RESUMO
STUDY QUESTION: How is the semen quality of sexually active men following recovery from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection? SUMMARY ANSWER: Twenty-five percent of the men with recent SARS-Cov-2 infections and proven healing were oligo-crypto-azoospermic, despite the absence of virus RNA in semen. WHAT IS KNOWN ALREADY: The presence of SARS-CoV-2 in human semen and its role in virus contagion and semen quality after recovery from coronavirus disease 2019 (COVID-19) is still unclear. So far, studies evaluating semen quality and the occurrence of SARS-CoV-2 in semen of infected or proven recovered men are scarce and included a limited number of participants. STUDY DESIGN, SIZE, DURATION: A prospective cross-sectional study on 43 sexually active men who were known to have recovered from SARS-CoV2 was performed. Four biological fluid samples, namely saliva, pre-ejaculation urine, semen, and post-ejaculation urine, were tested for the SARS-CoV-2 genome. Female partners were retested if any specimen was found to be SARS-CoV-2 positive. Routine semen analysis and quantification of semen leukocytes and interleukin-8 (IL-8) levels were performed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Questionnaires including International Index of Erectile Function and Male Sexual Health Questionnaire Short Form were administered to all subjects. The occurrence of virus RNA was evaluated in all the biological fluids collected by RT-PCR. Semen parameters were evaluated according to the World Health Organization manual edition V. Semen IL-8 levels were evaluated by a two-step ELISA method. MAIN RESULTS AND THE ROLE OF CHANCE: After recovery from COVID-19, 25% of the men studied were oligo-crypto-azoospermic. Of the 11 men with semen impairment, 8 were azoospermic and 3 were oligospermic. A total of 33 patients (76.7%) showed pathological levels of IL-8 in semen. Oligo-crypto-azoospermia was significantly related to COVID-19 severity (P < 0.001). Three patients (7%) tested positive for at least one sample (one saliva; one pre-ejaculation urine; one semen and one post-ejaculation urine), so the next day new nasopharyngeal swabs were collected. The results from these three patients and their partners were all negative for SARS-CoV-2. LIMITATIONS, REASONS FOR CAUTION: Although crypto-azoospermia was found in a high percentage of men who had recovered from COVID-19, clearly exceeding the percentage found in the general population, the previous semen quality of these men was unknown nor is it known whether a recovery of testicular function was occurring. The low number of enrolled patients may limit the statistical power of study. WIDER IMPLICATIONS OF THE FINDINGS: SARS-CoV-2 can be detected in saliva, urine, and semen in a small percentage of men who recovered from COVID-19. One-quarter of men who recovered from COVID-19 demonstrated oligo-crypto-azoospermia indicating that an assessment of semen quality should be recommended for men of reproductive age who are affected by COVID-19. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.
Assuntos
COVID-19 , SARS-CoV-2 , Estudos Transversais , Feminino , Humanos , Masculino , Estudos Prospectivos , RNA Viral , Sêmen , Análise do SêmenRESUMO
Whole-genome microarray analysis of Geobacter sulfurreducens grown on insoluble Fe(III) oxide or Mn(IV) oxide versus soluble Fe(III) citrate revealed significantly different expression patterns. The most upregulated genes, omcS and omcT, encode cell-surface c-type cytochromes, OmcS being required for Fe(III) and Mn(IV) oxide reduction. Other electron transport genes upregulated on both metal oxides included genes encoding putative menaquinolâ:âferricytochrome c oxidoreductase complexes Cbc4 and Cbc5, periplasmic c-type cytochromes Dhc2 and PccF, outer membrane c-type cytochromes OmcC, OmcG and OmcV, multicopper oxidase OmpB, the structural components of electrically conductive pili, PilA-N and PilA-C, and enzymes that detoxify reactive oxygen/nitrogen species. Genes upregulated on Fe(III) oxide encode putative menaquinolâ:âferricytochrome c oxidoreductase complexes Cbc3 and Cbc6, periplasmic c-type cytochromes, including PccG and PccJ, and outer membrane c-type cytochromes, including OmcA, OmcE, OmcH, OmcL, OmcN, OmcO and OmcP. Electron transport genes upregulated on Mn(IV) oxide encode periplasmic c-type cytochromes PccR, PgcA, PpcA and PpcD, outer membrane c-type cytochromes OmaB/OmaC, OmcB and OmcZ, multicopper oxidase OmpC and menaquinone-reducing enzymes. Genetic studies indicated that MacA, OmcB, OmcF, OmcG, OmcH, OmcI, OmcJ, OmcM, OmcV and PccH, the putative Cbc5 complex subunit CbcC and the putative Cbc3 complex subunit CbcV are important for reduction of Fe(III) oxide but not essential for Mn(IV) oxide reduction. Gene expression patterns for Geobacter uraniireducens were similar. These results demonstrate that the physiology of Fe(III)-reducing bacteria differs significantly during growth on different insoluble and soluble electron acceptors and emphasize the importance of c-type cytochromes for extracellular electron transfer in G. sulfurreducens.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Transporte de Elétrons , Compostos Férricos/metabolismo , Geobacter/enzimologia , Geobacter/metabolismo , Compostos de Manganês/metabolismo , Óxidos/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Perfilação da Expressão Gênica , Análise em MicrossériesRESUMO
OBJECTIVES: To evaluate the performance of an isothermal microcalorimetry (IMC) method for determining the MICs among extensively drug-resistant Gram-negative bacilli. METHODS: A collection of 320 clinical isolates (n = 80 of each) of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii from Sweden, Spain, Italy and the Netherlands were tested. The MICs were determined using the IMC device calScreener (Symcel, Stockholm, Sweden) and ISO-broth microdilution as the reference method. Essential agreement, categorical agreement, very major errors (VME), major errors (ME) and minor (mE) errors for each antibiotic were determined. RESULTS: Data from 316 isolates were evaluated. Four errors (two ME, one VME, one mE) among 80 K. pneumoniae, six errors (four ME, one VME, one mE) among 79 E. coli, 15 errors (seven VME, three ME, five mE) among 77 P. aeruginosa and 18 errors (12 VME, two ME, four mE) among 80 A. baumannii were observed. Average essential agreement and categorical agreement of the IMC method were 96.6% (95% confidence interval, 94.2-99) and 97.1% (95% confidence interval, 95.4-98.5) respectively when the MICs were determined at the end of 18 hours. Categorical agreement of the IMC method for prediction of MIC by the end of 8 hours for colistin, meropenem, amikacin, ciprofloxacin and piperacillin/tazobactam were 95%, 91.4%, 94%, 95.2% and 93.7% respectively. CONCLUSIONS: The IMC method could accurately determine the MICs among extensively drug-resistant clinical isolates of E. coli, K. pneumoniae, P. aeruginosa and A. baumannii isolates.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Calorimetria/métodos , Farmacorresistência Bacteriana Múltipla/fisiologia , Escherichia coli/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Acinetobacter baumannii/isolamento & purificação , Acinetobacter baumannii/metabolismo , Amicacina/farmacologia , Antibacterianos/farmacologia , Ciprofloxacina/farmacologia , Colistina/farmacologia , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Humanos , Itália , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/metabolismo , Meropeném/farmacologia , Testes de Sensibilidade Microbiana , Países Baixos , Combinação Piperacilina e Tazobactam/farmacologia , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismo , Espanha , SuéciaRESUMO
OBJECTIVES: The aim was to evaluate different methods for testing carbapenem susceptibility of Escherichia coli producing KPC-type carbapenemase. METHODS: Susceptibility to imipenem, meropenem and ertapenem was assayed using the reference broth microdilution method and several commercial methods (Vitek2, MicroScan, Etest, MIC Test Strip) starting from the same bacterial suspension. Susceptibility to imipenem and meropenem was also tested by Sensititre and disc diffusion (Bio-Rad). Results were interpreted according to EUCAST clinical breakpoints. Essential agreement (EA), category agreement (CA) and error rates were calculated as described by the International Organization for Standardization (ISO) guidelines and also considering the new EUCAST definitions. Genotypic diversity of isolates was evaluated with a RAPD profiling protocol. RESULTS: Of 54 KPC-positive E. coli isolates, 5.6%, 7.4% and 0% were susceptible standard dosing regimen (S), 55.6%, 72.2% and 0% susceptible increased exposure (I), and 38.9%, 20.4% and 100.0% resistant (R) to imipenem, meropenem and ertapenem, respectively, using the reference broth microdilution method. CA lower than 90% were observed with all systems for imipenem and meropenem using both the ISO and the modified EUCAST criteria. With ertapenem, CA >90% was observed with all methods except Vitek2. RAPD profiling revealed a remarkable genotypic diversity of the isolates, supporting that results were not biased by an oligoclonal nature of the collection. CONCLUSIONS: Commercial methods can be unreliable for testing susceptibility to carbapenems of KPC-producing E. coli. Susceptibility should be confirmed by reference broth microdilution.
Assuntos
Antibacterianos/farmacologia , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Enterobacteriáceas Resistentes a Carbapenêmicos/enzimologia , Carbapenêmicos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Enterobacteriáceas Resistentes a Carbapenêmicos/classificação , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Erros de Diagnóstico , Ertapenem/farmacologia , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Genótipo , Humanos , Imipenem/farmacologia , Meropeném/farmacologia , Testes de Sensibilidade Microbiana/métodos , Tipagem Molecular , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
OBJECTIVES: To evaluate a novel method, the colistin-MAC test, for phenotypic screening of acquired colistin resistance mediated by transferable mcr-1 resistance determinants, based on colistin MIC reduction in the presence of dipicolinic acid (DPA). METHODS: The colistin-MAC test consists in a broth microdilution method, in which colistin MIC is tested in the absence or presence of DPA (900 µg/mL). Overall, 74 colistin-resistant strains of Enterobacteriaceae (65 Escherichia coli and nine other species), including 61 strains carrying mcr-1-like genes and 13 strains negative for mcr genes, were evaluated with the colistin-MAC test. The presence of mcr-1-like and mcr-2-like genes was assessed by real-time PCR and end-point PCR. For 20 strains, whole-genome sequencing data were also available. RESULTS: A ≥8-fold reduction of colistin MIC in the presence of DPA was observed with 59 mcr-1-positive strains, including 53 E. coli of clinical origin, three E. coli transconjugants carrying MCR-1-encoding plasmids, one Enterobacter cloacae complex and two Citrobacter spp. Colistin MICs were unchanged, increased or at most reduced by twofold with the 13 mcr-negative colistin-resistant strains (nine E. coli and four Klebsiella pneumoniae), but also with two mcr-1-like-positive K. pneumoniae strains. CONCLUSIONS: The colistin-MAC test could be a simple phenotypic test for presumptive identification of mcr-1-positive strains among isolates of colistin-resistant E. coli, based on a ≥8-fold reduction of colistin MIC in the presence of DPA. Evaluation of the test with a larger number of strains, species and mcr-type resistance determinants would be of interest.
Assuntos
Antibacterianos/farmacologia , Colistina/farmacologia , Genes Bacterianos/genética , Testes de Sensibilidade Microbiana/métodos , Farmacorresistência Bacteriana , Escherichia coli/genética , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Humanos , FenótipoRESUMO
Na,K-ATPase, an essential transporter of mammalian cells, is an oligomeric transmembrane protein composed of two subunits, alpha and beta, of which there are several isoforms. In this study, we demonstrate that the alpha1 and alpha2 isoforms of the Na,K-ATPase alpha subunit are modified by the covalent attachment of ubiquitin polymers in COS-7 cells. We propose that polyubiquitination of the Na,K-ATPase alpha subunit may play a role in regulating its degradation.
Assuntos
ATPase Trocadora de Sódio-Potássio/metabolismo , Ubiquitinas/metabolismo , Animais , Células COS , Membrana Celular/enzimologia , Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma , Processamento de Proteína Pós-TraducionalRESUMO
AIM: To determine whether there has been an increase in deaths by overdose in Italy, as elsewhere in the Western world, over the past fifteen years. METHOD: This study's conclusions are based on analysis of official data on overdose deaths attributed to illicit drug addiction and abuse (ICD-9 codes 304 and 305) from 1984 to 2000, drawn from two archives of drug abuse information: the Direzione Centrale per i Servizi Antidroga (DADE) of the Italian Ministry of the Interior (1984-2000), and the Health Statistics held at the Italian Central Statistics Institute (ISTAT) (1984-1997). Mortality rates have been calculated for both genders in the following age groups: 15-24, 25-34, and 35-44 years. RESULTS: Official data indicate that there has been a steady increase in the number of deaths by overdose in Italy over recent 15 years. This trend has affected both genders, but is more evident among males. Over the whole period females had consistently lower overdose rates than males. In both genders the age group 35-44 was subject to the highest mortality rate increase over the study period, however, the highest overdose rates for both males and females were observed in the 25-34 age group. Consistently higher rates were witnessed in the northern regions of Italy with an overall increase across all latitudes. However, the greatest increase over the study period occurred in the South. In 5190 cases evaluated by the Italian Forensic Toxicology Group, 95.9% of deaths were attributed to heroin, but in about half of these, mixtures of three or more substances (heroin, benzodiazepines, cannabinoids, cocaine, methadone) were found in the deceased at doses that were likely to have contributed to death. CONCLUSIONS: The increase observed in the rates of death by overdose is likely to be a reflection of increased use of illicit drugs in the general population. Reporting practice by forensic pathologists might explain the extent of attribution of cause of death to heroin. Drop-out from addiction treatment is a commonly observed antecedent of fatal opioid overdose, therefore, caution is required when establishing treatment protocol for patients. Interventions that aim specifically at improving patient compliance with medical and psychiatric therapies should be favoured.
Assuntos
Drogas Ilícitas/efeitos adversos , Mortalidade/tendências , Adolescente , Adulto , Intervalos de Confiança , Overdose de Drogas/mortalidade , Feminino , Heroína/efeitos adversos , Humanos , Itália/epidemiologia , Modelos Lineares , Masculino , Fatores SexuaisRESUMO
Seasonal asymmetry in yearly suicide occurrence is a long-observed phenomenon in psychiatric, suicidological, and sociological research, and the effects of seasonal factors on suicidal behavior have been the focus of a number of earlier studies. Taking into account limitations of data and methods, these studies have generally favored interpretations based on psychosocial factors. Recent studies have challenged the widely held notion that seasonal effects on suicide are a unitary phenomenon, not influenced by age, gender, or circumstances of the act. In particular, the seasonal occurrence of suicides has been found to differ significantly between the young and the elderly, and differences have also been found between male and female cycles of occurrence. Suicides using violent methods have been shown to follow clearer seasonal patterns than suicides by less violent methods (such as drug or gas poisoning), possibly reflecting the greater impulsive component involved in the choice of a violent lethal means. In this paper, findings from Italy are used to illustrate the clinical implications of studies into the topic of season and suicide, with the aim of developing more effective preventative strategies.
Assuntos
Estações do Ano , Suicídio/tendências , Feminino , Humanos , Itália , MasculinoRESUMO
A case of simple intraparenchymal testicular cyst in a two and half months old child is reported. Dysplastic cysts of the testicle are exceedingly rare, in children only eight such cases have been described in the Literature, all, except one, treated by orchiectomy. In the herein reported case a conservative approach with cyst enucleation and sparing of the residual testicular parenchyma has been satisfactorily employed.
Assuntos
Cistos/diagnóstico , Doenças Testiculares/diagnóstico , Humanos , Lactente , Masculino , UltrassonografiaRESUMO
Fifteen cases of epididymitis in children are reviewed, six of these occurring in children under twelve months of age, and nine in older children. In five of the six cases occurring in infants a U.T.I. and a malformation of the urinary tract were found, while in children over one year only three infections and two urinary malformations were discovered. Authors recommend that in infants under 1 year presenting with epididymitis a thorough investigation of the urinary tract is performed.
Assuntos
Epididimite/etiologia , Infecções Urinárias/complicações , Sistema Urinário/anormalidades , Doença Aguda , Criança , Pré-Escolar , Humanos , Lactente , MasculinoRESUMO
Geobacter sulfurreducens is a well-studied representative of the Geobacteraceae, which play a critical role in organic matter oxidation coupled to Fe(III) reduction, bioremediation of groundwater contaminated with organics or metals, and electricity production from waste organic matter. In order to investigate G. sulfurreducens central metabolism and electron transport, a metabolic model which integrated genome-based predictions with available genetic and physiological data was developed via the constraint-based modeling approach. Evaluation of the rates of proton production and consumption in the extracellular and cytoplasmic compartments revealed that energy conservation with extracellular electron acceptors, such as Fe(III), was limited relative to that associated with intracellular acceptors. This limitation was attributed to lack of cytoplasmic proton consumption during reduction of extracellular electron acceptors. Model-based analysis of the metabolic cost of producing an extracellular electron shuttle to promote electron transfer to insoluble Fe(III) oxides demonstrated why Geobacter species, which do not produce shuttles, have an energetic advantage over shuttle-producing Fe(III) reducers in subsurface environments. In silico analysis also revealed that the metabolic network of G. sulfurreducens could synthesize amino acids more efficiently than that of Escherichia coli due to the presence of a pyruvate-ferredoxin oxidoreductase, which catalyzes synthesis of pyruvate from acetate and carbon dioxide in a single step. In silico phenotypic analysis of deletion mutants demonstrated the capability of the model to explore the flexibility of G. sulfurreducens central metabolism and correctly predict mutant phenotypes. These results demonstrate that iterative modeling coupled with experimentation can accelerate the understanding of the physiology of poorly studied but environmentally relevant organisms and may help optimize their practical applications.
Assuntos
Geobacter/metabolismo , Ferro/metabolismo , Aminoácidos/biossíntese , Transporte de Elétrons , Escherichia coli/metabolismo , Fumaratos/metabolismo , Geobacter/genética , Modelos Biológicos , Mutação , Oxirredução , Fenótipo , Prótons , Quinonas/metabolismo , Especificidade da EspécieRESUMO
The potential role of outer membrane proteins in electron transfer to insoluble Fe(III) oxides by Geobacter sulfurreducens was investigated because this organism is closely related to the Fe(III) oxide-reducing organisms that are predominant in many Fe(III)-reducing environments. Two of the most abundant proteins that were easily sheared from the outer surfaces of intact cells were c-type cytochromes. One, designated OmcS, has a molecular mass of ca. 50 kDa and is predicted to be an outer membrane hexaheme c-type cytochrome. Transcripts for omcS could be detected during growth on Fe(III) oxide, but not on soluble Fe(III) citrate. The omcS mRNA consisted primarily of a monocistronic transcript, and to a lesser extent, a longer transcript that also contained the downstream gene omcT, which is predicted to encode a second hexaheme outer membrane cytochrome with 62.6% amino acid sequence identity to OmcS. The other abundant c-type cytochrome sheared from the outer surface of G. sulfurreducens, designated OmcE, has a molecular mass of ca. 30 kDa and is predicted to be an outer membrane tetraheme c-type cytochrome. When either omcS or omcE was deleted, G. sulfurreducens could no longer reduce Fe(III) oxide but could still reduce soluble electron acceptors, including Fe(III) citrate. The mutants could reduce Fe(III) in Fe(III) oxide medium only if the Fe(III) chelator, nitrilotriacetic acid, or the electron shuttle, anthraquinone 2,6-disulfonate, was added. Expressing omcS or omcE in trans restored the capacity for Fe(III) oxide reduction. OmcT was not detected among the sheared proteins, and genetic studies indicated that G. sulfurreducens could not reduce Fe(III) oxide when omcT was expressed but OmcS was absent. In contrast, Fe(III) oxide was reduced when omcS was expressed in the absence of OmcT. These results suggest that OmcS and OmcE are involved in electron transfer to Fe(III) oxides in G. sulfurreducens. They also emphasize the importance of evaluating mechanisms for Fe(III) reduction with environmentally relevant Fe(III) oxide, rather than the more commonly utilized Fe(III) citrate, because additional electron transfer components are required for Fe(III) oxide reduction that are not required for Fe(III) citrate reduction.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Citocromos c/metabolismo , Compostos Férricos/metabolismo , Geobacter/metabolismo , Compostos de Manganês/metabolismo , Óxidos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Citocromos c/química , Citocromos c/genética , Primers do DNA , Cinética , Dados de Sequência Molecular , Oxirredução , Fragmentos de Peptídeos/químicaRESUMO
Ammonia is a bacterial metabolite which is commonly used to alter cytoplasmic and lysosomal pH of eukaryotic cells. Here we examine its effect on external N-formyl peptide receptors of human neutrophils. Ammonia does not affect the number of N-formyl peptide receptors on the cell surface, nor the association of the ligand-receptor complex with the cytoskeleton. However, ammonia causes a marked decrease in the affinity of the chemotactic peptide receptor for its ligand. The Kd of untreated cell for the chemotactic peptide was 0.65 +/- 0.06 nM, whereas that of ammonia treated cells was 1.02 +/- 0.10 nM (Mean +/- SEM, N = 6). These results suggest that ammonia can affect external as well as internal cellular components. Since ammonia is used to alter lysosomal and cytoplasmic pH, and is a metabolite of common bacterial pathogens, these results bear directly on its use in cell biology and on its potential as a virulence factor.
Assuntos
Amônia/farmacologia , Citoesqueleto/metabolismo , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Neutrófilos/metabolismo , Receptores Imunológicos/metabolismo , Sequência de Aminoácidos , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Fluoresceínas/metabolismo , Humanos , Cinética , Ligantes , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Receptores de Formil PeptídeoRESUMO
In the rat adipocyte, insulin increases potassium uptake by a preferential activation of the alpha2 isoform of the Na,K-ATPase. The question under consideration here is whether expression of the alpha2 isoform is sufficient to replicate its differential activation by insulin. Accordingly, we compared the effect of insulin on the activity of the ouabain resistant rat alpha1 and alpha2RD (alpha2L111R,N122D) isoforms in HeLa cells. In HeLa cells, in contrast to the rat adipocyte, insulin produces an increase of equal magnitude in the rate of 86Rb+/K+ uptake by the ouabain resistant rat alpha1 and rat alpha2RD subunits. We conclude that the mechanism of insulin activation of the alpha2RD isoform in HeLa cells differs from that of the wild type alpha2 isoform in the rat adipocyte.
Assuntos
Adipócitos/enzimologia , Células HeLa/enzimologia , Insulina/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Sequência de Aminoácidos , Animais , Inibidores Enzimáticos/farmacologia , Furosemida/farmacologia , Humanos , Isoenzimas/metabolismo , Dados de Sequência Molecular , Ouabaína/farmacologia , Potássio/metabolismo , Ratos , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
The Na,K-ATPase is an essential plasma membrane transporter of mammalian cells composed of two subunits, alpha and beta, of which there are several isoforms. We investigated the effect of a substitution, S364P, on the subcellular localization and enzymatic activity of the wild-type alpha2 and alpha2L111R,N122D (alpha2RD) subunits. The substitutions, L111R and N122D, lower the affinity of the alpha2 subunit for the inhibitor ouabain roughly one thousand-fold (E. A. Jewell and J. B. Lingrel, J. Biol. Chem. 266, 16925-16930, 1991) and were introduced into the alpha2 subunit to distinguish its enzymatic activity from that of the endogenous alpha1 subunit of COS-7 cells. The S364P substitution is located in the ATP binding site, only five residues from the aspartyl residue which is phosphorylated during the catalytic cycle of the Na,K-ATPase. This substitution dramatically decreases the amount of enzymatic activity associated with expression of the alpha2RD subunit. Despite the fact that S364P substitution does not block association of the alpha2RD subunit with the endogenous beta1 subunit, it prevents the alpha2 and alpha2RD subunits from accumulating in the plasma membrane and results in their localization in the endoplasmic reticulum.
Assuntos
Mutação , ATPase Trocadora de Sódio-Potássio/análise , ATPase Trocadora de Sódio-Potássio/genética , Animais , Sítios de Ligação/genética , Transporte Biológico , Western Blotting , Células COS , Fracionamento Celular , Retículo Endoplasmático/química , Genes Reporter/genética , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Orthomyxoviridae/química , Orthomyxoviridae/genética , Ouabaína/farmacologia , Fosforilação , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , TransfecçãoRESUMO
Geobacter sulfurreducens, previously classified as a strict anaerobe, tolerated exposure to atmospheric oxygen for at least 24 h and grew with oxygen as the sole electron acceptor at concentrations of 10% or less in the headspace. These results help explain how Geobacter species may survive in oxic subsurface environments, being poised to rapidly take advantage of the development of anoxic conditions.
Assuntos
Geobacter/crescimento & desenvolvimento , Geobacter/metabolismo , Aerobiose , Anaerobiose , Transporte de Elétrons , Microbiologia Ambiental , Oxigênio/metabolismoRESUMO
Microorganisms in the family Geobacteraceae are the predominant Fe(III)-reducing microorganisms in a variety of subsurface environments in which Fe(III) reduction is an important process, but little is known about the mechanisms for electron transport to Fe(III) in these organisms. The Geobacter sulfurreducens genome was found to contain a 10-kb chromosomal duplication consisting of two tandem three-gene clusters. The last genes of the two clusters, designated omcB and omcC, encode putative outer membrane polyheme c-type cytochromes which are 79% identical. The role of the omcB and omcC genes in Fe(III) reduction in G. sulfurreducens was investigated. OmcB and OmcC were both expressed during growth with acetate as the electron donor and either fumarate or Fe(III) as the electron acceptor. OmcB was ca. twofold more abundant under both conditions. Disrupting omcB or omcC by gene replacement had no impact on growth with fumarate. However, the OmcB-deficient mutant was greatly impaired in its ability to reduce Fe(III) both in cell suspensions and under growth conditions. In contrast, the ability of the OmcC-deficient mutant to reduce Fe(III) was similar to that of the wild type. When omcB was reintroduced into the OmcB-deficient mutant, the capacity for Fe(III) reduction was restored in proportion to the level of OmcB production. These results indicate that OmcB, but not OmcC, has a major role in electron transport to Fe(III) and suggest that electron transport to the outer membrane is an important feature in Fe(III) reduction in this organism.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Grupo dos Citocromos c/metabolismo , Deltaproteobacteria/metabolismo , Ferro/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Grupo dos Citocromos c/genética , Deltaproteobacteria/genética , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Família Multigênica , Mutação , Oxirredução , Homologia de Sequência de AminoácidosRESUMO
The Na,K-ATPase is specifically inhibited by the cardiac glycoside, ouabain. Via a largely undefined mechanism, the ouabain affinity of the Na,K-ATPase can be manipulated by mutating the residues at the borders of the first extracellular (M1-M2) loop of the alpha subunit [Price, E. M., Rice, D. A., and Lingrel, J. B. (1990) J. Biol. Chem. 265, 6638-6641]. To address this issue, we compared the effects of two combinations of charged residues at the M1-M2 loop border, R113, D124 and D113,R124 (numbered according to the rat alpha1 subunit), on the ouabain sensitivity of the alpha1 and alpha2 isoforms. We report that ouabain sensitivity is dependent not only upon the identity of the residues at the M1-M2 loop border but also upon the context into which they are introduced. Furthermore, at low concentrations of ATP, the identity of the residues at the M1-M2 loop border affects the regulation of ATP hydrolysis by potassium in an isoform-specific manner. Analysis of chimeric alpha subunits reveals that the effects of potassium are determined primarily by the interaction of the N-terminus and M1-M2 loop with the C-terminal third of the alpha subunit. M1-M2 loop border residues may, therefore, influence ouabain sensitivity indirectly by altering the stability or structure of the intermediate of the Na,K-ATPase catalytic cycle which is competent to bind ouabain.