteine per day. Patients were followed up clinically and biochemically for 20 days and compared with 37 healthy controls. Both glutathione and alpha-lipoic acid supplementation had positive effects on survival. Also, the blood glutathione concentrations correlated positively with survival rates. Furthermore, the initial skin lesions, glutathione and total protein concentrations were found to be strong predictors of survival. The data strongly suggest that a therapy restoring the antioxidative capacity by applying cysteine equivalents in the form of glutathione and/or alpha-lipoic acid is beneficial for biochemical and clinical recovery of kwashiorkor patients.
Proceedings of the National Academy of Sciences, 2003
Selenium, an essential trace element for mammals, is incorporated into a selected class of seleno... more Selenium, an essential trace element for mammals, is incorporated into a selected class of selenoproteins as selenocysteine. All known isoenzymes of mammalian thioredoxin (Trx) reductases (TrxRs) employ selenium in the C-terminal redox center -Gly-Cys-Sec-Gly-COOH for reduction of Trx and other substrates, whereas the corresponding sequence in Drosophila melanogaster TrxR is -Ser-Cys-Cys-Ser-COOH. Surprisingly, the catalytic competence of these orthologous enzymes is similar, whereas direct Sec-to-Cys substitution of mammalian TrxR, or other selenoenzymes, yields almost inactive enzyme. TrxRs are therefore ideal for studying the biology of selenocysteine by comparative enzymology. Here we show that the serine residues flanking the C-terminal Cys residues of Drosophila TrxRs are responsible for activating the cysteines to match the catalytic efficiency of a selenocysteine-cysteine pair as in mammalian TrxR, obviating the need for selenium. This finding suggests that the occurrence of selenoenzymes, which implies that the organism is selenium-dependent, is not necessarily associated with improved enzyme efficiency. Our data suggest that the selective advantage of selenoenzymes is a broader range of substrates and a broader range of microenvironmental conditions in which enzyme activity is possible.
Proceedings of the National Academy of Sciences, 1997
Thioredoxin reductase, lipoamide dehydrogenase, and glutathione reductase are members of the pyri... more Thioredoxin reductase, lipoamide dehydrogenase, and glutathione reductase are members of the pyridine nucleotide-disulfide oxidoreductase family of dimeric flavoenzymes. The mechanisms and structures of lipoamide dehydrogenase and glutathione reductase are alike irrespective of the source (subunit M(r) approximately 55,000). Although the mechanism and structure of thioredoxin reductase from Escherichia coli are distinct (M(r) approximately 35,000), this enzyme must be placed in the same family because there are significant amino acid sequence similarities with the other two enzymes, the presence of a redox-active disulfide, and the substrate specificities. Thioredoxin reductase from higher eukaryotes on the other hand has a M(r) of approximately 55,000 [Luthman, M. & Holmgren, A. (1982) Biochemistry 21, 6628-6633; Gasdaska, P. Y., Gasdaska, J. R., Cochran, S. & Powis, G. (1995) FEBS Lett 373, 5-9; Gladyshev, V. N., Jeang, K. T. & Stadtman, T.C. (1996) Proc. Natl. Acad. Sci. USA 93, 6146-6151]. Thus, the evolution of this family is highly unusual. The mechanism of thioredoxin reductase from higher eukaryotes is not known. As reported here, thioredoxin reductase from human placenta reacts with only a single molecule of NADPH, which leads to a stable intermediate similar to that observed in titrations of lipoamide dehydrogenase or glutathione reductase. Titration of thioredoxin reductase from human placenta with dithionite takes place in two spectral phases: formation of a thiolate-flavin charge transfer complex followed by reduction of the flavin, just as with lipoamide dehydrogenase or glutathione reductase. The first phase requires more than one equivalent of dithionite. This suggests that the penultimate selenocysteine [Tamura, T. & Stadtman, T.C. (1996) Proc. Natl. Acad. Sci. USA 93, 1006-1011] is in redox communication with the active site disulfide/dithiol. Nitrosoureas of the carmustine type inhibit only the NADPH reduced form of human thioredoxin reductase. These compounds are widely used as cytostatic agents, so this enzyme should be studied as a target in cancer chemotherapy. In conclusion, three lines of evidence indicate that the mechanism of human thioredoxin reductase is like the mechanisms of lipoamide dehydrogenase and glutathione reductase and differs fundamentally from the mechanism of E. coli thioredoxin reductase.
The pathophysiology of kwashiorkor, a severe edematous manifestation of malnutrition, is still po... more The pathophysiology of kwashiorkor, a severe edematous manifestation of malnutrition, is still poorly understood. The syndrome is, however, known to be associated with alterations in redox metabolism. To further elucidate the role of oxidative stress in kwashiorkor, we carried out a longitudinal study on the major blood antioxidants at the St. Joseph's Hospital, Jirapa, Ghana. All kwashiorkor patients (K) were followed up for 20 d. In comparison with local healthy controls (C), the plasma total antioxidant status was reduced to less than 50% in the patients (C, 0.87 +/- 0.21 mM; K, 0.40 +/- 0.20 mM; p<0.001). Similarly, the major plasma antioxidant albumin (C, 40.9 +/- 2.5 g/L; K, 19.1 +/- 7.4 g/L; p < 0.001) and erythrocyte glutathione (C, 2.39 +/- 0.28 mM; K, 1.01 +/- 0.33; p < 0.001) were decreased, whereas the levels of bilirubin and uric acid were not significantly altered. Nitrite and nitrate were found to be increased by a factor of 2 in kwashiorkor (C, 120 +/- 46 microM; K, 235 +/- 107 microM; p < 0.001). Over the observation period, the trends of albumin and glutathione levels were related to clinical outcome. These concentrations rose in patients who recovered and fell in patients who did not. Our study strongly supports the hypothesis that oxidative and nitrosative stress play a role in the pathophysiology of edematous malnutrition. Prophylactic and therapeutic strategies should aim at the careful correction of the reduced antioxidant status of the patients.
The use of selenocysteine (Sec) as the 21st amino acid in the genetic code has been described in ... more The use of selenocysteine (Sec) as the 21st amino acid in the genetic code has been described in all three major domains of life. However, within eukaryotes, selenoproteins are only known in animals and algae. In this study, we characterized selenoproteomes and Sec insertion systems in protozoan Apicomplexa parasites. We found that among these organisms, Plasmodium and Toxoplasma utilized Sec, whereas Cryptosporidium did not. However, Plasmodium had no homologs of known selenoproteins. By searching computationally for evolutionarily conserved selenocysteine insertion sequence (SECIS) elements, which are RNA structures involved in Sec insertion, we identified four unique Plasmodium falciparum selenoprotein genes. These selenoproteins were incorrectly annotated in PlasmoDB, were conserved in other Plasmodia and had no detectable homologs in other species. We provide evidence that two Plasmodium SECIS elements supported Sec insertion into parasite and endogenous selenoproteins when they were expressed in mammalian cells, demonstrating that the Plasmodium SECIS elements are functional and indicating conservation of Sec insertion between Apicomplexa and animals. Dependence of the plasmodial parasites on selenium suggests possible strategies for antimalarial drug development.
Methylene blue (MB) is known to have trypanocidal activity. We tested the interactions of MB with... more Methylene blue (MB) is known to have trypanocidal activity. We tested the interactions of MB with a number of trypanosomatid-specific molecules of the antioxidant metabolism. At pH 7, trypanothione and other (di)thiols were oxidized to disulfides by the phenothiazine drug. MB inhibited Trypanosoma cruzi trypanothione reductase (TR) (K(i)=1.9 microM), and served as a significant subversive substrate of this enzyme (K(M)=30 microM, k(cat)=4.9s(-1)). With lipoamide dehydrogenase, the second thiol-generating flavoenzyme of T. cruzi, the catalytic efficiency for MB reduction was found to be almost 10(6)M(-1)s(-1). When the system MB-enzyme-molecular oxygen acts as a NAD(P)H-driven redox cycler, a reactive oxygen species, H(2)O(2) or superoxide, is produced in each cycle. Since MB is an affordable, available, and accessible drug it might be tested--alone or in drug combinations--against trypanosomatid-caused diseases of animal and man.
Human thioredoxin reductase is a pyridine nucleotide-disulfide oxidoreductase closely related to ... more Human thioredoxin reductase is a pyridine nucleotide-disulfide oxidoreductase closely related to glutathione reductase but differing from the latter in having a Cys-SeCys (selenocysteine) sequence as an additional redox center. Because selenoproteins cannot be expressed yet in heterologous systems, we optimized the purification of the protein from placenta with respect to final yield (1-2 mg from one placenta), specific activity (42 units/mg), and selenium content (0.94 +/- 0.03 mol/mol subunit). The steady state kinetics showed that the enzyme operates by a ping-pong mechanism; the value of kcat was 3330 +/- 882 min-1, and the Km values were 18 microM for NADPH and 25 microM for Escherichia coli thioredoxin. The activation energy of the reaction was found to be 53.2 kJ/mol, which allows comparisons of the steady state data with previous pre-steady state measurements. In its physiological, NADPH-reduced form, the enzyme is strongly inhibited by organic gold compounds that are widely used in the treatment of rheumatoid arthritis; for auranofin, the Ki was 4 nM when measured in the presence of 50 microM thioredoxin. At 1000-fold higher concentrations, that is at micromolar levels, the drugs also inhibited human glutathione reductase and the selenoenzyme glutathione peroxidase.
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