RESUMO
Chikungunya virus (CHIKV) is a rapidly spreading re-emergent virus transmitted from mosquitoes to humans. The emergence of epidemic variants has been associated with changes in the viral genome, such as the duplication of repeated sequences in the 3' untranslated region (UTR). Indeed, blocks of repeated sequences seemingly favor RNA recombination, providing the virus with a unique ability to continuously change the 3'UTR architecture during host switching. In this work, we provide experimental data on the molecular mechanism of RNA recombination and describe specific sequence and structural elements in the viral 3'UTR that favor template switching of the viral RNA-dependent RNA polymerase on the 3'UTR. Furthermore, we found that a 3'UTR deletion mutant that exhibits markedly delayed replication in mosquito cells and impaired transmission in vivo, recombines in reference laboratory strains of mosquitoes. Altogether, our data provide novel experimental evidence indicating that RNA recombination can act as a nucleic acid repair mechanism to add repeated sequences that are associated to high viral fitness in mosquito during chikungunya virus replication.
Assuntos
Regiões 3' não Traduzidas , Vírus Chikungunya , Genoma Viral , RNA Viral , Recombinação Genética , Replicação Viral , Vírus Chikungunya/genética , Regiões 3' não Traduzidas/genética , RNA Viral/genética , RNA Viral/metabolismo , Animais , Replicação Viral/genética , Febre de Chikungunya/virologia , Febre de Chikungunya/genética , Febre de Chikungunya/transmissão , Humanos , Aedes/virologia , Aedes/genética , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Linhagem CelularRESUMO
Toxoplasmosis is the most prevalent parasitic zoonosis worldwide, causing ocular and neurological diseases. No vaccine has been approved for human use. We evaluated the response of peripheral blood mononuclear cells (PBMCs) to a novel construct of Toxoplasma gondii total antigen in maltodextrin nanoparticles (NP/TE) in individuals with varying infectious statuses (uninfected, chronic asymptomatic, or ocular toxoplasmosis). We analyzed the concentration of IFN-γ after NP/TE ex vivo stimulation using ELISA and the immunophenotypes of CD4+ and CD8+ cell populations using flow cytometry. In addition, serotyping of individuals with toxoplasmosis was performed by ELISA using GRA6-derived polypeptides. Low doses of NP/TE stimulation (0.9 µg NP/0.3 µg TE) achieved IFN-γ-specific production in previously exposed human PBMCs without significant differences in the infecting serotype. Increased IFN-γ expression in CD4+ effector memory cell subsets was found in patients with ocular toxoplasmosis with NP/TE but not with TE alone. This is the first study to show how T-cell subsets respond to ex vivo stimulation with a vaccine candidate for human toxoplasmosis, providing crucial insights for future clinical trials.
Assuntos
Antígenos de Protozoários , Interferon gama , Ativação Linfocitária , Nanopartículas , Polissacarídeos , Toxoplasma , Toxoplasmose , Humanos , Nanopartículas/química , Polissacarídeos/imunologia , Toxoplasma/imunologia , Antígenos de Protozoários/imunologia , Toxoplasmose/imunologia , Interferon gama/metabolismo , Interferon gama/imunologia , Ativação Linfocitária/imunologia , Feminino , Adulto , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Masculino , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Pessoa de Meia-IdadeRESUMO
Articular cartilage injuries are found in up to 60% of patients who undergo an arthroscopic knee procedure, and those that totally affect articular cartilage (grade IV) have limited regenerative capacity and extended time for recovery. 3-D scaffolds represent a novel solution to address this type of injury. Our purpose was to analyze the MRI findings and functional status of patients that underwent repair of chondral defects either by microfractures or Hyaluronan (HA) 3-D scaffolding. We conducted a retrospective study of patients with chondral defects. The outcomes analyzed in this study included anatomical changes evaluated by the Henderson score (based on MRI findings) at baseline, 6, and 12 months after surgery, and improvement in functionality evaluated by the Modified Cincinnati Knee Rating System (MCKRS) at baseline and 6 months after surgery. Clinical and demographic characteristics were similar for both groups. There was a statistically significant improvement in Henderson score for the 3-D scaffold-treated group at 6 months versus the microfracture group (p < 0.0001). Improvement in functionality, measured by the MCKRS, was more frequently found in the 3-D scaffold-treated group. In conclusion, the use of HA 3-D scaffolding was superior, with faster recovery evident 6 months after the surgery that progressed to full recovery in all patients a year after surgery. Future studies with a randomized design might help to support our findings. This study provides level III evidence.
RESUMO
This work analyzed exhaustion markers in CD8+ T-cell subpopulations in 21 samples of peripheral blood mononuclear cells (PBMCs) from individuals with ocular toxoplasmosis (n = 9), chronic asymptomatic toxoplasmosis (n = 7), and non-infected people (n = 5) by using RT-qPCR and flow cytometry techniques. The study found that gene expression of PD-1 and CD244, but not LAG-3, was higher in individuals with ocular toxoplasmosis versus individuals with asymptomatic infection or uninfected. Expression of PD1 in CD8+ central memory (CM) cells was higher in nine individuals with toxoplasmosis versus five uninfected individuals (p = .003). After ex vivo stimulation, an inverse correlation was found between the exhaustion markers and quantitative clinical characteristics (lesion size, recurrence index, and number of lesions). A total exhaustion phenotype was found in 55.5% (5/9) of individuals with ocular toxoplasmosis. Our results suggest that the CD8+ exhaustion phenotype is involved in the pathogenesis of ocular toxoplasmosis.
RESUMO
Resumen Objetivo: Describir los conocimientos, las actitudes y las prácticas acerca de la toxoplasmosis en dos comunas de Armenia, Quindío, con alta prevalencia de la infección. Metodología: Estudio descriptivo con población de dos comunas de Armenia, Quindío. Se aplicó un cuestionario autodiligenciado tipo conocimientos, actitudes y prácticas. Esta herramienta incluyó elementos sobre el parásito Toxoplasma gondii, sus vías de transmisión, aspectos clínicos, diagnósticos y de tratamiento generales, así como prácticas para evitar la infección. El instrumento se aplicó antes y después de una intervención educativa. Se describieron las frecuencias en el número de respuestas correctas antes y después de la intervención para cada comuna. Resultados: Participaron 27 personas, con una media de edad de 57 años. El 59 % fueron mujeres. El 48% había completado la educación media y el 40,7 % la primaria. El conocimiento del agente causal antes de la intervención fue del 22 %, mientras que posterior a la intervención fue del 92,3 % en la comuna 1 y del 81,8 % en la comuna 6. Posterior a la intervención, cerca del 90 % de los encuestados reconoció la retina como la principal estructura afectada y todos los encuestados reconocieron el consumo de agua hervida como factor protector. Conclusión: Los conocimientos sobre la toxoplasmosis en las dos comunas eran limitados. Luego de la intervención educativa, se evidenció un aumento en el porcentaje de respuestas correctas en la mayoría de las preguntas. Se recomienda realizar nuevas intervenciones educativas y en salud pública, para evaluar los efectos de estas a largo plazo.
Abstract Objective: To describe knowledge, attitudes and practices related to toxoplasmosis in two districts of high prevalence in Armenia, Quindío. Methodology: descriptive study; the population of two districts of Armenia, Quindío were engaged. A self-administered questionnaire regarding knowledge, attitudes, and practices was applied. This tool included elements related to Toxoplasma gondii, its transmission pathways, general clinical, diagnostic and treatment aspects, as well as practices to prevent infection. The instrument was applied before and after an educational intervention. Frequencies were described as the number of correct answers before and after the intervention for each district. Results: 27 people participated, with an average age of 57 years. 59 % were women; 48 % had completed high school and 40.7 % had completed elementary school. Before the intervention, the knowledge of the causal agent was 22 %, while after the intervention, it was 92.3 % in district 1 and 81.8 % in district 6. After the intervention, about 90 % of participants recognized the retina as the main compromised structure and all participants recognized the consumption of boiled water as a protective factor. Conclusion: The knowledge regarding toxoplasmosis in the two districts was limited. After the educational intervention, there was an increase in the percentage of correct answers in most of the questions. New educational and public health interventions are recommended to assess the long-term effects of these interventions.
Resumo Objetivos: O objetivo deste estudo é descrever os problemas, as ações e as práticas sobre a toxoplasmose nas comunidades da Armênia, Quindío, onde se nota alta prevalência da infecção. Metodologia: Este é um estudo descritivo que abrange uma população de duas comunas na Armênia, Quindío. Foi implementada uma ferramenta, que consiste em questionários destinados àquela população e aplicáveis pelos seus próprios membros, que correspondem a conhecimentos, atitudes e práticas. Essa ferramenta inclui elementos sobre o parasito Toxoplasma gondii, suas vias de transmissão, aspectos clínicos gerais, diagnósticos e tratamento, e práticas de prevenção. O instrumento foi aplicado antes e após uma intervenção educativa exata. Foram descritas as frequências do número de acertos, antes e depois da intervenção para cada comuna. Resultados: participaram 27 pessoas, com média de idade de 57 anos, das quais 59 % eram mulheres e 48 % tinham ensino médio completo e 40,7 % ensino primário. O conhecimento do agente causal antes da intervenção havia em 22 %, enquanto que após a intervenção, passou para 92,3 % na comuna 1, e 81,8 % na comuna 6. Após a intervenção, cerca de 90 % dos entrevistados reconheceram a retina como a estrutura mais afetada e todos os entrevistados reconheceram que o consumo de água fervida é um fator de proteção. Conclusão: Desconhecimento sobre a toxoplasmose nas duas comunas. Após a intervenção educativa, houve evidência de aumento do percentual de acertos na maioria das questões. Recomenda-se a realização de novas intervenções educacionais e de saúde pública, para avaliar os efeitos destas a longo prazo na populacão.
RESUMO
Thiol peroxidase from Escherichia coli (EcTPx) is a peroxiredoxin that catalyzes the reduction of different hydroperoxides. During the catalytic cycle of EcTPx, the peroxidatic cysteine (CP) is oxidized to a sulfenic acid by peroxide, then the resolving cysteine (CR) condenses with the sulfenic acid of CP to form a disulfide bond, which is finally reduced by thioredoxin. Purified EcTPx as dithiol and disulfide behaves as a monomer under near physiological conditions. Although secondary structure rearrangements are present when comparing different redox states of the enzyme, no significant differences in unfolding free energies are observed under reducing and oxidizing conditions. A conformational change denominated fully folded (FF) to locally unfolded (LU) transition, involving a partial unfolding of αH2 and αH3, must occur to enable the formation of the disulfide bond since the catalytic cysteines are 12 Å apart in the FF conformation of EcTPx. To explore this process, the FF â LU and LU â FF transitions were studied using conventional molecular dynamics simulations and an enhanced conformational sampling technique for different oxidation and protonation states of the active site cysteine residues CP and CR. Our results suggest that the FF â LU transition has a higher associated energy barrier than the refolding LU â FF process in agreement with the relatively low experimental turnover number of EcTPx. Furthermore, in silico designed single-point mutants of αH3 enhanced locally unfolding events, suggesting that the native FF interactions in the active site are not evolutionarily optimized to fully speed-up the conformational transition of wild-type EcTPx.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Simulação de Dinâmica Molecular , Proteínas Periplásmicas/química , Peroxidases/química , Dobramento de Proteína , Simulação por Computador , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação/genética , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/metabolismo , Peroxidases/genética , Peroxidases/metabolismo , Conformação ProteicaRESUMO
Toxoplasma gondii engenders the common parasitic disease toxoplasmosis in almost all warm-blooded animals. Being a critical secretory protein, ROP18 is a major virulence factor of Toxoplasma. There are no reports about ROP18 detection in human serum samples with different clinical manifestations. New aptamers against ROP18 protein were developed through Systematic Evolution of Ligands by Exponential enrichment (SELEX). An Enzyme-Linked Aptamer Assay (ELAA) platform was developed using SELEX-derived aptamers, namely AP001 and AP002. The ELAA was used to evaluate total antigen from T. gondii RH strain (RH Ag) and recombinant protein of ROP18 (rROP18). The results showed that the ELAA presented higher affinity and specificity to RH Ag and rROP18, compared to negative controls. Detection limit of rROP18 protein in serum samples was measured by standard addition method, achieving a lower concentration of 1.56 µg/mL. Moreover, 62 seropositive samples with different clinical manifestations of toxoplasmosis and 20 seronegative samples were tested. A significant association between ELAA test positive for human serum samples and severe congenital toxoplasmosis was found (p = 0.006). Development and testing of aptamers-based assays opens a window for low-cost and rapid tests looking for biomarkers and improves our understanding about the role of ROP18 protein on the pathogenesis of human toxoplasmosis.
Assuntos
Ensaio de Imunoadsorção Enzimática , Proteínas Serina-Treonina Quinases/imunologia , Técnica de Seleção de Aptâmeros , Toxoplasma/imunologia , Toxoplasmose/diagnóstico , Toxoplasmose/parasitologia , Aptâmeros de Peptídeos , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Humanos , Proteínas Serina-Treonina Quinases/sangue , Proteínas de Protozoários , Proteínas Recombinantes , Sensibilidade e Especificidade , Toxoplasmose/imunologiaRESUMO
Toxoplasma gondii ROP16 and ROP18 proteins have been identified as important virulence factors for this parasite. Here, we describe the effect of ROP16 and ROP18 proteins on peripheral blood mononuclear cells (PBMCs) from individuals with different clinical status of infection. We evaluated IFN-γ, IL-10, and IL-1ß levels in supernatants from PBMCs cultures infected with tachyzoites of the T. gondii wild-type RH strain or with knock-out mutants of the rop16 and rop18 encoding genes (RHΔrop16 and RHΔrop18). Cytokine secretion was compared between PBMCs obtained from seronegative individuals (n = 10), with those with chronic asymptomatic (n = 8), or ocular infection (n = 12). We also evaluated if polymorphisms in the genes encoding for IFN-γ, IL-10, IL-1ß, Toll-like receptor 9 (TLR9), and purinoreceptor P2RX7 influenced the production of the encoded proteins after ex vivo stimulation. In individuals with chronic asymptomatic infection, only a moderate effect on IL-10 levels was observed when PBMCs were infected with RHΔrop16, whereas a significant difference in the levels of inflammatory cytokines IFN-γ and IL-1ß was observed in seronegative individuals, but this was also dependent on the host's cytokine gene polymorphisms. Infection with ROP16-deficient parasites had a significant effect on IFN-γ production in previously non-infected individuals, suggesting that ROP16 which is considered as a virulence factor plays a role during the primary infection in humans, but not in the secondary immune response.
Assuntos
Leucócitos Mononucleares/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Tirosina Quinases/imunologia , Proteínas de Protozoários/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Toxoplasmose/parasitologia , Citocinas/metabolismo , Fibroblastos , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/imunologia , Humanos , Leucócitos Mononucleares/metabolismo , Polimorfismo Genético , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT6/metabolismo , Toxoplasma/patogenicidade , Toxoplasmose/genética , Virulência , Fatores de Virulência/imunologiaRESUMO
OBJECTIVE: To describe the long term safety and efficacy of pegvisomant (PEGV), and the predictors of treatment response in patients with acromegaly in the real life setting. SUBJECTS AND METHODS: We retrospectively reviewed the clinical, hormonal and radiological data of acromegalic patients treated with PEGV in 17 Argentine centers. RESULTS: Seventy-five patients (age range 22-77, 51 females) with acromegaly have been treated with PEGV for up to 118 months (median 27 months). Before PEGV, 97.3% of patients had been treated with medical therapy, surgery and/or radiotherapy, two patients had no previous treatment. At that time, all patients had an IGF-1 above the upper normal limit (ULN) (mean 2.4 x ULN ± 0.98, range 1.25-7). At diagnosis of acromegaly 84% presented macroadenomas, prior to PEGV only 23,5% of patients remained with tumor remnant > 1 cm, the remaining showed normal or less than 1 cm images. Disease control (IGF-1 ≤ 1.2 x ULN) was achieved in 62.9% of patients with a mean dose of 11.8 mg/day. Thirty-four patients (45%) received PEGV monotherapy, while 41 (55%) received combined therapy with either somatostatin analogues and/or cabergoline. Adverse events related to PEGV were: local injection site reaction in 5.3%, elevated liver enzymes in 9.3%, and tumor size growth in 9.8%. Pre-PEGV IGF-I level was the only predictor of treatment response: 2.1 x ULN vs 2.8 x ULN in controlled and uncontrolled patients respectively (p < 0.001). CONCLUSION: this long term experience indicates PEGV treatment was highly effective and safe in our series of Argentine patients with acromegaly refractory to standard therapies. Arch Endocrinol Metab. 2019;63(4):320-7.
Assuntos
Acromegalia/tratamento farmacológico , Cabergolina/uso terapêutico , Agonistas de Dopamina/uso terapêutico , Hormônio do Crescimento Humano/análogos & derivados , Somatostatina/análogos & derivados , Adulto , Idoso , Argentina , Cabergolina/administração & dosagem , Agonistas de Dopamina/administração & dosagem , Quimioterapia Combinada , Feminino , Seguimentos , Hormônio do Crescimento Humano/administração & dosagem , Hormônio do Crescimento Humano/uso terapêutico , Humanos , Fator de Crescimento Insulin-Like I/análise , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Retrospectivos , Somatostatina/administração & dosagem , Somatostatina/uso terapêutico , Resultado do Tratamento , Adulto JovemRESUMO
ABSTRACT Objective To describe the long term safety and efficacy of pegvisomant (PEGV), and the predictors of treatment response in patients with acromegaly in the real life setting. Subjects and methods We retrospectively reviewed the clinical, hormonal and radiological data of acromegalic patients treated with PEGV in 17 Argentine centers. Results Seventy-five patients (age range 22-77, 51 females) with acromegaly have been treated with PEGV for up to 118 months (median 27 months). Before PEGV, 97.3% of patients had been treated with medical therapy, surgery and/or radiotherapy, two patients had no previous treatment. At that time, all patients had an IGF-1 above the upper normal limit (ULN) (mean 2.4 x ULN ± 0.98, range 1.25-7). At diagnosis of acromegaly 84% presented macroadenomas, prior to PEGV only 23,5% of patients remained with tumor remnant > 1 cm, the remaining showed normal or less than 1 cm images. Disease control (IGF-1 ≤ 1.2 x ULN) was achieved in 62.9% of patients with a mean dose of 11.8 mg/day. Thirty-four patients (45%) received PEGV monotherapy, while 41 (55%) received combined therapy with either somatostatin analogues and/or cabergoline. Adverse events related to PEGV were: local injection site reaction in 5.3%, elevated liver enzymes in 9.3%, and tumor size growth in 9.8%. Pre-PEGV IGF-I level was the only predictor of treatment response: 2.1 x ULN vs 2.8 x ULN in controlled and uncontrolled patients respectively (p < 0.001). Conclusion this long term experience indicates PEGV treatment was highly effective and safe in our series of Argentine patients with acromegaly refractory to standard therapies. Arch Endocrinol Metab. 2019;63(4):320-7
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Adulto Jovem , Acromegalia/tratamento farmacológico , Somatostatina/análogos & derivados , Agonistas de Dopamina/uso terapêutico , Hormônio do Crescimento Humano/análogos & derivados , Cabergolina/uso terapêutico , Argentina , Fator de Crescimento Insulin-Like I/análise , Valor Preditivo dos Testes , Estudos Retrospectivos , Seguimentos , Resultado do Tratamento , Agonistas de Dopamina/administração & dosagem , Hormônio do Crescimento Humano/administração & dosagem , Hormônio do Crescimento Humano/uso terapêutico , Quimioterapia Combinada , Cabergolina/administração & dosagemRESUMO
Metal-fluoride complexes have been used to induce E2P-like states with the aim of studying the events that occur during E2P hydrolysis in P-type ATPases. In the present work, we compared the E2P-like state induced by a beryllium fluoride complex (BeFx) with the actual E2P state formed through backdoor phosphorylation of the Na,K-ATPase. Formation of E2P and E2P-like states were investigated employing the styryl dye RH421. We found that BeFx is the only fluorinated phosphate analog that, like Pi, increases the RH421 fluorescence. The observed rate constant, kobs, for the formation of E2P decreases with [Pi] whereas that of E2BeFx increases with [BeFx]. This might wrongly be taken as evidence of a mechanism where the binding of BeFx induces a conformational transition. Here, we rather propose that, like for Pi, binding of BeFx follows a conformational-selection mechanism, i.e. it binds to the E2 conformer forming a complex that is much more stable than E2P, as seen from its impaired capacity to return to E1 upon addition of Na+. Although E2P and E2BeFx are able to form states with 2 occluded Rb+, both enzyme complexes differ in that the affinity for the binding and occlusion of the second Rb+ is much lower in E2BeFx than in E2P. The higher rates of Rb+ occlusion and deocclusion observed for E2BeFx, as compared to those observed for other E2P-like transition and product states suggest a more open access to the cation transport sites, supporting the idea that E2BeFx mimics the E2P ground state.
Assuntos
Berílio/farmacologia , Fluoretos/farmacologia , Rubídio/metabolismo , ATPase Trocadora de Sódio-Potássio/química , Animais , Fluorescência , Imidazóis/farmacologia , Cinética , Modelos Biológicos , Fosfatos/metabolismo , Conformação Proteica , ATPase Trocadora de Sódio-Potássio/metabolismo , Suínos , Fatores de TempoRESUMO
A comprehensive study of the interaction between Na(+) and K(+) with the Na(+)/K(+)-ATPase requires dissecting the incidence of alternative cycling modes on activity measurements in which one or both of these cations are absent. With this aim, we used membrane fragments containing pig-kidney Na(+)/K(+)-ATPase to perform measurements, at 25°C and pH=7.4, of ATPase activity and steady-state levels of (i) intermediates containing occluded Rb(+) at different [Rb(+)] in media lacking Na(+), and (ii) phosphorylated intermediates at different [Na(+)] in media lacking Rb(+). Most relevant results are: (1) Rb(+) can be occluded through an ATPasic cycling mode that takes place in the absence of Na(+) ions, (2) the kinetic behavior of the phosphoenzyme formed by ATP in the absence of Na(+) is different from the one that is formed with Na(+), and (3) binding of Na(+) to transport sites during catalysis is not at random unless rapid equilibrium holds.
Assuntos
Trifosfato de Adenosina/metabolismo , Medula Renal/enzimologia , Rubídio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sódio/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Biocatálise/efeitos dos fármacos , Relação Dose-Resposta a Droga , Cinética , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Rubídio/farmacologia , Sódio/farmacologia , SuínosRESUMO
This work presents a detailed kinetic study that shows the coupling between the E2âE1 transition and Rb(+) deocclusion stimulated by Na(+) in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb(+) and the increase in eosin fluorescence (the formation of E1) as a function of time. The E2âE1 transition and Rb(+) deocclusion are described by the sum of two exponential functions with equal amplitudes, whose rate coefficients decreased with increasing [Rb(+)]. The rate coefficient values of the E2âE1 transition were very similar to those of Rb(+)-deocclusion, indicating that both processes are simultaneous. Our results suggest that, when ATP is absent, the mechanism of Na(+)-stimulated Rb(+) deocclusion would require the release of at least one Rb(+) ion through the extracellular access prior to the E2âE1 transition. Using vanadate to stabilize E2, we measured occluded Rb(+) in equilibrium conditions. Results show that, while Mg(2+) decreases the affinity for Rb(+), addition of vanadate offsets this effect, increasing the affinity for Rb(+). In transient experiments, we investigated the exchange of Rb(+) between the E2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the E2âE1 transition caused by Na(+) without significantly affecting the rate of Rb(+) deocclusion. On the other hand, we found the first evidence of a very low rate of Rb(+) occlusion in the enzyme-vanadate complex, suggesting that this complex would require a change to an open conformation in order to bind and occlude Rb(+).
Assuntos
Rim/metabolismo , Rubídio/farmacologia , ATPase Trocadora de Sódio-Potássio/química , Vanadatos/farmacologia , Trifosfato de Adenosina/química , Animais , Biofísica/métodos , Amarelo de Eosina-(YS)/química , Cinética , Magnésio/química , Modelos Biológicos , Ligação Proteica , Conformação Proteica , Rubídio/química , Suínos , Fatores de Tempo , Vanadatos/químicaRESUMO
Annular lipid-protein stoichiometry in native pig kidney Na+/K+ -ATPase preparation was studied by [125I]TID-PC/16 labeling. Our data indicate that the transmembrane domain of the Na+/K+ -ATPase in the E1 state is less exposed to the lipids than in E2, i.e., the conformational transitions are accompanied by changes in the number of annular lipids but not in the affinity of these lipids for the protein. The lipid-protein stoichiometry was 23 ± 2 (α subunit) and 5.0 ± 0.4 (ß subunit) in the E1 conformation and 32 ± 2 (α subunit) and 7 ± 1 (ß subunit) in the E2 conformation.
Assuntos
Rim/enzimologia , Lipídeos/química , ATPase Trocadora de Sódio-Potássio/química , Animais , Sítios de Ligação , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Conformação Proteica , ATPase Trocadora de Sódio-Potássio/metabolismo , SuínosRESUMO
Despite its similarity with the Na(+)/K(+)-ATPase, it has not been possible so far to isolate a K(+)-occluded state in the H(+)/K(+)-ATPase at room temperature. We report here results on the time course of formation of a state containing occluded Rb(+) (as surrogate for K(+)) in H(+)/K(+)-ATPase from gastric vesicles at 25°C. Alamethicin (a pore-forming peptide) showed to be a suitable agent to open vesicles, allowing a more efficient removal of Rb(+) ions from the intravesicular medium than C(12)E(8) (a non-ionic detergent). In the presence of vanadate and Mg(2+), the time course of [(86)Rb]Rb(+) uptake displayed a fast phase due to Rb(+) occlusion. The specific inhibitor of the H(+)/K(+)-ATPase SCH28080 significantly reduces the amount of Rb(+) occluded in the vanadate-H(+)/K(+)-ATPase complex. Occluded Rb(+) varies with [Rb(+)] according to a hyperbolic function with K(0.5)=0.29±0.06mM. The complex between the Rb(+)-occluded state and vanadate proved to be very stable even after removal of free Mg(2+) with EDTA. Our results yield a stoichiometry lower than one occluded Rb(+) per phosphorylation site, which might be explained assuming that, unlike for the Na(+)/K(+)-ATPase, Mg(2+)-vanadate is unable to recruit all the Rb(+)-bound to the Rb(+)-occluded form of the Rb(+)-vanadate-H(+)/K(+)-ATPase complex.
Assuntos
ATPase Trocadora de Hidrogênio-Potássio/química , Rubídio/química , Estômago/enzimologia , Vanadatos/química , Alameticina/química , Alameticina/farmacologia , Animais , Detergentes/química , Inibidores Enzimáticos/farmacologia , Íons , Ligantes , Peptídeos/química , Fosforilação , Suínos , Temperatura , Fatores de TempoRESUMO
We used suspensions of partially purified Na(+)/K(+)-ATPase from pig kidney to compare the effects of Rb(+), as a K(+) congener, on the time course and on the equilibrium values of eosin fluorescence and of Rb(+) occlusion. Both sets of data were collected under identical conditions in the same enzyme preparations. The incubation media lacked ATP so that all changes led to an equilibrium distribution between enzyme conformers with and without bound eosin and with and without bound or occluded Rb(+). Results showed that as Rb(+) concentration was increased, the equilibrium value of fluorescence decreased and occlusion increased along rectangular hyperbolas with similar half-maximal values. The time courses of attainment of equilibrium showed an initial phase which was so quick as to fall below the time resolution of our rapid-mixing apparatus. This phase was followed by the sum of at least two exponential functions of time. In the case of fluorescence the fast exponential term accounted for a larger fraction of the time course than in the case of occlusion. Comparison between experimental and simulated results suggests that fluorescence changes express a process that is coupled to Rb(+) occlusion but that is completed before occlusion reaches equilibrium.
Assuntos
Amarelo de Eosina-(YS)/química , Rubídio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Algoritmos , Animais , Amarelo de Eosina-(YS)/metabolismo , Fluorescência , Transporte de Íons/efeitos dos fármacos , Cinética , Modelos Químicos , Ligação Proteica , Rubídio/metabolismo , Rubídio/farmacologia , ATPase Trocadora de Sódio-Potássio/química , SuínosRESUMO
We report a study on the effect of the fluorescent probe eosin on some of the reactions involved in the conformational transitions that lead to the occlusion of the K(+)-congener Rb(+) in the Na(+)/K(+)-ATPase. Eosin decreases the equilibrium levels of occluded Rb(+), this effect being fully attributable to a decrease in the apparent affinity of the enzyme for Rb(+) since the capacity for occlusion remains independent of eosin concentration. The results can be quantitatively described by a model that assumes that two molecules of eosin are able to bind to the Na(+)/K(+)-ATPase, both to the Rb(+)-free and to the Rb(+)-occluded enzyme regardless of the degree of cation occlusion. Concerning the effect on the affinity for Rb(+) occlusion, transient state experiments show that eosin reduces the initial velocity of occlusion, and that, like ATP, it increases the velocity of deocclusion of Rb(+). Interactions between eosin and ATP on Rb(+)-release experiments seem to indicate that eosin binds to the low-affinity site of ATP from which it exerts effects that are similar to those of the nucleotide.
Assuntos
Amarelo de Eosina-(YS)/química , Amarelo de Eosina-(YS)/farmacocinética , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Radioisótopos de Rubídio/farmacocinética , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/farmacocinética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/farmacocinética , Animais , Sítios de Ligação , Modelos Químicos , Dinâmica não Linear , Ligação Proteica , Conformação Proteica , Suínos , Termodinâmica , Fatores de TempoRESUMO
Occlusion of K(+) or its congeners in the Na(+)/K(+)-ATPase occurs after K(+)-dependent dephosphorylation (physiological route) or in media lacking ATP and Na(+) (direct route). The effects of P(i) or ATP on the kinetics of deocclusion of the K(+)-congener Rb(+) formed by each of the above mentioned routes was independent of the route of occlusion, which suggests that both routes lead to the same enzyme intermediate. The time course of occlusion via the direct route can be described by the sum of two exponential functions plus a small component of very high velocity. At equilibrium, occluded Rb(+) is a hyperbolic function of free [Rb(+)] suggesting that the direct route results in enzyme states holding either one or two occluded Rb(+). Release of occluded Rb(+) follows the sum of two decreasing exponential functions of time, corresponding to two phases with similar sizes. These phases are not caused by independent physical compartments. The rate constant of one of the phases is reduced up to 30 times by free Rb(+). When Rb(+) is the only pump ligand, the kinetics of occlusion and deocclusion through the direct route are consistent with an ordered-sequential process with additional independent step(s) interposed between the uptake or the release of each occluded Rb(+).
Assuntos
Rubídio/metabolismo , ATPase Trocadora de Sódio-Potássio/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Rim/enzimologia , Cinética , Modelos Teóricos , Fosfatos/metabolismo , Fosfatos/farmacologia , Análise de Regressão , ATPase Trocadora de Sódio-Potássio/metabolismo , SuínosRESUMO
Rats chronically fed (15 weeks) a sucrose-rich diet (SRD) developed hypertriglyceridemia (hyperTg), increased plasma free fatty acids (FFA), impaired glucose homeostasis and insulin insensitivity. An increase of Tg and glycogen (Gly) in heart muscle was also observed. HyperTg with altered glucose metabolism could have profound effects on myocardial glucose utilization. To test this hypothesis male Wistar rats were fed a semi-synthetic SRD (w/w: 62.5% sucrose, 8% corn-oil, 17% protein), and the control group (CD) received the same semi-synthetic diet, except that sucrose was replaced with starch for 90 days. At that time, the hearts from these animals were isolated and perfused for 30 min in the presence or absence of insulin (30 mU/ml). Levels of the exogenous substrates were similar to those found in the plasma of the animal in vivo in both dietary groups (glucose 8.5 mM, palmitate 0.8 mM in SRD and glucose 5-5 mM, palmitate 0.3 mM in CD). In the absence of insulin glucose uptake was reduced (40%) and lactate release was increased (50%) in SRD hearts. Glucose oxidation was depressed mainly due to both, an increase of PDH kinase and a decrease of 60% of PDHa (active form of PDHc). Insulin in the perfusion medium improved only glucose uptake. The results suggest that at least two different mechanisms might contribute to insulin resistance and to impaired glucose metabolism in the perfused hearts of dyslipemic SRD fed rats: 1) reduced basal and insulin-stimulated glucose uptake and its utilization and 2) increased availability and oxidation of lipids (low PDHa and PDH kinase activities), which in turn decreased glucose uptake and utilization. Thus, this experimental model may be useful to study how impaired glucose homeostasis, increased plasma FFA and hyperTg could contribute to heart tissue malfunction.
Assuntos
Animais , Masculino , Ratos , Glucose , Hiperlipidemias , Insulina , Miocárdio , Análise de Variância , Modelos Animais de Doenças , Ácidos Graxos , Resistência à Insulina , Miocárdio , Complexo Piruvato Desidrogenase , Piruvato Quinase , Ratos Wistar , Aumento de PesoRESUMO
Rats chronically fed (15 weeks) a sucrose-rich diet (SRD) developed hypertriglyceridemia (hyperTg), increased plasma free fatty acids (FFA), impaired glucose homeostasis and insulin insensitivity. An increase of Tg and glycogen (Gly) in heart muscle was also observed. HyperTg with altered glucose metabolism could have profound effects on myocardial glucose utilization. To test this hypothesis male Wistar rats were fed a semi-synthetic SRD (w/w: 62.5% sucrose, 8% corn-oil, 17% protein), and the control group (CD) received the same semi-synthetic diet, except that sucrose was replaced with starch for 90 days. At that time, the hearts from these animals were isolated and perfused for 30 min in the presence or absence of insulin (30 mU/ml). Levels of the exogenous substrates were similar to those found in the plasma of the animal in vivo in both dietary groups (glucose 8.5 mM, palmitate 0.8 mM in SRD and glucose 5-5 mM, palmitate 0.3 mM in CD). In the absence of insulin glucose uptake was reduced (40%) and lactate release was increased (50%) in SRD hearts. Glucose oxidation was depressed mainly due to both, an increase of PDH kinase and a decrease of 60% of PDHa (active form of PDHc). Insulin in the perfusion medium improved only glucose uptake. The results suggest that at least two different mechanisms might contribute to insulin resistance and to impaired glucose metabolism in the perfused hearts of dyslipemic SRD fed rats: 1) reduced basal and insulin-stimulated glucose uptake and its utilization and 2) increased availability and oxidation of lipids (low PDHa and PDH kinase activities), which in turn decreased glucose uptake and utilization. Thus, this experimental model may be useful to study how impaired glucose homeostasis, increased plasma FFA and hyperTg could contribute to heart tissue malfunction. (AU)