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1.
Emerg Microbes Infect ; 8(1): 1406-1415, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31544591

RESUMO

Vibrio vulnificus is a halophilic estuarine bacterium causing severe opportunistic infections. To successfully establish an infection, V. vulnificus must adapt to redox fluctuations in vivo. In the present study, we show that deletion of V. vulnificus fexA gene caused hypersensitivity to acid and reactive oxygen species. The ΔfexA mutant exhibited severe in vivo survival defects. For deeper understanding the role of fexA gene on the successful V. vulnificus infection, we analyzed differentially expressed genes in ΔfexA mutant in comparison with wild type under aerobic, anaerobic or in vivo culture conditions by genome-scale DNA microarray analyses. Twenty-two genes were downregulated in the ΔfexA mutant under all three culture conditions. Among them, cydAB appeared to dominantly contribute to the defective phenotypes of the ΔfexA mutant. The fexA deletion induced compensatory point mutations in the cydAB promoter region over subcultures, suggesting essentiality. Those point mutations (PcydSMs) restored bacterial growth, motility, cytotoxicity ATP production and mouse lethality in the ΔfexA mutant. These results indicate that the cydAB operon, being regulated by FexA, plays a crucial role in V. vulnificus survival under redox-fluctuating in vivo conditions. The FexA-CydAB axis should serve an Achilles heel in the development of therapeutic regimens against V. vulnificus infection.


Assuntos
Proteínas de Bactérias/genética , Grupo dos Citocromos d/genética , Regulação Bacteriana da Expressão Gênica , Oxirredutases/genética , Vibrio vulnificus/genética , Ácidos/farmacologia , Animais , Animais Recém-Nascidos , Regulação para Baixo , Deleção de Genes , Peróxido de Hidrogênio/farmacologia , Dose Letal Mediana , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Mutação Puntual , Ratos , Vibrioses/microbiologia , Vibrio vulnificus/efeitos dos fármacos , Vibrio vulnificus/crescimento & desenvolvimento
2.
MBio ; 7(6)2016 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-27999164

RESUMO

In the course of an infection, Salmonella enterica occupies diverse anatomical sites with various concentrations of oxygen (O2) and nitric oxide (NO). These diatomic gases compete for binding to catalytic metal groups of quinol oxidases. Enterobacteriaceae express two evolutionarily distinct classes of quinol oxidases that differ in affinity for O2 and NO as well as stoichiometry of H+ translocated across the cytoplasmic membrane. The investigations presented here show that the dual function of bacterial cytochrome bd in bioenergetics and antinitrosative defense enhances Salmonella virulence. The high affinity of cytochrome bd for O2 optimizes respiratory rates in hypoxic cultures, and thus, this quinol oxidase maximizes bacterial growth under O2-limiting conditions. Our investigations also indicate that cytochrome bd, rather than cytochrome bo, is an intrinsic component of the adaptive antinitrosative toolbox of Salmonella Accordingly, induction of cytochrome bd helps Salmonella grow and respire in the presence of inhibitory NO. The combined antinitrosative defenses of cytochrome bd and the flavohemoglobin Hmp account for a great part of the adaptations that help Salmonella recover from the antimicrobial activity of NO. Moreover, the antinitrosative defenses of cytochrome bd and flavohemoglobin Hmp synergize to promote Salmonella growth in systemic tissues. Collectively, our investigations indicate that cytochrome bd is a critical means by which Salmonella resists the nitrosative stress that is engendered in the innate response of mammalian hosts while it concomitantly allows for proper O2 utilization in tissue hypoxia. IMPORTANCE: It is becoming quite apparent that metabolism is critically important to the virulence potential of pathogenic microorganisms. Bacterial cells use a variety of terminal electron acceptors to power electron transport chains and metabolic processes. Of all the electron acceptors available to bacteria, utilization of O2 yields the most energy while diversifying the type of substrates that a pathogen can use. Recent investigations have demonstrated important roles for bd-type quinol oxidases with high affinity for O2 in bacterial pathogenesis. The investigations presented here have revealed that cytochrome bd potentiates virulence of a clinically relevant bacterial pathogen by fueling bioenergetics of prokaryotic cells while protecting the respiratory chain against NO toxicity. The adaptive antinitrosative defenses afforded by cytochrome bd synergize with other NO-detoxifying systems to preserve cellular bioenergetics, thereby promoting bacterial virulence in tissue hypoxia.


Assuntos
Grupo dos Citocromos b/metabolismo , Grupo dos Citocromos d/metabolismo , Metabolismo Energético , Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Salmonella enterica/metabolismo , Salmonella enterica/patogenicidade , Animais , Complexo de Proteínas da Cadeia de Transporte de Elétrons , Humanos , Hipóxia , Imunidade Inata , Oxirredução , Oxirredutases/metabolismo , Consumo de Oxigênio , Salmonella enterica/crescimento & desenvolvimento , Estresse Fisiológico
4.
Science ; 352(6285): 583-6, 2016 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-27126043

RESUMO

The cytochrome bd oxidases are terminal oxidases that are present in bacteria and archaea. They reduce molecular oxygen (dioxygen) to water, avoiding the production of reactive oxygen species. In addition to their contribution to the proton motive force, they mediate viability under oxygen-related stress conditions and confer tolerance to nitric oxide, thus contributing to the virulence of pathogenic bacteria. Here we present the atomic structure of the bd oxidase from Geobacillus thermodenitrificans, revealing a pseudosymmetrical subunit fold. The arrangement and order of the heme cofactors support the conclusions from spectroscopic measurements that the cleavage of the dioxygen bond may be mechanistically similar to that in the heme-copper-containing oxidases, even though the structures are completely different.


Assuntos
Proteínas de Bactérias/química , Grupo dos Citocromos d/química , Citocromos b/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Geobacillus/enzimologia , Oxigênio/química , Proteínas de Bactérias/ultraestrutura , Grupo dos Citocromos d/ultraestrutura , Citocromos b/ultraestrutura , Complexo IV da Cadeia de Transporte de Elétrons/ultraestrutura , Dobramento de Proteína , Estrutura Secundária de Proteína
5.
J Ind Microbiol Biotechnol ; 38(6): 667-77, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20730594

RESUMO

Agrobacterium sp. ATCC 31749 (formerly named Alcaligenes faecalis var. myxogenes) is a non-pathogenic aerobic soil bacterium used in large scale biotechnological production of curdlan. However, little is known about its genomic information. DNA partial sequence of electron transport chains (ETCs) protein genes were obtained in order to understand the components of ETC and genomic-specificity in Agrobacterium sp. ATCC 31749. Degenerate primers were designed according to ETC conserved sequences in other reported species. DNA partial sequences of ETC genes in Agrobacterium sp. ATCC 31749 were cloned by the PCR method using degenerate primers. Based on comparative genomic analysis, nine electron transport elements were ascertained, including NADH ubiquinone oxidoreductase, succinate dehydrogenase complex II, complex III, cytochrome c, ubiquinone biosynthesis protein ubiB, cytochrome d terminal oxidase, cytochrome bo terminal oxidase, cytochrome cbb (3)-type terminal oxidase and cytochrome caa (3)-type terminal oxidase. Similarity and phylogenetic analyses of these genes revealed that among fully sequenced Agrobacterium species, Agrobacterium sp. ATCC 31749 is closest to Agrobacterium tumefaciens C58. Based on these results a comprehensive ETC model for Agrobacterium sp. ATCC 31749 is proposed.


Assuntos
Rhizobium/genética , beta-Glucanas/metabolismo , Grupo dos Citocromos d/genética , Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Genes Bacterianos , Genoma Bacteriano , Genômica , Oxirredutases/genética , Filogenia , Rhizobium/classificação , Rhizobium/enzimologia , Succinato Desidrogenase/genética
6.
J Biosci Bioeng ; 110(1): 42-7, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20541114

RESUMO

Rhodococcus rhodochrous is an active soil bacterium belonging to the Nocardia group of high GC gram-positive bacteria. It is rich in various enzymes and thus important in the industrial production of chemicals and bioremediation. In this work, the respiratory chain of this aerobic organism was investigated and characterized. Grown under highly aerobic conditions, the membrane fraction of R. rhodochrous cells only contained a-, b- and c-type cytochromes, suggesting that it is the cytochrome bcc-aa(3)-type pathway that mainly operates under these conditions. In contrast, the d-type cytochrome was also present under microaerobic conditions, indicating that the alternative pathway of the bd-type oxidase works in these circumstances. In addition, the results of H(+)/O ratio measurements indicate that these two pathways have different energy efficiencies.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Rhodococcus/enzimologia , Aerobiose/fisiologia , Membrana Celular/química , Grupo dos Citocromos c/metabolismo , Grupo dos Citocromos d/metabolismo , Citocromos/análise , Citocromos/metabolismo , Transporte de Elétrons/fisiologia , Complexo IV da Cadeia de Transporte de Elétrons/química , Metabolismo Energético/fisiologia , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Oxirredução , Oxirredutases/metabolismo , Oxigênio/metabolismo , Rhodococcus/crescimento & desenvolvimento
7.
J Biol Chem ; 285(24): 18464-72, 2010 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-20392690

RESUMO

Escherichia coli possesses cytochrome bo' (CyoABCDE), cytochrome bd-I (CydAB), and cytochrome bd-II (AppBC) quinol oxidases, all of which can catalyze the terminal step in the aerobic respiratory chain, the reduction of oxygen by ubiquinol. Although CydAB has a role in the generation of DeltapH, AppBC has been proposed to alleviate the accumulation of electrons in the quinone pool during respiratory stress via electroneutral ubiquinol oxidation. A cydB mutant strain exhibited lower respiration rates while maintaining a wild type growth rate. Transcriptomic analysis revealed a dramatic up-regulation of AppBC in the cydB strain, accompanied by the induction of genes involved in glutamate/gamma-aminobutyric acid (GABA) antiport, the GABA shunt, the glyoxylate shunt, respiration (including appBC), motility, and osmotic stress. Transcription factor modeling suggests that the underpinning regulation is largely controlled by H-NS, GadX, FlhDC, and AppY. The transcriptional adaptations imply that cydB cells contribute to the proton motive force via consumption of intracellular protons and glutamate/GABA antiport. Indeed, supplementation of culture medium with l-glutamate stimulates growth in a cydB strain. Phenotype analyses of the cydB strain confirm decreased motility and elevated acid resistance and also an elevated cytochrome d spectroscopic signal in cells grown at low pH. We propose a mechanism via which E. coli can compensate for the loss of cytochrome bd-I activity; cytochrome bd-II-mediated quinol oxidation prevents the accumulation of NADH, whereas GABA synthesis/antiport maintains the proton motive force for ATP production.


Assuntos
Grupo dos Citocromos d/genética , Citocromos b/genética , Escherichia coli/enzimologia , Ácido Glutâmico/metabolismo , Consumo de Oxigênio , Movimento Celular , Respiração Celular , Eletrodos , Regulação Bacteriana da Expressão Gênica , Glutamatos/química , Concentração de Íons de Hidrogênio , Cinética , Modelos Estatísticos , Oxigênio/química , Espectrofotometria/métodos
8.
J Bacteriol ; 192(2): 391-9, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19897650

RESUMO

The Arc (anoxic redox control) two-component signal transduction system, consisting of the ArcB sensor kinase and the ArcA response regulator, allows adaptive responses of Escherichia coli to changes of O(2) availability. The arcA gene was previously known as the dye gene because null mutants were growth sensitive to the photosensitizer redox dyes toluidine blue and methylene blue, a phenotype whose molecular basis still remains elusive. In this study we report that the toluidine blue O (TBO) effect on the arc mutants is light independent and observed only during aerobic growth conditions. Moreover, 16 suppressor mutants with restored growth were generated and analyzed. Thirteen of those possessed insertion elements upstream of the cydAB operon, rendering its expression ArcA independent. Also, it was found that, in contrast to cythocrome d, cythocrome o was not able to confer toluidine blue resistance to arc mutants, thereby representing an intriguing difference between the two terminal oxidases. Finally, a mechanism for TBO sensitivity and resistance is discussed.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Grupo dos Citocromos b/metabolismo , Grupo dos Citocromos d/metabolismo , Citocromos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas Repressoras/genética , Cloreto de Tolônio/farmacologia , Anaerobiose , Proteínas da Membrana Bacteriana Externa/metabolismo , Sequência de Bases , Carotenoides/metabolismo , Catalase/metabolismo , Corantes/farmacologia , Grupo dos Citocromos b/genética , Grupo dos Citocromos d/genética , Citocromos/genética , Escuridão , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Glucose/farmacologia , Luz , Dados de Sequência Molecular , Mutação/genética , Oxirredutases/genética , Regiões Promotoras Genéticas/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/metabolismo , Homologia de Sequência do Ácido Nucleico , Superóxido Dismutase/metabolismo
9.
Anaerobe ; 14(3): 145-56, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18457966

RESUMO

In the anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough genes were found encoding membrane terminal oxygen reductases of two types: a cytochrome c oxidase and a cytochrome bd oxidase, both enzymes are terminal oxidases typical of facultative or aerobic microorganisms (Heidelberg JF, et al., The genome sequence of the anaerobic, sulfate-reducing bacterium D. vulgaris Hildenborough. Nat Biotechnol 2004; 22: 554-9). To apprehend the presence of both oxidases in other sulfate-reducing bacteria (SRB), several assays were performed on isolates recovered from salt-marsh sediments in Portugal, representative of the different phylogenetic groups identified. Hybridization and PCR experiments for DNA sequencing were performed on the chosen isolates. Primers were selected to amplify conserved regions of cytochrome c oxidases and cytochrome bd oxidases taking into consideration alignment of corresponding subunit I sequences. The results showed that both oxidase genes are present on the chromosome of several isolates characterized as Desulfovibrio. These genes were shown to be transcribed, as demonstrated by Reverse Transcriptase-PCR experiments on total RNA. In order to assess the relative contribution of each oxidase to oxygen consumption, oxygen uptake was measured for each isolate and further characterized by the effect of cyanide on oxygen consumption. It was concluded that cytochrome bd oxidase was the terminal membrane oxygen reductase allowing oxygen consumption. In addition, it was observed that isolates containing cytochrome bd oxidase had higher resistance to air exposure, suggesting an important role of this enzyme in survival to air exposure. The pattern for the presence of oxygen reductase genes was compared to the physiological pattern of substrate use, which was determined for each isolate. Salinity tolerance, pH and temperature growth of each isolate were also analyzed.


Assuntos
Desulfovibrio vulgaris/enzimologia , Sedimentos Geológicos/microbiologia , Oxirredutases/metabolismo , Oxigênio/metabolismo , Água do Mar/microbiologia , Bactérias Redutoras de Enxofre/enzimologia , Anaerobiose , Grupo dos Citocromos d/genética , Grupo dos Citocromos d/metabolismo , Citocromos b/genética , Citocromos b/metabolismo , Desulfovibrio vulgaris/genética , Desulfovibrio vulgaris/crescimento & desenvolvimento , Desulfovibrio vulgaris/isolamento & purificação , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Dados de Sequência Molecular , Oxirredutases/genética , Oxigênio/farmacologia , Consumo de Oxigênio , Filogenia , Portugal , Análise de Sequência de DNA , Bactérias Redutoras de Enxofre/genética , Bactérias Redutoras de Enxofre/crescimento & desenvolvimento , Bactérias Redutoras de Enxofre/isolamento & purificação
10.
Biochim Biophys Acta ; 1777(6): 488-95, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18420022

RESUMO

PSII activity was inhibited after Spirulina platensis cells were incubated with different salt concentrations (0-0.8 M NaCl) for 12 h. Flash-induced fluorescence kinetics showed that in the absence of DCMU, the half time of the fast and slow components decreased while that of the middle component increased considerably with increasing salt concentration. In the presence of DCMU, fluorescence relaxation was dominated by a 0.6s component in control cells. After salt stress, this was partially replaced by a faster new component with half time of 20-50 ms. Thermoluminescence measurements revealed that S(2)Q(A)(-) and S(2)Q(B)(-) recombinations were shifted to higher temperatures in parallel and the intensities of the thermoluminescence emissions were significantly reduced in salt-stressed cells. The period-four oscillation of the thermoluminescence B band was highly damped. There were no significant changes in contents of CP47, CP43, cytochrome c550, and D1 proteins. However, content of the PsbO protein in thylakoid fraction decreased but increased significantly in soluble fraction. The results suggest that salt stress leads to a modification of the Q(B) niche at the acceptor side and an increase in the stability of the S(2) state at the donor side, which is associated with a dissociation of the PsbO protein.


Assuntos
Proteínas de Bactérias/metabolismo , Grupo dos Citocromos c/metabolismo , Grupo dos Citocromos d/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Cloreto de Sódio/farmacologia , Spirulina/metabolismo , Proteínas de Bactérias/química , Grupo dos Citocromos c/química , Grupo dos Citocromos d/química , Fluorescência , Temperatura Alta , Cinética , Complexos de Proteínas Captadores de Luz/química , Pressão Osmótica , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteína do Fotossistema II/química , Spirulina/química
11.
Biochemistry ; 46(39): 11177-84, 2007 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-17784736

RESUMO

Cytochrome bd from Azotobacter vinelandii is a respiratory quinol oxidase that is highly efficient in reducing intracellular oxygen concentration, thus enabling nitrogen fixation under ambient aerobic conditions. Equilibrium measurements of O2 binding to ferrous heme d in the one-electron-reduced form of the A. vinelandii enzyme give Kd(O2) = 0.5 microM, close to the value for the Escherichia coli cytochrome bd (ca. 0.3 microM); thus, both enzymes have similar, high affinity for oxygen. The reaction of the A. vinelandii cytochrome bd in the one-electron-reduced and fully reduced states with O2 is extremely fast approaching the diffusion-controlled limit in water. In the fully reduced state, the rate of O2 binding depends linearly on the oxygen concentration consistently with a simple, single-step process. In contrast, in the one-electron-reduced state the rate of oxygen binding is hyperbolic, implying a more complex binding pattern. Two possible explanations for the saturation kinetics are considered: (A) There is a spectroscopically silent prebinding of oxygen to an unidentified low-affinity saturatable site followed by the oxygen transfer to heme d. (B) Oxygen binding to heme d requires an "activated" state of the enzyme in which an oxygen channel connecting heme d to the bulk is open. This channel is permanently open in the fully reduced enzyme (hence no saturation behavior) but flickers between the open and closed states in the one-electron-reduced enzyme.


Assuntos
Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/metabolismo , Citocromos/metabolismo , Oxigênio/metabolismo , Proteínas de Bactérias/química , Ligação Competitiva , Grupo dos Citocromos b/química , Grupo dos Citocromos b/metabolismo , Grupo dos Citocromos d/química , Grupo dos Citocromos d/metabolismo , Citocromos/química , Heme/análogos & derivados , Heme/química , Cinética , Oxirredução , Oxigênio/química , Ligação Proteica
13.
Encephale ; 32(2 Pt 1): 263-9, 2006.
Artigo em Francês | MEDLINE | ID: mdl-16910628

RESUMO

INTRODUCTION: Depression is common in people with schizophrenia and is associated with substantial morbidity explaining also the considerable attention and recognition of this entity as suggested by the inclusion of the post-psychotic depression in DSM IV and ICD 10. The prevalence of this disorder varies according to the type of approach used (range between 7% to 75%). Prescription of antidepressants plus antipsychotic treatment is frequent in clinical practice (11 to 43%). BACKGROUND: Pharmacokinetic and metabolic interactions have been identified. The cytochrome P450 has been identified as being implicated in the metabolism of most psychotropics, mainly through the CYP1A2, CYP2C19, CYP2D6, CYP3A4 isoenzymes. Tricyclic antidepressants are likely to increase chlorpromazine plasma levels. Similarly, antipsychotics such as perphenazine, chlorpromazine or haloperidol can increase antidepressant plasma levels, through the inhibition of CYP 450 isoenzymes (CYP2D6). Most of the Specific Serotonin Recapture Inhibitors (SSRIs) are likely to inhibit one or several CYP450 isoenzymes. The inhibition is moderate to marked for CYP1A2 (fluvoxamine and fluoxetine), CYP2C19 (fluoxetine, fluvoxamine and sertraline), CYP2D6 (paroxetine, fluoxetine and sertraline), and CYP3A4 (fluvoxamine, fluoxetine and sertraline). In the US, one-fourth of psychiatrists report the use of depression-rating scales in schizophrenic patients. Non specific scales (Hamilton Depression Rating Scale or Beck Depression Inventory) are the most commonly used in spite of the fact that these scales do not allow the distinction of depressive from negative symptoms in schizophrenic patients. LITERATURE FINDINGS: Due to these limitations, more specific assessment tools for depressive symptoms in schizophrenia are required. Two specific scales for assessing depressive symptoms in schizophrenic patients have been constructed and validated. The Calgary Depression Scale (CDS) is a nine item scale, each item scored from 0 to 3. This scale was derived from the HDRS and the Present State Examination. Factor analysis showed that the CDS is unidimensional, has high internal consistency, and significant strong correlation with scores on the HDRS, Beck and BPRS depression scales. The CDS has been validated in different languages (Brazilian, Danish, French...). It has been shown that there is no overlap between negative or extrapyramidal and depressive symptoms assessed by the PDS in schizophrenic patients. The Psychotic Depression Scale (PDS) is a 32 item scale derived from the HDRS, PANSS, CPRS and AMDP, each item being rated from 0 to 7. A principal component analysis of the PDS items using a Varimax rotation disclosed 8 orthogonal components that account for 71% of the variance. These components involved the following dimensions : depressive mood, inhibition, vegetative signs, paranoid signs, strangeness of thought, inverse vegetative signs, guilt feelings and cognitive signs. The analysis revealed that the 'depressive mood' factor of the PDS was correlated with the 'depressive' factor and was slightly correlated with the cognitive factor of the PANSS. This first factor was not correlated with either the "negative" factor of the PANSS, or the Positive or Excitement factor of the PANSS. Hence, this PDS, factor distinguished depressive signs from negative symptoms. Due to their metrologic properties, specific scales should be preferred. However, only one open trial (of an antipsychotic) and two double blind controlled trials (one comparison of 2 antipsychotics and one comparison of an cholinesterase inhibitor versus placebo) have been published using the CDS. Likewise, only one double blind controlled trial using the PDS (comparison of 2 antipsychotics) has been published. No study of the effect of antidepressants in depressed schizophrenic patients has been published, using either the CDS or the PDS assessment criteria. DISCUSSION: These specific scales are rarely used in clinical practice. Only about 1% of the US psychiatrists reported the use of the Calgary Depression Scale. Several open clinical trials have assessed the efficacy of antidepressant agents added to antipsychotic in patients with schizophrenia. They have produced inconsistent results but have suffered from methodological limits (short duration of the studies, non homogeneous inclusion criteria, heterogeneous assessment methods...). Due to the lack of a reference drug, double blind placebo-controlled trial are necessary. A recent meta-analysis has been performed on results of trials that have investigated the clinical efficacy of antidepressant medication (either tricyclics, SSRIs or others) in the treatment of depression in schizophrenic patients. In a subset of 5 trials (209 patients), the proportion improved in the antidepressant group was 26% (95% CI 10 to 42) higher than in the placebo group. The estimated number needed to treat was 4. In a subgroup of 6 studies (267 patients), the standardized mean difference on the HDRS score was -0.27 (95% CI - 0.7 to -0.2). There was no evidence that antidepressant treatment induced a deterioration of psychotic symptoms in these trials. CONCLUSION: The results provide weak evidence for the efficacy of antidepressants in patients with schizophrenia and depression. Today, the only SSRI tested in the treatment of depression in schizophrenic patients is sertraline. One study led to positive results. Since the meta-analysis, one additional study has been performed comparing sertraline to placebo. No difference between the 2 treatment groups was demonstrated but the power of the trial was rather low. Further research is required to determine the best approach towards treating depression in patients with schizophrenia, with clinical trials performed for longer periods, using appropriate assessment criteria such as depressive symptoms and quality of life.


Assuntos
Antidepressivos/uso terapêutico , Transtorno Depressivo Maior/tratamento farmacológico , Transtorno Depressivo Maior/epidemiologia , Esquizofrenia/epidemiologia , Inibidores de Captação de Serotonina/uso terapêutico , Antidepressivos/farmacocinética , Sistema Enzimático do Citocromo P-450/metabolismo , Grupo dos Citocromos d/metabolismo , Transtorno Depressivo Maior/diagnóstico , Humanos , Prevalência , Testes Psicológicos , Esquizofrenia/enzimologia , Inibidores de Captação de Serotonina/farmacocinética
14.
Curr Microbiol ; 52(4): 274-81, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16550467

RESUMO

Although classified as anaerobic, Desulfovibrio gigas contains a functional canonical membrane respiratory chain, including a cytochrome bd quinol oxidase as its terminal element. In the present study, we report the identification of the operon cydAB encoding the two subunits of cytochrome bd from this bacterium. Two hypothetical promoter regions and sequences resembling transcriptional regulators-binding sites have been identified. Amino acid sequence analysis revealed a high similarity to cytochrome bd from other organisms, presenting the conserved residues typical from these proteins. Reverse transcription polymerase chain reaction (RT-PCR) and Northern blot analysis confirmed the operon transcription. Gene expression was assessed by real-time RT-PCR in cells grown in different media and under exposure to oxygen and nitric oxide. mRNA levels were slightly enhanced in the presence of 150 microM: NO. However, in the presence of 10 microM: NO, a decrease was observed of the steady-state population of cydAB mRNA. No considerable effect was observed in the presence of fumarate/sulfate medium, 60 microM: O2 or 10 microM: NO.


Assuntos
Proteínas de Bactérias/metabolismo , Desulfovibrio gigas/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Grupo dos Citocromos d/metabolismo , Citocromos b/metabolismo , Desulfovibrio gigas/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Dados de Sequência Molecular , Óxido Nítrico/fisiologia , Óperon , Oxigênio/fisiologia , Regiões Promotoras Genéticas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Análise de Sequência de DNA
15.
Extremophiles ; 9(3): 247-53, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15844013

RESUMO

The genes of cytochrome bd-encoding cydAB were identified from a deep-sea bacterium Shewanella violacea DSS12. These showed significant homologies with known cydAB gene sequences from various organisms. Additionally, highly conserved regions that are important for the enzymatic function were also conserved in cydA of S. violacea. Based on the results, transcriptional analysis of cydAB operon and cydDC operon (required for assembly of cytochrome bd) of S. violacea in microaerobic condition was performed under the growth condition of various pressures. The gene of cydA was expressed even under the condition of atmospheric pressure and its expression was enhanced with pressurization. On the other hand, the expression of cydC was strongly depressed under the condition of atmospheric pressure compared with the case under high pressure. It appeared spectrophotometrically that loss of cytochrome bd in S. violacea under atmospheric pressure shown in previous study is caused mainly by the loss of cydDC. Further, under the growth condition of atmospheric pressure, either less amount or no d-type cytochrome was expressed compared with the case of high-pressure condition even if the organism was grown under alkaline condition or in the presence of uncoupler, which are the inducible condition of d-type cytochrome in Escherichia coli. These results suggested that the significant amount of d-type cytochrome expression is specific event under the growth condition of high pressure.


Assuntos
Citocromos/biossíntese , Água do Mar/microbiologia , Shewanella/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Sequência Conservada , Grupo dos Citocromos d/biossíntese , Grupo dos Citocromos d/genética , Citocromos/genética , Citocromos b/biossíntese , Citocromos b/genética , Primers do DNA , Dados de Sequência Molecular , Óperon , Filogenia , Reação em Cadeia da Polimerase , Pressão , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
16.
Biotechnol Bioeng ; 79(5): 558-67, 2002 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-12209827

RESUMO

The function of the reversible oxygen-binding hemoprotein from Vitreoscilla (VHb), which enhances oxygen-limited cell growth and recombinant protein production when functionally expressed in Escherichia coli, was investigated in wild-type E. coli and in E. coli mutants lacking one of the two terminal oxidases, cytochrome o complex (aerobic terminal oxidase, Cyo) or cytochrome d complex (microaerobic terminal oxidase, Cyd). Deconvolution of VHb, cytochrome o, and cytochrome d bands from in vivo absorption spectra revealed a 5-fold enhancement in cytochrome o content and a 1.5-fold increment in cytochrome d by VHb under microaerobic environments (dissolved oxygen less than 2% air saturation). Based upon oxygen uptake kinetics measurements of these mutants, the apparent oxygen affinity of the Cyo(+), Cyd(-) E. coli was increased in the presence of VHb, but no difference in the apparent K(m) was observed for the Cyo(-), Cyd(+) strain. Results suggest that the expression of VHb in E. coli increases the level and activity of terminal oxidases and thereby improves the efficiency of microaerobic respiration and growth.


Assuntos
Proteínas de Bactérias/metabolismo , Grupo dos Citocromos b , Citocromos/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Hemoglobinas/metabolismo , Oxirredutases/metabolismo , Aerobiose , Proteínas de Bactérias/genética , Reatores Biológicos , Respiração Celular , Células Cultivadas , Clonagem Molecular , Grupo dos Citocromos d/genética , Grupo dos Citocromos d/metabolismo , Citocromos/genética , Metabolismo Energético , Escherichia coli/classificação , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Hemoglobinas/genética , Oxirredução , Oxigênio/metabolismo , Consumo de Oxigênio , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , Especificidade da Espécie , Hemoglobinas Truncadas
17.
Biochim Biophys Acta ; 1506(1): 1-11, 2001 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-11418092

RESUMO

This investigation focused on the kinetics of cyanide binding to oxidized and reduced cytochrome d in Salmonella typhimurium intact cells, spheroplasts, membrane fragments and solubilized enzyme, and on the effect of pH on this binding. Cyanide bound to the oxidized form of cytochrome d under all experimental conditions, inducing a trough at 649 nm in the oxidized-cyanide-minus-oxidized difference absorption spectra. V(max) of cyanide binding to oxidized cytochrome d at pH 7.0 was 14.0+/-2.0 pmol/min/mg protein (prot.) in intact cells, 37.0+/-3.5 pmol/min/mg prot. in spheroplasts, 125.0+/-6.0 pmol/min/mg prot. in membrane fragments, and 538.0+/-8.5 pmol/min/mg prot. in solubilized cytochrome d. The pseudo-first order rate constants were 0.004 s(-1) for intact cells, 0.005 s(-1) for spheroplasts, 0.007 s(-1) for membrane fragments and 0.025 s(-1) for the solubilized enzyme. The V(max) value was highest at pH 7.0 for intact cells and solubilized cytochrome d and at pH 8.0 for both spheroplasts and membrane fragments. The K(s) of binding at pH 7.0 was around 4 mM in intact cells, spheroplasts and membrane fragments, but was 10.5 mM in solubilized cytochrome d. This difference between the K(s) values suggested a change in conformation, upon solubilization, leading to a decrease in the affinity of cyanide for the solubilized enzyme. The K(s) value was nearly the same at all pH investigated (pH 5-10). Cyanide was found to also bind to the reduced form of cytochrome d in membrane fragments (K(s)=18+/-3 mM, V(max)=377+/-28 pmol/min/mg prot. at pH 7) and the solubilized enzyme (K(s)=18+/-1.2 mM, V(max)=649+/-45 pmol/min/mg prot. at pH 7) with a lower affinity of cyanide for the reduced cytochrome d than for the oxidized enzyme. Pseudo-first order rate constants were 0.025 s(-1) and 0.042 s(-1) respectively for membrane fragments and solubilized enzyme. The value of V(max) for cyanide binding to the reduced cytochrome d, whether membrane-bound or solubilized, increased slightly with pH (for pH 6-10) while the K(s) value dropped significantly with increasing pH. The pH dependence observed here might be interpretable as a possible role for conformational transition associated with energy transduction. Finally, this investigation pointed to the influence of the microenvironment of a protein within the cell on its reactivity.


Assuntos
Cianetos/química , Grupo dos Citocromos d/metabolismo , Salmonella typhimurium/metabolismo , Esferoplastos/metabolismo , Cianetos/metabolismo , Grupo dos Citocromos d/química , Grupo dos Citocromos d/isolamento & purificação , Concentração de Íons de Hidrogênio , Cinética , Octoxinol , Oxirredução , Cianeto de Potássio/química , Cianeto de Potássio/farmacologia , Salmonella typhimurium/enzimologia , Espectrofotometria , Frações Subcelulares/metabolismo
18.
Mol Microbiol ; 38(5): 1061-73, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11123679

RESUMO

The Escherichia coli cydAB operon, encoding the subunits of the high-affinity cytochrome d oxidase, is maximally transcribed in microaerobiosis as a result of the combined action of the oxygen-responsive regulators Fnr and ArcA. Here, we report that the histone-like protein H-NS is an aerobic repressor of cydAB expression. ArcA is shown to antagonize H-NS action to render cydAB expression insensitive to H-NS repression in anaerobiosis. The targets for H-NS-mediated aerobic repression are the four oxygen-regulated promoters, designated P1, P2, P3 and P4. H-NS control is the result of H-NS binding to an extended region within the cydAB promoter element, including sequences upstream from and overlapping the four regulated promoters. We propose a regulatory model in which oxygen control of cydAB transcription is mediated by three alternative protein-DNA complexes that are assembled sequentially on the promoter region as the cells are shifted from aerobic to microaerobic and to anaerobic conditions. According to this model, ArcA-P plays a central role in cydAB regulation by antagonizing H-NS repression of cydAB transcription when oxygen becomes limiting. This allows peak gene expression and subsequent repression by Fnr under fully anaerobic conditions.


Assuntos
Proteínas de Bactérias , Grupo dos Citocromos d/genética , Escherichia coli/enzimologia , Óperon , Oxigênio/metabolismo , Sequência de Bases , Primers do DNA , Proteínas de Ligação a DNA/fisiologia , Regiões Promotoras Genéticas , Transcrição Genética/fisiologia
19.
Microbiology ; 146 ( Pt 2): 527-36, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10708391

RESUMO

The mechanism(s) that bacteria use to transport haem into and across the cytoplasmic membrane to complete the assembly of periplasmic cytochromes is unknown. The authors have tested directly the role(s) of two ATP-binding cassette (ABC) transporters - the cydDC and ccmAB gene products - in Escherichia coli by measuring haem uptake in everted (inside-out) membrane vesicles. If haem is exported to the periplasm in vivo, the same process should result in active accumulation in such everted vesicles. [14C]Haemin (chloride) with bovine serum albumin (BSA) as a carrier protein was accumulated in intact everted membrane vesicles by an energy-independent mechanism. The kinetics of this process were biphasic: rapid uptake/binding was followed by a slower uptake of haem, which was inhibited by a large excess of unlabelled haemin-BSA, but not by BSA. However, accumulated haemin was not chased out of the vesicles by unlabelled haemin-BSA, suggesting specific binding of haemin with the membrane or transport into the lumen of the vesicle. Neither ATP nor a protonmotive force (delta(p)) generated by lactate oxidation was required for haemin binding or subsequent transport, and carbonyl cyanide m-chlorophenylhydrazone (CCCP), sodium vanadate and monensin had no effect on haemin transport. The rate of haemin uptake following the initial rapid binding was proportional to the external haemin concentration, suggesting that the uptake process was driven by the haemin concentration gradient across the cell membrane. The kinetics of [14C]haemin uptake were similar in wild-type and cydD1 or delta(ccmA) mutants, suggesting that the activity of neither the CydDC nor CcmAB transporters is essential for haem export to the periplasm. Cytochrome d levels were unaffected by mutations in trxB (encoding thioredoxin reductase), trxA (thioredoxin), or grx (glutaredoxin), suggesting that the CydDC transporter does not export these components of reducing pathways for cytochrome assembly.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Membrana Celular/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Heme/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Transporte Biológico , Radioisótopos de Carbono/metabolismo , Bovinos , Grupo dos Citocromos b , Grupo dos Citocromos d/metabolismo , Citocromos/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Hemina/metabolismo , Lactose/metabolismo , Oxirredutases/metabolismo , Periplasma/enzimologia , Soroalbumina Bovina/metabolismo
20.
Microbiology ; 144 ( Pt 8): 2271-80, 1998 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9720050

RESUMO

A ferrous oxygenated form of cytochrome d is characteristic of all cytochrome bd-type oxidases so far examined, but its participation in enzyme turnover is unclear. It is relatively stable, occurs in aerated cell suspensions and predominates during enzyme preparation. In this study, diode-array reflectance spectrophotometry was used to assess the redox poise and oxygenation of cytochrome bd in vivo, in the aerobic diazotroph Azotobacter vinelandii. Mutants either lacking or overproducing the cytochrome bd oxidase were used to confirm the reliability of the optical configuration. Changes in absorbance attributed to cytochromes b, c and d were followed as the O2 supply was altered either in suspensions of harvested cells or during steady-state growth. In washed cell suspensions, three states of cytochrome d, which differed in absorbance characteristics, were seen: (1) an oxygenated form that absorbs at 650 nm, (2) a form which has little absorbance at either 650 or 630 nm and (3) the reduced form that absorbs at 630 nm. The transition between states 2 and 3, but not 1 and 2, correlated with the changes in the redox states of cytochromes b595 and b560. The dissolved O2 concentration at which this transition occurred coincided approximately with the apparent O2 affinity for the oxidase in vivo (approx. 5 microM). During steady-state growth, the cytochromes were partially reduced and the oxygenated form of cytochrome d was undetected. These in situ measurements support the view that an oxygenated form of cytochrome d (absorbing at 650 nm) in the one-electron-reduced cytochrome bd-type oxidase does not take part in enzyme turnover.


Assuntos
Azotobacter vinelandii/enzimologia , Citocromos/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons , Proteínas de Escherichia coli , Oxirredutases/metabolismo , Azotobacter vinelandii/crescimento & desenvolvimento , Azotobacter vinelandii/metabolismo , Grupo dos Citocromos b , Grupo dos Citocromos d/metabolismo , Compostos Ferrosos/metabolismo , Oxirredução , Oxigênio/metabolismo , Oxigênio/farmacologia , Soluções , Espectrofotometria Ultravioleta
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