RESUMO
Macrophage migration inhibitory factor (MIF) is a cytokine associated with tissue damage in multiple autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis and psoriatic arthritis. The role of MIF in multiple sclerosis (MS) and the contribution of its polymorphisms are unknown in our population. Therefore, we decided to investigate the genetic association of -794 CATT5-8 (rs5844572) and -173 G>C (rs755622) MIF polymorphisms with MS, clinical variables and MIF serum levels in the population of western Mexico. 230 MS patients diagnosed according to McDonald criteria and 248 control subjects (CS) were recruited for this study, both polymorphisms were genotyped by PCR and PCR-RFLP and MIF serum levels were measured by ELISA kit. Severity and progression of MS were evaluated by EDSS and MSSS scores, respectively. Genotypes carrying the 5 repeats alleles of -794 CATT5-8MIF polymorphism present higher MIF serum levels in comparison with no carriers, and the presence of 5,7 heterozygous genotype contribute to the increase of disease severity and damage progression in MS patients. Notably when we stratified by sex, an effect of risk alleles (7 repeats and -173*C) of both MIF polymorphisms on EDSS and MSSS scores on males was found (pâ¯<â¯0.01). This study suggests that polymorphic alleles of MIF polymorphisms could act as sex-specific disease modifiers that increase the severity and progression of MS in male Mexican-Mestizo western population.
Assuntos
Predisposição Genética para Doença/genética , Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/genética , Esclerose Múltipla/genética , Caracteres Sexuais , Adulto , Progressão da Doença , Feminino , Genótipo , Humanos , Masculino , México , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BACKGROUND: Low cognitive performance has been associated with a wide array of adverse health-related outcomes in elderly populations. Recently, the effect of vitamin D on cognition has been studied; however, its benefits are still controversial. Moreover, most studies have been carried out on North-American and European populations where vitamin D deficiency could represent a greater public-health issue when compared to Latin American ones. OBJECTIVE: To investigate the association between 25-OH-vitamin D and cognitive performance in Mexican community-dwelling elderly. DESIGN, SETTING AND PARTICIPANTS: Cross-sectional study sample of 331 community-dwelling elderly aged 70 and older, participating in the Mexican Study of Nutritional and Psychosocial Markers of Frailty. MEASUREMENTS: Serum 25-OH-vitamin D, cognitive performance as per the Mini-Mental State Examination (MMSE) and the IST (Isaacs Set Test), as well as several elements from the comprehensive geriatric assessment. RESULTS: Mean age of participants was 79.3 years (SD 5.9), 54.1% were women. The mean serum 25-OH-vitamin D level was 59.0 (SD 23.3) nmol/L while mean MMSE score was 22.3 (SD 3.4) and mean IST score was 37.1 (SD 9.1). Although 25-OH-vitamin D levels were lower across all the definitions of low cognitive perfomance, the difference between groups was not statistically significant in any of them. CONCLUSION: No association between 25-OH-vitamin D level and cognitive performance was found in this population of Mexican community-dwelling elderly. Further investigation is required in order to clarify its existence and if so, to delineate its characteristics.
RESUMO
Tumor necrosis factor-alpha (TNF-alpha) plays a central role in inflammation, and it has been directly implicated in the pathogenesis of rheumatoid arthritis (RA). TNF-alpha activity is mediated through TNFRI and TNFRII cell surface receptors, which act as physiological attenuators of TNF-alpha activity. We recruited 190 RA patients and 190 healthy subjects (HS) in order to associate the -383A>C TNFRI polymorphism with sTNFRI levels and DAS28 score in RA. In results, sTNFRI levels were higher in RA patients than HS (P = 0.04). The -383A>C TNFRI polymorphism did not show significant differences in both studied groups. However, in the RA group the sTNFRI levels were significantly elevated (P = 0.004) in A/A genotype carriers. In addition, the A/A genotype carriers had the higher DAS28 score than A/C genotype (P = 0.02). These data suggest that -383A>C TNFRI polymorphism is not a susceptibility marker in RA, whereas the increased levels of sTNFRI could reflect the clinical activity in RA patients.
Assuntos
Artrite Reumatoide/genética , Polimorfismo Genético , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/etnologia , Artrite Reumatoide/imunologia , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Masculino , México/epidemiologia , Pessoa de Meia-Idade , Fenótipo , Receptores Tipo I de Fatores de Necrose Tumoral/sangue , Medição de Risco , Fatores de Risco , Índice de Gravidade de Doença , Adulto JovemRESUMO
OBJECTIVE: To measure levels of soluble tumour necrosis factor alpha (TNFalpha) receptor type I (sTNFRI) and type II (sTNFRII) in order to correlate them with C-reactive protein (CRP), rheumatoid factor (RF), erythrocyte sedimentation rate (ESR), and disease activity score (DAS28) in RA patients. METHODS: We recruited 41 RA patients classified according to American College of Rheumatology (ACR) criteria and 38 healthy subjects (HS). sTNFRI and sTNFRII were measured using an enzyme-linked immunosorbent assay (ELISA) kit. Clinical activity in RA patients was evaluated using the Disease Activity Score using 28 joint counts (DAS28). The statistical analysis was realized using SPSS version 10.0. RESULTS: Soluble TNFRI and TNFRII levels were higher in RA patients (p = 0.04 and 0.001, respectively) than HS. Serum levels of sTNFRI correlated with sTNFRII (r = 0.699, p < 0.0001). sTNFRII correlated with DAS28 (r = 0.375, p = 0.017), RF (r = 0.505, p = 0.004), and ESR (r = 0.323, p = 0.042). CONCLUSION: The increased levels of both sTNFRI and sTNFRII suggest a secondary event related to the inflammatory state observed in RA, whereas the correlation of sTNFRII with RF, ESR, and DAS28 reflects the preferential TNFRII shedding induced by TNFalpha. sTNFRII may be useful as an additional inflammatory marker in RA.
Assuntos
Artrite Reumatoide/sangue , Receptores Tipo II do Fator de Necrose Tumoral/sangue , Receptores Tipo I de Fatores de Necrose Tumoral/sangue , Índice de Gravidade de Doença , Adulto , Biomarcadores/sangue , Sedimentação Sanguínea , Proteína C-Reativa/metabolismo , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fator Reumatoide/sangueRESUMO
En nuestro país la tuberculosis es aún una enfermedad muy prevalente. La tuberculosis puede comprometer cualquier órgano, uno de ellos es la piel. Este compromiso dérmico, se puede evidenciar por la formación de inmunocomplejos que se depositan en la piel y se manifiestan como vasculitis leucocitoclástica. Se presenta el caso de un paciente de 34 años, con un tiempo de enfermedad de cuatro meses, cuyas manifestaciones clínicas iniciales fueron púrpura palpable en miembros inferiores y poliartralgias, quien recibió antinflamatorios no esteroideos durante un mes, presentando leve mejoría de dichas lesiones; luego de un periodo subclínico reinicia sintomatología con fiebre, astenia y dolor torácico. Se evidenció derrame pleural izquierdo, el cual dio como resultado un exudado mononuclear y adenosin deaminasa elevada. En la biopsia pleural se observó granulomas y se evidenció BK positivo. En la biopsia de piel se evidenció una vasculitis leucocitoclástica. Recibió tratamiento específico esquema I, con evolución clínica favorable a los diez días.
In our country tuberculosis is still a very prevalent disease. Tuberculosis can affect any organ, included the skin. This cutaneous disorder can be evidenced by the formation of immunocomplex that are deposited at the skin and manifested as leukocytoclastic vasculitis. We describe the case of a 34 years old patient, with an illness time of four months, whose initial clinical manifestations were palpable purpura on the lower limbs and polyarthralgia, received non-steroidalanti inflammatory drugs for a month, showing slight improvement from such injury; after a subclinical period its symptoms restarts with fever, fatigue and pain chest. There was evidence of left pleural effusion, which resulted in an mononuclear exudate and elevationa of adenosine deaminase. Pleural biopsy showed granulomas and positive BK. Skin biopsy showed leukocytoclastic vasculitis. The patient received first line antituberculosis drugs with favorable clinical outcome after ten days.
Assuntos
Humanos , Masculino , Adulto , Tuberculose Pleural , Vasculite Leucocitoclástica CutâneaRESUMO
BACKGROUND: We describe a family with a 7-year-old proband case diagnosed with systemic lupus erythematosus (SLE) plus secondary anti-phospholipid syndrome (APS) as well as two affected paternal aunts. We compared the frequency of these polymorphisms with healthy controls. OBJECTIVES: To evaluate the mode of inheritance in this familial case of APS and SLE and the possible association of plasminogen activator inhibitor-1 (PAI-1) -675 4G/5G and PAI-2 Ser(413)/Cys polymorphisms. To compare the genotype frequency of these polymorphisms with the results found in a Mexican Mestizo population. METHODS: PAI-1 -675 4G/5G and PAI-2 Ser(413)/Cys were determined by the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technique using Bsl I and Mwo I on four generations of the family studied. PAI-2 Ser(413)/Cys polymorphism was also determined in 50 healthy individuals of Mexican Mestizo origin. RESULTS: The family pedigree demonstrated that this family did not follow a Mendelian inheritance pattern. When the PAI-2 Ser(413)/Cys polymorphism was examined, we found that 60% (3/5) of the relatives homozygous to Ser(413)/Ser were affected with SLE and/or APS (p = 0.027). The proband case was 4G/5G genotype for the PAI-1 -675 4G/5G polymorphism. No differences between healthy controls of the Mexican Mestizo population and the family studied for the PAI-2 Ser(413)/Cys polymorphism or PAI-1 -675 4G/5G polymorphisms were found. CONCLUSIONS: Our data indicate that this family did not follow the Mendelian inheritance pattern. The Ser(413)/Ser genotype demonstrated in 60% of the affected members (3/5) of this family might increase the risk for autoimmune syndromes such as APS or SLE.
Assuntos
Síndrome Antifosfolipídica/genética , Lúpus Eritematoso Sistêmico/genética , Inibidor 2 de Ativador de Plasminogênio/genética , Polimorfismo Genético , Criança , Feminino , Genótipo , Humanos , Masculino , LinhagemRESUMO
AIMS: To achieve reliable detection of methicillin resistance in clinical isolates of coagulase-negative staphylococci. METHODS AND RESULTS: Strains (105) were evaluated by normatized antimicrobial susceptibility methods, and for the presence of the methicillin resistance-determining mecA gene, using the polymerase chain reaction. Correlation between phenotypic and genotypic methods was obtained in 87.6% of the samples. Six strains, classified as methicillin-susceptible by phenotypic assays, revealed the presence of the mecA gene, indicating that methicillin resistance expression was probably repressed. Another seven isolates failed to show mecA amplification after displaying methicillin resistance in phenotypic evaluations. The susceptibility of the methicillin-resistant isolates to other antimicrobial agents was variable. CONCLUSION: Genotypic determination of the mecA gene proved to be the most reliable method for detection of methicillin resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: Correct assessment of methicillin resistance, such as that attained through genotyping, is essential for defining therapeutic strategies, particularly when treating severely compromised patients.
Assuntos
Proteínas de Bactérias , Coagulase/metabolismo , Hexosiltransferases , Resistência a Meticilina/genética , Meticilina/farmacologia , Peptidil Transferases , Staphylococcus/efeitos dos fármacos , Staphylococcus/genética , Proteínas de Transporte/genética , Genes Bacterianos/genética , Genótipo , Meticilina/uso terapêutico , Testes de Sensibilidade Microbiana , Muramilpentapeptídeo Carboxipeptidase/genética , Proteínas de Ligação às Penicilinas , Fenótipo , Reação em Cadeia da Polimerase , Staphylococcus/classificação , Staphylococcus/enzimologiaRESUMO
The stability of chloroplastic glutamine synthetase (GS; EC 6.3.1.2) was investigated under photooxidative stress using wheat (Triticum aestivum L.) leaves, chloroplasts, and chloroplast lysates. Illuminated seedlings sprayed with the superoxide radical (O-(2)) propagator methyl viologen showed rapid GS decline dependent on MV concentration and exposure time. Degradation products of approximately 39 and 31 kD were detected when chloroplast lysates containing both stroma and thylakoids were illuminated in the presence of MV or H(2)O(2). In all cases, GS cleavage was prevented by the addition of the electron transport inhibitor 3-(3, 4-dichlorophenyl)-1,1-dimethylurea. Full protection against degradation could also be obtained by the incorporation of chelators or antioxidant enzymes. Maximal rates of degradation required the presence of transition metals and reducing compounds such as NADPH or dithiothreitol. Similar patterns of GS cleavage were obtained when seedlings were exposed to high doses of irradiation. The results indicate that chloroplastic GS is extremely prone to oxidative cleavage, and that reduced transition metals, presumably resulting from the destruction of iron-sulfur clusters by light-generated O-(2), play a crucial role in the degradation process. The physiological implications of GS lability to oxidative stress are discussed.
RESUMO
Genetic complementation of a sodA sodB Escherichia coli mutant strain was used to clone Rhodobacter capsulatus genes involved in detoxification of superoxide radicals. After sequence analysis, 1 of the 16 identical clones obtained by this selection procedure was shown to contain an open reading frame with sequence similarity to that coding for Fe-containing superoxide dismutases (SodB). The R. capsulatus sodB gene was expressed in E. coli, and the nature of the metal ligand was confirmed by inhibitor sensitivity assays with lysates from both bacterial species. Activity staining of cleared Rhodobacter lysates resolved by polyacrylamide gel electrophoresis indicated that SodB was the only superoxide dismutase present in this phototrophic organism. The sodB gene was expressed at low levels in R. capsulatus cells grown under anaerobic or semiaerobic conditions, but expression was strongly induced upon exposure of the bacteria to air or to methyl viologen. Attempts to construct a sodB mutant in this organism by allelic exchange of the chromosomal copy of the gene with a suicide plasmid containing a mutated sodB gene were unsuccessful, strongly suggesting that the encoded superoxide dismutase is essential for viability of R. capsulatus in aerobic cultures.
Assuntos
Proteínas de Bactérias/genética , Genes Bacterianos , Ferro , Metaloproteínas/genética , Rhodobacter capsulatus/genética , Superóxido Dismutase/genética , Aerobiose/genética , Sequência de Aminoácidos , Clonagem Molecular , Resistência a Medicamentos , Regulação Bacteriana da Expressão Gênica , Genes Essenciais , Teste de Complementação Genética , Dados de Sequência Molecular , Oxidantes/farmacologia , Estresse Oxidativo , Rhodobacter capsulatus/enzimologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Superóxidos/farmacologiaRESUMO
The DnaK system is required for the productive folding of pea chloroplast ferredoxin-NADP+ reductase (FNR) expressed in Escherichia coli. The formation of a mature active enzyme was severely impaired in E. coli dnaK, dnaJ or grpE mutants expressing either the cytosolic precursor of the reductase (preFNR) or the mature apoenzyme, and these forms aggregated extensively in these cells. Coexpression of dnaK from a multicopy plasmid in the dnaK-null mutants restored preFNR processing and folding of FNR, rendering a mature-sized active enzyme. Overexpression of GroESL chaperonins failed to prevent preFNR aggregation, but it restored productive folding of FNR in dnaK-null mutants expressing the mature enzyme. Expression of preFNR in OmpT-protease-deficient E. coli cells resulted in the accumulation of the unprocessed precursor in the soluble fraction of the cells. The interaction of this soluble preFNR, but not the mature reductase, with DnaK and GroEL was evidenced by immunoprecipitation studies. We conclude that, in addition to the GroE chaperonins [Carrillo, N., Ceccarelli, E. A., Krapp, A. R., Boggio, S., Ferreyra, R. G. & Viale, A. M. (1992) J. Biol. Chem. 267, 15537-15541], the DnaK chaperone system plays a crucial role in the folding pathway of FNR.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/fisiologia , Ferredoxina-NADP Redutase/biossíntese , Ferredoxina-NADP Redutase/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Óperon , Pisum sativum/enzimologia , Dobramento de Proteína , Alelos , Proteínas de Bactérias/genética , Sítios de Ligação , Chaperoninas , Cloroplastos/enzimologia , Clonagem Molecular , Escherichia coli/genética , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/químicaRESUMO
Ferredoxin-NADP+ reductases (FNR) participate in cellular defense against oxidative damage. Escherichia coli mutants deficient in FNR are abnormally sensitive to methyl viologen and hydrogen peroxide. Tolerance to these oxidants was regained by expression of plant FNR, superoxide dismutase, or catalase genes in the mutant cells. FNR contribution to the concerted defense against viologen toxicity under redox-cycling conditions was similar to that of the two major E. coli superoxide dismutases together, as judged by the phenotypes displayed by relevant mutant strains. However, FNR expression in sodA sodB strains failed to increase their tolerance to viologens, indicating that the FNR target is not the superoxide radical. Sensitivity of FNR-deficient cells to oxidants is related to extensive DNA damage. Incubation of the mutant bacteria with iron chelators or hydroxyl radical scavengers provided significant protection against viologens or peroxide, suggesting that oxidative injury in FNR-deficient cells was mediated by intracellular iron through the formation of hydroxyl radicals in situ. The NADP(H)-dependent activities of the reductase were necessary and sufficient for detoxification, without participation of either ferredoxin or flavodoxin in the process. Possible mechanisms by which FNR may exert its protective role are discussed.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/fisiologia , Ferredoxina-NADP Redutase/metabolismo , Genes de Plantas , Estresse Oxidativo/fisiologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catalase/biossíntese , Catalase/metabolismo , Cloroplastos/enzimologia , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Ferredoxina-NADP Redutase/biossíntese , Ferredoxina-NADP Redutase/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Proteínas Ferro-Enxofre/biossíntese , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Modelos Biológicos , Modelos Estruturais , Oxigênio/toxicidade , Paraquat/farmacologia , Pisum sativum/enzimologia , Pisum sativum/genética , Conformação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Superóxido Dismutase/biossíntese , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
Ferredoxin-NADP+ (oxido)reductase (EC 1.18.1.2, FNR) is an FAD-containing enzyme that catalyzes the reversible electron transfer between NADP(H) and electron carrier proteins such as ferredoxin and flavodoxin. Isoforms of this flavoprotein are present in chloroplasts, mitochondria, and bacteria in which they participate in a wide variety of redox metabolic pathways. Although ferredoxin-NADP+ reductases have been thoroughly investigated and their properties reviewed on several occasions, considerable advances in the understanding of these flavoenzymes have occurred in the last few years, including the characterization of cDNA and genomic clones encoding FNR proteins from plants, algae, vertebrates, and bacteria, determination of the atomic structure of a plant FNR at high resolution, and the expression of functional reductases in microorganisms like Escherichia coli and Saccharomyces cerevisiae. The aim of this article is to summarize information gained through these recent developments, including the phylogenetic relationships among ferredoxin reductases and the key structural features of the plant FNR family. Other aspects such as the catalytic mechanism of FNR and the molecular events underlying biogenesis, intracellular sorting, folding, and holoenzyme assembly of this important flavoenzyme are also discussed in some detail. Ferredoxin-NADP+ reductases display several outstanding properties that make them excellent model proteins to address broad biological questions.
Assuntos
Ferredoxina-NADP Redutase/química , Ferredoxina-NADP Redutase/fisiologia , Plantas/enzimologia , Bactérias/enzimologia , Relação Estrutura-AtividadeRESUMO
INTRODUCTION AND OBJECTIVES: Because left ventricular mass is associated with an increase in the risk of morbidity and mortality of cardiovascular diseases in the general population having the electrocardiogram as an accessible and inexpensive method for the diagnosis of left ventricular hypertrophy, we decided to calculate the sensitivity and specificity of 5 electrocardiographic criteria for the diagnosis of left ventricular hypertrophy and to compare the results of the original authors to ours. PATIENTS AND METHODS: 135 patients were evaluated; 46 patients were excluded by the following criteria: chronic obstructive pulmonary disease, complete left or right bundle branch block, cardiovascular ischemic disease or Wolf-Parkinson-White Syndrome. 89 patients remained and had an electrocardiogram performed applying the following criteria: Romhilt-Estes Point-Score system. Sokolow-Lyon (SV1 + RV5 or V6 > 3.5 mV) and (RaVL > 1.1 mV), Cornell and Rodríguez Padial. Left ventricular hypertrophy was defined by the Penn Convention Criteria. RESULTS: In our study we obtained the following results: a) Romhilt-Estes had a sensitivity of 12% and a specificity of 87%; b) Sokolow-Lyon (SV1 + RV5 or V6) had a sensitivity of 22% and a specificity of 79%; c) Sokolow-Lyon (RaVL) has a sensitivity of 18% and a specificity of 92%; d) Cornel had a sensitivity of 31% and a specificity of 87%, and e) Rodríguez Padial had a sensitivity of 82% and a specificity of 8%. There are similarities between our results and the authors's original ones. However, there are significant statistical differences between them (p < or = 0.01). CONCLUSION: Our conclusion is that these criteria have a low diagnostic value in the isolated interpretation of patients with left ventricular hypertrophy, and we need to integrate them with the whole medical history and physical examination.
Assuntos
Eletrocardiografia/métodos , Hipertrofia Ventricular Esquerda/diagnóstico , Adulto , Eletrocardiografia/normas , Feminino , Humanos , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Pessoa de Meia-Idade , Sensibilidade e EspecificidadeRESUMO
The flavoenzyme ferredoxin-NADP+ reductase (FNR) is a member of the cellular defense barrier against oxidative damage in Escherichia coli. We evaluated the responses of chloroplast FNR to methyl viologen, a superoxide radical propagator, in wheat (Triticum aestivum L.) plants and chloroplasts. Treatments with the herbicide showed little effect on the levels of FNR protein or transcripts, indicating that expression of this reductase is not upregulated by oxidants in plants. Viologens and peroxides caused solubilization of active FNR from the thylakoids into the stroma, converting the enzyme from a membrane-bound NADPH producer to a soluble NADPH consumer. This response appeared specific for FNR, since other thylakoid proteins were unaffected by the treatments. The reductase-binding protein was released together with FNR, suggesting that it might be the target of oxidative modification. Stromal accumulation of a functional NADPH reductase in response to oxidative stress is formally analogous to the induction of FNR synthesis observed in E. coli under similar conditions. FNR solubilization may be playing a crucial role in maintaining the NADPH/NADP+ homeostasis of the stressed plastid. The unchecked accumulation of NADPH might otherwise increase the risks of oxidative damage through a rise in the Mehler reaction rates and/or the production of hydroxyl radicals.
Assuntos
Cloroplastos/enzimologia , Ferredoxina-NADP Redutase/metabolismo , Membranas Intracelulares/enzimologia , Organelas/enzimologia , Estresse Oxidativo , Paraquat/farmacologia , Triticum/enzimologia , Clonagem Molecular , Escherichia coli , Ferredoxina-NADP Redutase/biossíntese , Ferredoxina-NADP Redutase/química , Modelos Biológicos , Folhas de Planta , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
The cytosolic precursor of the chloroplast flavoprotein ferredoxin-NADP+ reductase was expressed in Escherichia coli rendering a soluble protein that contained bound FAD and could be imported by isolated chloroplasts. The mechanism of plastid translocation was studied under defined conditions using this recombinant precursor holoprotein and intact pea chloroplasts. The first step in the import pathway, namely, binding of the reductase precursor to isolated chloroplasts, was saturable at about 2000 molecules/plastid, and showed a high-affinity interaction with a dissociation constant Kd of approximately 5 nM. Binding was not affected by the addition of soluble leaf extracts or by prior denaturation of the precursor with urea. Analysis of the initial import rates at different precursor concentrations indicated the existence of a single translocation system for this protein. Inclusion of leaf extracts in the assay resulted in a three-fold increase of the maximal import rates to 14,000 molecules . min-(1).chloroplast-(1), with a concomitant decrease in the apparent Km for the recombinant precursor, from 1 microM to 100-150 nM. Comparison of Km and Kd values under various conditions indicated that the binding step of the translocation process is largely irreversible, favouring import and processing. In the absence of extract, a denatured precursor obtained by incubation with urea was a better substrate for plastid import than the holoprotein. Treatment of the precursor with either extract or urea resulted in similar increases in import efficiency (V/Km), suggesting that stimulation by leaf extracts is probably related to unfolding of the precursor prior to translocation.
Assuntos
Cloroplastos/metabolismo , Ferredoxinas/química , Ferredoxinas/metabolismo , NADP/metabolismo , Transporte Biológico , Cloroplastos/enzimologia , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , NADP/química , Folhas de Planta/química , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Fatores de Tempo , Ureia/químicaRESUMO
Epidemic cholera reached Guatemala in July 1991. By mid-1993, Guatemala ranked third in the hemisphere in reported cases of cholera. We conducted a case-control study with two age-, sex-, and neighbourhood-matched controls per patient in periurban Guatemala City. Twenty-six patients hospitalized for cholera and 52 controls were enrolled. Seven (47%) of 15 stool cultures obtained after admission yielded toxigenic Vibrio cholerae O1. All seven were resistant to furazolidone, sulfisoxazole, and streptomycin, and differed substantially by pulsed-field gel electrophoresis from the Latin American epidemic strain dominant in the hemisphere since 1991. In univariate analysis, illness was associated with consumption of left-over rice (odds ratio [OR] = 7.0, 95% confidence interval [CI] = 1.4-36), flavored ices (-helados') (OR = 3.6, CI = 1.1 - 12), and street-vended non-carbonated beverages (OR = 3.8, CI = 1.2-12) and food items (OR = 11.0, CI = 2.3-54). Street-vended food items remained significantly associated with illness in multivariate analysis (OR = 6.5, CI = 1.4-31). Illness was not associated with drinking municipal tap water. Maintaining water safety is important, but slowing the epidemic in Guatemala City and elsewhere may also require improvement in street vendor food handling and hygiene.
Assuntos
Cólera/transmissão , Surtos de Doenças , Microbiologia de Alimentos , Vibrio cholerae/classificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Cólera/epidemiologia , Cólera/microbiologia , Feminino , Guatemala/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Vibrio cholerae/isolamento & purificação , Abastecimento de Água/análiseRESUMO
The contribution made by tyrosine 308 to the stability of pea ferredoxin-NADP+ reductase was investigated using site-directed mutagenesis. The phenol side chain of the invariant carboxyl terminal tyrosine is stacked coplanar to the isoalloxazine moiety of the FAD cofactor. Fluorescence measurements indicate that this interaction plays a significant role in FAD fluorescent quenching by the reductase apoprotein. Replacement of the tyrosine by tryptophan or phenylalanine caused only a minor increase in the quantum yields of bound FAD, whereas nonaromatic substitutions to serine and glycine resulted in a large fluorescent rise. Results from NADP+ titration experiments support a recent hypothesis [Karplus et al. (1991) Science 251, 60-66], suggesting that the phenol ring of Tyr 308 may fill the nicotinamide binding pocket in the absence of the nucleotide. The stability of the site-directed mutants, judged by thermal- and urea-induced denaturation studies, was lowered with respect to the wild-type enzyme. FNR variants harboring nonaromatic substitutions displayed more extensive destabilization. The decrease in thermodynamic stability correlated with the impairment of catalytic activities [Orellano et al. (1993) J. Biol. Chem 268, 19267-19273]. The results indicate that the presence of the electron-rich aromatic side chain adjacent to the isoalloxazine ring is essential for maximum stabilization of the FNR holoenzyme, resulting in a flavin conformation which optimizes electron flow between the prosthetic group and its redox partners.
Assuntos
Ferredoxina-NADP Redutase/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Pisum sativum/enzimologia , Tirosina/metabolismo , Sítios de Ligação , Estabilidade Enzimática , Ferredoxina-NADP Redutase/química , Ferredoxina-NADP Redutase/genética , Temperatura Alta , Mutação , Desnaturação Proteica , Espectrometria de Fluorescência , Tirosina/químicaRESUMO
The precursor of the chloroplast flavoprotein ferredoxin-NADP+ reductase from pea was expressed in Escherichia coli as a carboxyl-terminal fusion to glutathione S-transferase. The fused protein was soluble, and the precursor could be purified in a few steps involving affinity chromatography on glutathione-agarose, cleavage of the transferase portion by protease Xa, and ion exchange chromatography on DEAE-cellulose. The purified prereductase contained bound FAD but displayed marginally low levels of activity. Removal of the transit peptide by limited proteolysis rendered a functional protease-resistant core exhibiting enzymatic activity. The FAD-containing precursor expressed in E. coli was readily transported into isolated pea chloroplasts and was processed to the mature size, both inside the plastid and by incubation with stromal extracts in a plastid-free reaction. Import was dependent on the presence of ATP and was stimulated severalfold by the addition of plant leaf extracts.
Assuntos
Precursores Enzimáticos/metabolismo , Ferredoxina-NADP Redutase/metabolismo , Pisum sativum/enzimologia , Sequência de Bases , Transporte Biológico Ativo , Cloroplastos/enzimologia , DNA Complementar/genética , DNA de Plantas/genética , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Escherichia coli/genética , Ferredoxina-NADP Redutase/química , Ferredoxina-NADP Redutase/genética , Flavina-Adenina Dinucleotídeo/química , Vetores Genéticos , Dados de Sequência Molecular , Pisum sativum/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Escherichia coli cells carrying the mvrA mutation are unable to grow aerobically in the presence of the radical propagator methyl viologen (MV). Resistance against MV toxicity could be restored by the introduction of cloned DNA sequences encoding pea chloroplast ferredoxin-NADP+ reductase (FNR), a member of a class of flavoenzymes involved in redox pathways in bacteria, plants and animals. Complementation was strictly dependent on the accumulation of a functional transgenic FNR, since mutated reductases showing decreased enzymatic activities only partially rescued the MV-resistant phenotype. These results support recent observations suggesting that the E. coli mvrA gene encodes a ferredoxin (flavodoxin)-NADP+ reductase (V. Bianchi et al. (1993) J. Bacteriol. 175, 1590-1595). The mvrA mutant cells showed a moderate decrease in the flavodoxin-dependent activation of enzymes essential for anaerobic growth of E. coli. This effect is prevented by expression of a functional pea FNR in the mutant cells.