RESUMO
Nitric oxide (NO) is a reactive gas that participates in many physiological as well as pathogenic processes in higher eukaryotic organisms. Inflammatory responses elicit higher levels of this molecule. Nevertheless, there are many technical challenges to accurately measure the amount of NO produced. Previously, a method using whole-cell extracts from Escherichia coli was able to generate the conversion of nitrate into nitrite to measure the amount of nitrate or indirectly the NO present in a sample using the Griess reaction. Here we present an improvement to this method, by using E. coli whole-cell extracts lacking one of the two nitrite reductases, rendered a more precise measurement when coupled with the Griess reaction than our previous report. Alternatively, osmotic stress showed to downregulate the expression of both nitrate reductases, which can be an alternative for indirect nitrate and NO reduction. The results presented here show an easy method for nitrate and NO reduction to nitrite and avoid the reconversion to nitrate, also as an alternative for other analytical methods that are based on cadmium, purified nitrate reductase enzyme, or salicylic methods to reduce NO. This method can be widely used for measuring NO production in living organisms, soil, and other relevant microbiological samples.
Assuntos
Escherichia coli/metabolismo , Macrófagos/metabolismo , Óxido Nítrico/análise , Nitritos/análise , Animais , Grupo dos Citocromos c/genética , Escherichia coli/genética , Ativação de Macrófagos , Macrófagos/imunologia , Camundongos , Mutação , Nitrato Redutase/metabolismo , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Oxirredução , Células RAW 264.7 , Sensibilidade e EspecificidadeRESUMO
Two domain copper-nitrite reductases (NirK) contain two types of copper centers, one electron transfer (ET) center of type 1 (T1) and a catalytic site of type 2 (T2). NirK activity is pH-dependent, which has been suggested to be produced by structural modifications at high pH of some catalytically relevant residues. To characterize the pH-dependent kinetics of NirK and the relevance of T1 covalency in intraprotein ET, we studied the biochemical, electrochemical, and spectroscopic properties complemented with QM/MM calculations of Bradyrhizobium japonicum NirK (BjNirK) and of its electron donor cytochrome c550 (BjCycA). BjNirK presents absorption spectra determined mainly by a S(Cys)3pπ â Cu2+ ligand-to-metal charge-transfer (LMCT) transition. The enzyme shows low activity likely due to the higher flexibility of a protein loop associated with BjNirK/BjCycA interaction. Nitrite is reduced at high pH in a T1-decoupled way without T1 â T2 ET in which proton delivery for nitrite reduction at T2 is maintained. Our results are analyzed in comparison with previous results found by us in Sinorhizobium meliloti NirK, whose main UV-vis absorption features are determined by S(Cys)3pσ/π â Cu2+ LMCT transitions.
Assuntos
Proteínas de Bactérias/metabolismo , Bradyrhizobium/metabolismo , Grupo dos Citocromos c/metabolismo , Nitrito Redutases/metabolismo , Proteínas de Bactérias/genética , Bradyrhizobium/genética , Clonagem Molecular , Cobre/metabolismo , Grupo dos Citocromos c/genética , Nitrito Redutases/genética , Oxirredução , Regulação para CimaRESUMO
Accurate quantification depends on normalization of the measured gene expression data. In particular, gene expression studies with exposure to metals are challenging due their toxicity and redox-active properties. Here, we assessed the stability of potential reference genes in three cell lines commonly used to study metal cell metabolism: Caco-2 (colon), HepG2 (liver) and THP-1 (peripheral blood) under copper (Cu) or zinc (Zn) exposure. We used combined statistical tools to identify the best reference genes from a set of eleven candidates, which included traditional "housekeeping" genes such as GAPDH and B-ACTIN, in cell lines exposed to high and low, Zn and Cu concentrations. The expression stabilities of ATP5B (ATP synthase) and CYC1 (subunits of the cytochrome) were the highest considering the effect of Zn and Cu treatments whereas SDHA (succinate dehydrogenase) was found to be the most unstable gene. Even though the transcriptional effect of Zn and Cu is very different in term of redox properties, the same best reference genes were identified when Zn or Cu treatments were analyzed together. Our results indicate that ATP5B/CYC1 are the best candidates for reference genes after metal exposure, which can be used as a suitable starting point to evaluate gene expression with other metals or in different cell types in human models.
Assuntos
Cobre/farmacologia , Grupo dos Citocromos c/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Zinco/farmacologia , Linhagem Celular , Grupo dos Citocromos c/metabolismo , Grupo dos Citocromos c/normas , Perfilação da Expressão Gênica , Humanos , ATPases Mitocondriais Próton-Translocadoras/metabolismo , ATPases Mitocondriais Próton-Translocadoras/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de ReferênciaRESUMO
Chemolithoautotrophic acidophilic bacteria, which belong to the genus Leptospirillum, can only grow with Fe(II) as electron donor and oxygen as an electron acceptor. Members of this genus play an important role in bioleaching sulfide ores. We used nearly complete genome sequences of Leptospirillum ferrooxidans (group I), Leptospirillum rubarum, Leptospirillum '5-way CG' (group II) and Leptospirillum ferrodiazotrophum (group III) to identify cytochromes that are likely involved in electron transfer chain(s). The results show the presence of genes encoding a number of c-type cytochromes (18-20 genes were identified in each species), as well as bd and cbb3 oxidases. Genes encoding cbb3 oxidase are clustered, with predicted genes involved in cbb3 maturation proteins. Duplication of cbb3 encoding genes (ccoNO) was detected in all four genomes. Interestingly, these micro-organisms also contain genes that potentially encode bc1 and b6f-like complexes organized into two putative operon structures. To date, the Leptospirillum genus includes the only organisms reported to have genes coding for two different bc complexes. This study provides detailed insights into the components of electron transfer chains of Leptospirillum spp., revealing their conservation among leptospirilla groups and suggesting that there may be a single common pathway for electron transport between Fe(II) and oxygen.
Assuntos
Bactérias/genética , Grupo dos Citocromos c/genética , Genoma Bacteriano , Bactérias/classificação , Bactérias/enzimologia , Hibridização Genômica Comparativa , Grupo dos Citocromos c/metabolismo , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Compostos Férricos/metabolismo , Duplicação Gênica , Óperon , Oxirredução , Oxirredutases/metabolismo , Oxigênio/metabolismo , Periplasma/enzimologia , FilogeniaRESUMO
Acidithiobacillus ferrooxidans is a gram-negative bacterium that obtains energy from the oxidation of ferrous iron or reduced sulfur compounds. In this bacterium, the proteins encoded by the rus operon are involved in electron transfer from Fe(II) to O(2), and the first two proteins in this pathway also participate in the electron transfer pathway from Fe(II) to NAD(P). In this work we analyzed the expression, by real-time PCR, of the eight genes from the rus operon when A. ferrooxidans LR was grown in the presence of iron (control) and then kept in contact with chalcopyrite (CuFeS(2)) and covellite (CuS). A small decrease in rus operon gene expression was observed in the presence of chalcopyrite, while in the presence of covellite the expression of these genes showed a remarkable decrease. These results can be explained by the absence of ferrous iron in covellite. To explain the expression difference observed between the gene cyc1 and the gene rus, we investigated the information content presented at the Translation Initiation Site (TIS) of both genes. cyc1 showed a highly information content (8.4 bits) that can maximize translation, and rus showed a less favorable context (5.5 bits). Our hypothesis is that the energetic metabolism in A. ferrooxidans may be controlled at the transcriptional and posttranscriptional level by different mechanisms.
Assuntos
Acidithiobacillus/crescimento & desenvolvimento , Azurina/metabolismo , Cobre/farmacologia , Grupo dos Citocromos c/metabolismo , Regulação Bacteriana da Expressão Gênica , Óperon , Acidithiobacillus/classificação , Acidithiobacillus/genética , Acidithiobacillus/metabolismo , Azurina/genética , Meios de Cultura , Grupo dos Citocromos c/genética , Transporte de Elétrons/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Reação em Cadeia da Polimerase , Transcrição GênicaRESUMO
The regulation of the expression of the rus operon, proposed to encode an electron transfer chain from the outer to the inner membrane in the obligate acidophilic chemolithoautroph Acidithiobacillus ferrooxidans, has been studied at the RNA and protein levels. As observed by Northern hybridization, real-time PCR and reverse transcription analyses, this operon was more highly expressed in ferrous iron- than in sulfur-grown cells. Furthermore, it was shown by immunodetection that components of this respiratory chain are synthesized in ferrous iron- rather than in sulfur-growth conditions. Nonetheless, weak transcription and translation products of the rus operon were detected in sulfur-grown cells at the early exponential phase. The results strongly support the notion that rus-operon expression is induced by ferrous iron, in agreement with the involvement of the rus-operon-encoded products in the oxidation of ferrous iron, and that ferrous iron is used in preference to sulfur.
Assuntos
Acidithiobacillus/metabolismo , Azurina , Azurina/análogos & derivados , Proteínas de Bactérias/metabolismo , Regulação da Expressão Gênica , Óperon , Acidithiobacillus/enzimologia , Acidithiobacillus/crescimento & desenvolvimento , Azurina/genética , Azurina/metabolismo , Proteínas de Bactérias/genética , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Compostos Ferrosos/metabolismo , Ferro/metabolismo , OxirreduçãoRESUMO
DNA sequence analysis and bioinformatic interpretations have identified two adjacent clusters of genes potentially involved in the formation of a bc1 complex and in the maturation of a cytochrome c-type protein in two strains (ATCC 19859 and ATCC 33020) of the acidophilic, chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans). Reverse transcriptase-PCR experiments suggest that the two clusters are organized as operons, and +1 start sites of transcription for the operons have been determined by primer extension experiments. Potential promoters have been identified. The presence of these operons lends support to a recent model of reverse electron flow and is consistent with previous reports of phenotypic switching in this bacterium.
Assuntos
Proteínas de Bactérias/genética , Grupo dos Citocromos c/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Gammaproteobacteria/genética , Óperon , Proteínas de Bactérias/metabolismo , Sequência de Bases , Grupo dos Citocromos c/genética , Complexo III da Cadeia de Transporte de Elétrons/genética , Gammaproteobacteria/metabolismo , Dados de Sequência MolecularRESUMO
We have used RNA in situ hybridization to analyze the expression of transcripts encoding cytochrome c in different tissues and organs of sunflower (Helianthus annuus). Although northern-blot hybridization experiments indicate that the relative abundance of transcripts does not vary greatly, we have detected important changes in localization during flower development. Enhanced expression is observed in floral meristems as soon as they are discernible from the central portion of the capitulum containing the inflorescence meristem. As flowers develop, labeling is observed in all developing floral organ primordia. Later in development, expression in petals is reduced, and only the central portion of the flower becomes labeled. During the process of stamen formation, hybridization signals were obtained mainly in anthers. Less developed flowers at this stage showed expression through the archesporial tissue. During meiosis, the label was observed mainly in tapetal cells. Specific expression patterns, similar to those obtained for sunflower, were observed when Arabidopsis flowers were analyzed with a homologous cytochrome c probe. Specific patterns of expression were also observed in young sunflower roots. In this case, enhanced expression was detected in developing endodermis and pericycle and in protoxylem initials. We conclude that cell-specific mechanisms operate to regulate the abundance of cytochrome c encoding transcripts in different plant tissues. The overlap between the expression patterns of the nuclear encoded cytochrome c gene and some mitochondrial genes suggests the existence of coordinated mechanisms of expression.
Assuntos
Grupo dos Citocromos c/genética , Regulação da Expressão Gênica de Plantas , Helianthus/genética , Transcrição Gênica , Regulação da Expressão Gênica no Desenvolvimento , Helianthus/citologia , Helianthus/crescimento & desenvolvimento , Hibridização In Situ , Raízes de Plantas/citologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Caules de Planta/citologia , Caules de Planta/crescimento & desenvolvimento , Caules de Planta/metabolismo , RNA Mensageiro/genéticaRESUMO
Although salamanders are characteristic amphibians in Holarctic temperate habitats, in tropical regions they have diversified evolutionarily only in tropical America. An adaptive radiation centered in Middle America occurred late in the history of a single clade, the supergenus Bolitoglossa (Plethodontidae), and large numbers of species now occur in diverse habitats. Sublineages within this clade decrease in number from the northern to southern parts of Middle America, and in Costa Rica, there are but three. Despite this phylogenetic constraint, Costa Rica has many species; the number of salamander species on one local elevational transect in the Cordillera de Talamanca may be the largest for any such transect in the world. Extraordinary variation in sequences of the mitochondrial gene cytochrome b within a clade of the genus Bolitoglossa in Costa Rica reveals strong phylogeographic structure within a single species, Bolitoglossa pesrubra. Allozymic variation in 19 proteins reveals a pattern largely concordant with the mitochondrial DNA phylogeography. More species exist than are currently recognized. Diversification occurs in restricted geographic areas and involves sharp geographic and elevational differentiation and zonation. In their degree of genetic differentiation at a local scale, these species of the deep tropics exceed the known variation of extratropical salamanders, which also differ in being less restricted in elevational range. Salamanders display "tropicality" in that although speciose, they are usually local in distribution and rare. They display strong ecological and physiological differentiation that may contribute importantly to morphological divergence and species formation.
Assuntos
Variação Genética , Urodelos/genética , Altitude , Animais , Evolução Biológica , Costa Rica , Grupo dos Citocromos c/genética , DNA Mitocondrial/genética , Ecologia , Geografia , Dados de Sequência Molecular , FilogeniaRESUMO
The expression of the Chlamydomonas reinhardtii cytochrome c gene was studied at the steady-state mRNA level. The inclusion of acetate under illumination produced a marked increase in cytochrome c transcripts. This effect was not affected by two inhibitors of mitochondrial energy metabolism. Three different obligate photoautotrophic mutants with defective mitochondria showed normal levels of induction, suggesting that utilization of acetate for respiration is not required for this process. Light, in the presence or absence of acetate, also promoted an increase in cytochrome c transcript levels. This effect could be abolished by treatment of the cells with an inhibitor of the photosynthetic electron transport chain, suggesting that light acts through photosynthesis to promote the induction. In addition, a genomic clone encompassing the Chlamydomonas cytochrome c gene has been isolated and analyzed. The gene contains three introns, two of which are located at positions similar to those in the rice and Arabidopsis cytochrome c genes, indicating the existence of an evolutionary link. It is concluded that the cytochrome c gene from C. reinhardtii is subject to metabolic regulation through a mechanism that responds to the intracellular level of either acetate or a compound derived from its metabolization through a pathway different from mitochondrial respiration.