RESUMO
OBJECTIVE: COVID-19 due to SARS-CoV-2 virus is a new cause of severe acute respiratory syndrome (SARS). Little is known about the short-term cognitive prognosis for these patients. We prospectively evaluated basic cognitive functions shortly after care in the intensive care unit (ICU) and three months later in post-ICU COVID-19 patients. MATERIAL AND METHODS: We performed a prospective single-center study in our institution in Paris. Patients with SARS-CoV-2 SARS were prospectively recruited via our ICU. Patients were evaluated using standardized cognitive tests at baseline and at three months' follow-up. Our primary endpoint was the evolution of the following five global tests: MMSE, FAB, oral naming test, Dubois five words test and MADRS. RESULTS: We explored 13 patients at baseline and follow-up. All patients had cognitive impairment at baseline but they all improved at three months, significantly on two of the five global tests after Bonferroni correction for multiple testing: MMSE (median 18 (IQR [15-22]) and 27 (IQR [27-29]) respectively, P=0.002) and FAB test (median 14 (IQR [14-17]) and 17 (IQR [17,18]) respectively, P=0.002). CONCLUSIONS: We report here the first longitudinal data on short-term cognitive impairment after intensive care in COVID-19 patients. We found acute and short-term cognitive impairment but significant improvement at three months. This pattern does not seem to differ from other causes of post-intensive care syndrome.
Assuntos
COVID-19 , Disfunção Cognitiva , COVID-19/complicações , COVID-19/epidemiologia , Disfunção Cognitiva/diagnóstico , Disfunção Cognitiva/epidemiologia , Disfunção Cognitiva/etiologia , Estado Terminal , Humanos , Unidades de Terapia Intensiva , Prevalência , SARS-CoV-2RESUMO
OBJECTIVE: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Delta variant was classified as a variant of concern in May 2021 due to its increased transmissibility. It became dominant in Europe during the summer, raising concerns on the effectiveness of vaccines. We assessed the vaccine effectiveness (VE) of mRNA BNT162b2 (BioNTech-Pfizer) against SARS-CoV-2 Delta variant during an outbreak affecting long-term care facility (LTCF) residents in southern France, May 2021. MATERIALS AND METHODS: We conducted a retrospective cohort study among LTCF residents. We described sex, age, dependency level, reverse transcription PCR and sequencing results, clinical evolution, vaccination status. We compared attack rates of SARS-CoV-2 infection, symptomatic coronavirus disease 2019 (COVID-19), and severe COVID-19 (respiratory support, hospitalization, and/or death) by vaccination status (two doses administered vs. none) to estimate VE (1 - Relative Risk [RR]) with 95% confidence intervals (CI). VE was adjusted by age (Poisson regression). RESULTS: Among 72 LTCF residents, 75.0% (n=54) were women, mean age was 88.7 (SD 8.1) years, 69% (n=49/71) were severely dependent. SARS-CoV-2 infections were identified in 39 residents (54.2%), 11 with symptomatic, and eight with severe COVID-19. All sequenced samples (n=19, 48.7%) had the same Delta variant genomic sequence. Age-adjusted BNT162b2 VE against SARS-CoV-2 Delta variant infection was 11.2% (95% CI: 0.0-61.1%), it was 88.4% (95% CI: 59.9-96.7%) against symptomatic, and 93.5% (95% CI: 67.2-98.7%) against severe COVID-19. CONCLUSIONS: We found a high BNT162b2 VE against symptomatic and severe COVID-19 caused by SARS-CoV-2 Delta variant among LTCF elderly residents, but not against Delta variant infection. This supports vaccination rollout and the implementation of control measures for close contacts among vaccinated LTCF elderly residents.
Assuntos
COVID-19 , SARS-CoV-2 , Idoso , Idoso de 80 Anos ou mais , Vacina BNT162 , COVID-19/epidemiologia , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Feminino , França/epidemiologia , Humanos , Assistência de Longa Duração , Masculino , RNA Mensageiro , Estudos Retrospectivos , SARS-CoV-2/genéticaRESUMO
This pilot prospective study assessed the association between the faecal relative abundance of extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-PE) and the occurrence of ESBL-PE related infections. Twenty-four patients were included. The median ESBL relative abundance was 32.4%. The mean ESBL-PE relative abundance (ESBL-PE-RA) was more than five-fold higher in patients exposed during the last three months to antibiotics (P = 0.002). Furthermore, the mean ESBL relative abundance was more than two-fold higher in patients colonized with non-E. coli strains (P = 0.044). The mean ESBL-PE-RA was more than 10-fold higher for the concordant patients than for the discordant patients (59.1% vs 4.9%; P < 0.001).
Assuntos
Antibacterianos , beta-Lactamases , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Fezes , Humanos , Unidades de Terapia Intensiva , Projetos Piloto , Estudos ProspectivosRESUMO
We report the cases of 2 patients hospitalized in our intensive care unit with confirmed coronavirus disease 2019 infection in whom brain MR imaging showed an unusual DWI pattern with nodular and ring-shaped lesions involving the periventricular and deep white matter. We discuss the possible reasons for these findings and their relationship to the infection.
Assuntos
Encéfalo/diagnóstico por imagem , Encéfalo/patologia , COVID-19/complicações , Adulto , Encéfalo/virologia , Humanos , Leucoencefalopatias/diagnóstico por imagem , Leucoencefalopatias/patologia , Leucoencefalopatias/virologia , Imageamento por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Neuroimagem/métodos , Síndrome do Desconforto Respiratório/virologia , SARS-CoV-2RESUMO
We report a case of a 62-year-old man treated for Streptococcus pneumoniae meningitis by ceftriaxone and dexamethasone. After neurological improvement, neurological degradation by vasculitis occurred, despite effective concentrations of ceftriaxone in the serum and cerebrospinal fluid (CSF). S. pneumoniae with increased MICs to third-generation-cephalosporins (3GC) was isolated from the ventricular fluid 10 days after the isolation of the first strain. Isolate analysis showed that a mutation in the penicillin-binding protein 2X (PBP2X) has occurred under treatment.
Assuntos
Ceftriaxona/uso terapêutico , Meningite Pneumocócica/tratamento farmacológico , Ceftriaxona/sangue , Ceftriaxona/farmacocinética , Cefalosporinas/sangue , Cefalosporinas/farmacocinética , Cefalosporinas/uso terapêutico , Dexametasona/sangue , Dexametasona/farmacocinética , Dexametasona/uso terapêutico , Humanos , Masculino , Meningite Pneumocócica/sangue , Meningite Pneumocócica/metabolismo , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/patogenicidadeRESUMO
Boron nitride nanotubes (BNNTs) are electrically insulating nanoparticles that display highly competitive elastic modulus and thermal conductivity. Long presented as potential fillers for nanocomposite applications, their poor dispersibility in most commodity polymers has, however, limited their spread. In this work, the chemical affinity of purified BNNTs, measured in terms of Hansen solubility parameters (HSP), were obtained through sedimentation tests in a wide set of organic solvents, taking into account relative sedimentation time. The parameters obtained were {δ d; δ p; δ h} = {16.8; 10.7; 14.7} ± {0.3; 0.9; 0.3} MPa1/2, with a Hildebrand parameter, δ t = 24.7 MPa1/2 and a sphere radius of 5.4 MPa1/2. The solubility parameters were determined considering complete dispersion of the purified nanomaterial, as well as the viscosity and density of the host solvent. These factors, combined with the high purity of the BNNTs, are crucial to minimize the uncertainty of the HSP characterization. Such refined values provide necessary insights both to optimize the solvent casting of unmodified BNNTs, and to orient the surface modification efforts that would be needed to integrate these nanomaterials into a wider range of host matrices.
Assuntos
Antibacterianos/uso terapêutico , Ceftriaxona/uso terapêutico , Infecções Comunitárias Adquiridas/microbiologia , Dexametasona/uso terapêutico , Meningite/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus/isolamento & purificação , Líquido Cefalorraquidiano/microbiologia , Rinorreia de Líquido Cefalorraquidiano/cirurgia , Derivações do Líquido Cefalorraquidiano , Infecções Comunitárias Adquiridas/líquido cefalorraquidiano , Infecções Comunitárias Adquiridas/diagnóstico , Infecções Comunitárias Adquiridas/tratamento farmacológico , DNA Bacteriano/líquido cefalorraquidiano , Quimioterapia Combinada , Feminino , Fístula/complicações , Fístula/cirurgia , Humanos , Imageamento por Ressonância Magnética , Meningite/líquido cefalorraquidiano , Meningite/diagnóstico , Meningite/tratamento farmacológico , Pessoa de Meia-Idade , Fístula do Sistema Respiratório/complicações , Fístula do Sistema Respiratório/cirurgia , Ribotipagem , Sinusite Esfenoidal/complicações , Sinusite Esfenoidal/diagnóstico por imagem , Sinusite Esfenoidal/microbiologia , Sinusite Esfenoidal/cirurgia , Infecções Estreptocócicas/líquido cefalorraquidiano , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/tratamento farmacológico , Espaço Subaracnóideo , Tomografia Computadorizada por Raios XRESUMO
PURPOSE: To assess preferences among individuals aged ≥80 years for a future hypothetical critical illness requiring life-sustaining treatments. METHODS: Observational cohort study of consecutive community-dwelling elderly individuals previously hospitalised in medical or surgical wards and of volunteers residing in nursing homes or assisted-living facilities. The participants were interviewed at their place of residence after viewing films of scenarios involving the use of non-invasive mechanical ventilation (NIV), invasive mechanical ventilation (IMV), and renal replacement therapy after a period of invasive mechanical ventilation (RRT after IMV). Demographic, clinical, and quality-of-life data were collected. Participants chose among four responses regarding life-sustaining treatments: consent, refusal, no opinion, and letting the physicians decide. RESULTS: The sample size was 115 and the response rate 87 %. Mean participant age was 84.8 ± 3.5 years, 68 % were female, and 81 % and 71 % were independent for instrumental activities and activities of daily living, respectively. Refusal rates among the elderly were 27 % for NIV, 43 % for IMV, and 63 % for RRT (after IMV). Demographic characteristics associated with refusal were married status for NIV [relative risk (RR), 2.9; 95 % confidence interval (95 %CI), 1.5-5.8; p = 0.002] and female gender for IMV (RR, 2.4; 95 %CI, 1.2-4.5; p = 0.01) and RRT (after IMV) (RR, 2.7; 95 %CI, 1.4-5.2; p = 0.004). Quality of life was associated with choices regarding all three life-sustaining treatments. CONCLUSIONS: Independent elderly individuals were rather reluctant to accept life-sustaining treatments, especially IMV and RRT (after IMV). Their quality of life was among the determinants of their choices.
Assuntos
Atitude Frente a Saúde , Unidades de Terapia Intensiva , Cuidados para Prolongar a Vida/psicologia , Preferência do Paciente , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Humanos , Masculino , Admissão do PacienteRESUMO
PURPOSE: To assess physician decisions about ICU admission for life-sustaining treatments (LSTs). METHODS: Observational simulation study of physician decisions for patients aged ≥80 years. Each patient was allocated at random to four physicians who made decisions based on actual bed availability and existence of an additional bed before and after obtaining information on patient preferences. The simulations involved non-invasive ventilation (NIV), invasive mechanical ventilation (IMV), and renal replacement therapy after a period of IMV (RRT after IMV). RESULTS: The physician participation rate was 100/217 (46 %); males without religious beliefs predominated, and median ICU experience was 9 years. Among participants, 85.7, 78, and 62 % felt that NIV, IMV, or RRT (after IMV) was warranted, respectively. By logistic regression analysis, factors associated with admission were age <85 years, self-sufficiency, and bed availability for NIV and IMV. Factors associated with IMV were previous ICU stay (OR 0.29, 95 % CI 0.13-0.65, p = 0.01) and cancer (OR 0.23, 95 % CI 0.10-0.52, p = 0.003), and factors associated with RRT (after IMV) were living spouse (OR 2.03, 95 % CI 1.04-3.97, p = 0.038) and respiratory disease (OR 0.42, 95 % CI 0.23-0.76, p = 0.004). Agreement among physicians was low for all LSTs. Knowledge of patient preferences changed physician decisions for 39.9, 56, and 57 % of patients who disagreed with the initial physician decisions for NIV, IMV, and RRT (after IMV) respectively. An additional bed increased admissions for NIV and IMV by 38.6 and 13.6 %, respectively. CONCLUSIONS: Physician decisions for elderly patients had low agreement and varied greatly with bed availability and knowledge of patient preferences.
Assuntos
Atitude do Pessoal de Saúde , Unidades de Terapia Intensiva , Padrões de Prática Médica , Terapia de Substituição Renal , Respiração Artificial , Triagem , Idoso de 80 Anos ou mais , Tomada de Decisões , Feminino , Humanos , MasculinoRESUMO
Bed bugs are hematophagous insects responsible for a re-emerging and challenging indoor pest in many countries. Bed bugs infestations may have health consequences including nuisance biting, cutaneous and systemic reactions. This resurgence can probably be attributed to factors such as increased international travel and development of resistance against insecticides. Resistance against pyrethroids has been reported several times from the USA and rarely in Europe. In France, very few data on bed bugs are available. The present study aimed to assess the infestation by bed bugs of a complex of two high-rise apartment buildings in the suburb of Paris and to evaluate their susceptibility to pyrethroid insecticides. We inspected for bed bugs 192 out of 198 apartments units (97%) and interviewed their residents. 76 (39.6%) apartments were infested. Among the 97 residents living in infested apartments, 53 (54.6%) reported bed bug bites. A total of 564 bed bugs were collected in the infested units. Bioassays showed that 54 out of 143 bed bugs were resistant to pyrethroids (37.8%; 95% confidence interval: 29.9-45.7%). DNA sequencing showed that all bed bugs tested (n=124) had homozygous L925I kdr-like gene mutation. The level of pyrethroid resistance found indicates that this phenomenon was already established in the site and prompts the need to reevaluate the wide use of pyrethroids to control bed bugs.
Assuntos
Percevejos-de-Cama , Habitação , Mordeduras e Picadas de Insetos/etiologia , Piretrinas , Animais , Sequência de Bases , Percevejos-de-Cama/classificação , Percevejos-de-Cama/genética , Roupas de Cama, Mesa e Banho/parasitologia , Bioensaio , DNA/química , DNA/isolamento & purificação , Técnicas de Genotipagem , Habitação/normas , Humanos , Mordeduras e Picadas de Insetos/epidemiologia , Resistência a Inseticidas , Dados de Sequência Molecular , Paris/epidemiologia , Polimorfismo de Nucleotídeo Único , Alinhamento de SequênciaRESUMO
We report the case of an athletic 49-year-old female who has run the 2011 Marathon of Paris and was addressed to the hospital for a confusion. The investigations revealed a cerebral edema complicating a severe hyponatremia secondary to an exercise-associated hyponatremia (EAH). Using 3% hypertonic saline solution, the evolution the patient rapidly improve allowing discharge after 7 days. We then discuss the importance of EAH in long-term efforts.
RESUMO
Two mAbs generated against rhodopsin kinase (RK) were characterized for their epitopes. Both antibodies recognize short peptide sequences, overlapping but distinct, close to the carboxyl terminus. Binding of RK to the antibodies is slow. Attempts were made to use the antibodies immobilized on protein A-Sepharose beads to bind and purify the enzyme. Time-dependent inactivation of the enzyme occurred after its binding to the antibodies. Studies using different conditions to maintain the enzyme in the active form during binding or to reactivate the purified inactivated enzyme were unsuccessful.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas do Olho , Inibidores de Proteínas Quinases , Sequência de Aminoácidos , Animais , Sítios de Ligação de Anticorpos , Western Blotting , Células COS , Receptor Quinase 1 Acoplada a Proteína G , Dados de Sequência Molecular , Proteínas Quinases/química , Proteínas Quinases/imunologiaRESUMO
A suitable system for expression of the rhodopsin kinase (RK) gene and its mutants is needed for structure-function studies of RK. Previously, investigation of the baculovirus system showed satisfactory production of RK, but posttranslational isoprenylation was deficient. We now report on a comparative study of expression of the RK gene in yeast (Pichia pastoris), COS-1 cells and in an HEK293 stable cell line. Expression in COS-1 cells, by using pCMV5 vector, is the most satisfactory. A two-step procedure for purification of the expressed enzyme with an N-terminal histidine tag has been developed. The purified enzyme has correct posttranslational modifications and shows a somewhat broader pH vs. catalytic activity profile than the wild-type enzyme.
Assuntos
Proteínas do Olho , Proteínas Quinases/genética , Trifosfato de Adenosina/metabolismo , Animais , Células COS , Bovinos , Linhagem Celular , Cromatografia Líquida , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Receptor Quinase 1 Acoplada a Proteína G , Humanos , Concentração de Íons de Hidrogênio , Proteínas Quinases/isolamento & purificação , Proteínas Quinases/metabolismo , Prenilação de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Retina/enzimologiaRESUMO
Previously, we reported that the carboxyl-reacting reagent DCCD, and its fluorescent derivative NCD-4 binds covalently to aspartate-160 localized in amphipathic helix cd of the CD loop connecting membrane-spanning helices C and D of cytochrome b (Wang et al., 1995). We have investigated the fluorescent properties of NCD-4 to probe possible changes in the cd helix resulting from the binding of exogenous ubiquinol analogues to the bc1 complex. Preincubation of the bc1 complex with the reduced substrate analogues, DQH2, DBH2, and Q6H2 resulted in 20-40% increase in the fluorescence emission intensity of NCD-4 and a 10-20% increase in the binding of [14C]DCCD to the bc1 complex. By contrast, preincubation with the oxidized analogues DQ. DB, and Q6 resulted in a 20-40% decrease in the fluorescence emission intensity of NCD-4 and a 20-40% decrease in the binding of [14C]DCCD to the bc1 complex. Moreover, addition of the reduced ubiquinols to the bc1 complex preincubated with NCD-4 resulted in a blue shift in the fluorescence emission spectrum. In addition, incubation of the cytochrome bc1 complex reconstituted into proteoliposomes with both reduced and oxidized ubiquinol analogues resulted in changes in the quenching of NCD-4 fluorescence by CAT-16, the spin-label probe that intercalates at the membrane surface. These results indicate that the addition of exogenous ubiquinol to the bc1 complex may result in changes in the cd helix leading to a more hydrophobic environment surrounding the NCD-4 binding site. By contrast, preincubation with the inhibitors of electron transfer through the bc1 complex had no effect on the binding of NCD-4 to the bc1 complex or on the fluorescent emission spectra, which suggests that the binding of the inhibitors does not result in changes in the environment of the NCD-4 binding site.
Assuntos
Ácido Aspártico/metabolismo , Proteínas de Bactérias , Carbodi-Imidas/metabolismo , Grupo dos Citocromos b/metabolismo , Ferritinas/metabolismo , Corantes Fluorescentes/metabolismo , Ubiquinona/análogos & derivados , Sequência de Aminoácidos , Dicicloexilcarbodi-Imida/metabolismo , Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Ubiquinona/química , LevedurasRESUMO
Structure-function studies of rhodopsin kinase (RK; EC 2.7.1.125) require a variety of mutants. Therefore, there is need for a suitable system for the expression of RK mutant genes. Here we report on a study of expression of the RK gene in baculovirus-infected Sf21 cells and characterization of the enzyme produced as purified to near homogeneity. Particular attention has been paid to the post-translational modifications, autophosphorylation and isoprenylation, found in the native bovine RK. The protein produced has been purified using, successively, heparin-Sepharose, Mono Q, and Mono S FPLC (fast protein liquid chromatography) and was obtained in amounts of about 2 mg from 1 liter of cell culture. The enzyme from the last step of purification was obtained in two main fractions that differ in the level of phosphorylation. The protein peak eluted first carries two phosphate groups per protein, whereas the second protein peak is monophosphorylated. Further, while both peaks are isoprenylated, the isoprenyl groups consist of mixtures of C5, C10, C15, and C20 isoprenyl moieties. From these results, we conclude that the above expression system is suitable for some but not all aspects of structure-function studies.
Assuntos
Baculoviridae/genética , Proteínas do Olho , Proteínas Quinases/genética , Processamento de Proteína Pós-Traducional , Animais , Bovinos , Linhagem Celular , Clonagem Molecular , Receptor Quinase 1 Acoplada a Proteína G , Concentração de Íons de Hidrogênio , Fosforilação , Proteínas Quinases/metabolismo , Prenilação de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera , TemperaturaRESUMO
A cDNA carrying the Rip1 gene, which encodes the Rieske iron-sulfur protein of Schizosaccharomyces pombe, has been cloned by complementing the respiratory deficiency of a Saccharomyces cerevisiae strain in which the endogenous copy of the RIP1 gene has been deleted. The deduced amino acid sequences of the S. pombe and S. cerevisiae iron-sulfur proteins are 50% identical, with the highest region of identity being in the C termini of the proteins, where the 2Fe:2S cluster is bound. When expressed in the S. cerevisiae deletion strain, the S. pombe iron-sulfur protein restores 25-30% of the ubiquinol-cytochrome c reductase activity. The kinetics of cytochrome c reduction, the effects of inhibitors which act at defined sites in the cytochrome bc1 complex, and the optical properties of cytochrome b in membranes from the S. cerevisiae deletion strain complemented with S. pombe iron-sulfur protein indicate that the S. pombe protein interacts with cytochrome b to restore an apparently normal ubiquinol oxidase site, but that interaction between the iron-sulfur protein and cytochrome c1 is partially impaired. This is the first heterologous replacement of an electron transfer protein in a respiratory enzyme complex in S. cerevisiae.
Assuntos
Proteínas Ferro-Enxofre/genética , Saccharomyces cerevisiae/enzimologia , Schizosaccharomyces/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Genes Fúngicos , Teste de Complementação Genética , Dados de Sequência Molecular , Oxirredução , Estrutura Secundária de Proteína , Schizosaccharomyces/enzimologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Análise EspectralRESUMO
We have investigated the function of subunit 8 of the cytochrome bc1 complex by generating six site-directed mutants, F46C, R51S, P62V, G64A, R91N, and W69-stop, in the cloned QCR8 gene and expressing the mutated genes in a Saccharomyces cerevisiae strain in which the chromosomal copy of QCR8 is deleted. The W69-stop mutation impairs assembly of the bc1 complex and growth of yeast on nonfermentable carbon sources as does deletion of QCR8 [Maarse, A. C., De Haan, M., Schoppink, P. J., Berden J. A., and Grivell, L. A. (1988) Eur. J. Biochem. 172, 179-184], implying that the C-terminus of subunit 8 is important for assembly and/or the stability of the bc1 complex. The F46C, R51S, P62V, G64A, and R91N mutations do not affect the growth of yeast on nonfermentable carbon sources, not do they lower the activity or alter the inhibitor sensitivity of the bc1 complex. Rather, some of the mutations increase the cytochrome C reductase activity of the bc1 complex by as much as 40%. However, succinate-ubiquinone reductase activity was consistently reduced 40-60% in mitochondrial membranes from these mutants, while NADH-ubiquinone reductase activity was not affected. In addition, the activation of succinate-ubiquinone reductase activity by succinate was diminished by the F46C, R51S, P62V, and G64A mutations. These results indicate that the cytochrome bc1 complex participates in electron transfer from succinate to ubiquinone in situ and also suggest an interaction between succinate-ubiquinone reductase and cytochrome bc1 complex which involves subunit 8 of the bc1 complex.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexos Multienzimáticos/metabolismo , Oxirredutases/metabolismo , Saccharomyces cerevisiae/metabolismo , Succinato Desidrogenase/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Transporte de Elétrons , Complexo II de Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/genética , Ativação Enzimática , Genes Fúngicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenótipo , Conformação Proteica , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/genética , Deleção de SequênciaRESUMO
Four mutations in the mitochondrial cytochrome b of S. cerevisiae have been characterized with respect to growth capacities, catalytic properties, ATP/2e- ratio, and transmembrane potential. The respiratory-deficient mutant G137E and the three pseudo-wild type revertants E137 + I147F, E137 + C133S, and E137 + N256K were described previously (Tron and Lemesle-Meunier, 1990; Di Rago et al., 1990a). The mutant G137E is unable to grow on respiratory substrates but its electron transfer activity is partly conserved and totally inhibited by antimycin A. The secondary mutations restore the respiratory growth at variable degree, with a phosphorylation efficiency of 12-42% as regards the parental wild type strain, and result in a slight increase in the various electron transfer activities at the level of the whole respiratory chain. The catalytic efficiency for ubiquinol was slightly (G137E) or not affected (E137 + I147F, E137 + C133S, and E137 + N256K) in these mutants. Mutation G137E induces a decrease in the ATP/2e- ratio (50% of the W.T. value) and transmembrane potential (60% of the W.T. value) at the bc1 level, whereas the energetic capacity of the cytochrome oxidase is conserved. Secondary mutations I147F, C133S, and N256K partly restore the ATP/2e- ratio and the transmembrane potential at the bc1 complex level. The results suggest that a partial decoupling of the bc1 complex is induced by the cytochrome b point mutation G137E. In the framework of the protonmotive Q cycle, this decoupling can be explained by the existence of a proton wire connecting centers P and N in the wild type bc1 complex which may be amplified or uncovered by the G137E mutation when the bc1 complex is functioning.
Assuntos
Grupo dos Citocromos b/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Genes Fúngicos , Mutação Puntual , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Grupo dos Citocromos b/química , Grupo dos Citocromos b/genética , Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons/genética , Membranas Intracelulares/fisiologia , Cinética , Potenciais da Membrana , Mitocôndrias/enzimologia , Mitocôndrias/fisiologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Consumo de Oxigênio , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genéticaRESUMO
Trp-142 is a highly conserved residue of the cytochrome b subunit in the bc1 complexes. To study the importance of this residue in the quinol oxidation catalyzed by the bc1 complex, we characterized four yeast mutants with arginine, lysine, threonine, and serine at position 142. The mutant W142R was isolated previously as a respiration-deficient mutant unable to grow on non-fermentable carbon sources (Lemesle-Meunier, D., Brivet-Chevillotte, P., di Rago, J.-P, Slonimski, P.P., Bruel, C., Tron, T., and Forget, N. (1993) J. Biol. Chem. 268, 15626-15632). The mutants W142K, W142T, and W142S were obtained here as respiration-sufficient revertants from mutant W142R. Mutant W142R exhibited a decreased complex II turnover both in the presence and absence of antimycin A; this suggests that the structural effect of W142R in the bc1 complex probably interferes with the correct assembly of the succinate-ubiquinone reductase complex. The mutations resulted in a parallel decrease in turnover number and apparent Km, with the result that there was no significant change in the second-order rate constant for ubiquinol oxidation. Mutants W142K and W142T exhibited some resistance toward myxothiazol, whereas mutant W142R showed increased sensitivity. The cytochrome cc1 reduction kinetics were found to be severely affected in mutants W142R, W142K, and W142T. The respiratory activities and the amounts of reduced cytochrome b measured during steady state suggest that the W142S mutation also modified the quinol-cytochrome c1 electron transfer pathway. The cytochrome b reduction kinetics through center P were affected when Trp-142 was replaced with arginine or lysine, but not when it was replaced with threonine or serine. Of the four amino acids tested at position 142, only arginine resulted in a decrease in cytochrome b reduction through center N. These findings are discussed in terms of the structure and function of the quinol oxidation site and seem to indicate that Trp-142 is not critical to the kinetic interaction of ubiquinol with the reductase, but plays an important role in the electron transfer reactions that intervene between ubiquinol oxidation and cytochrome c1 reduction.