RESUMO
Background and Objectives: The hemoadsorption device CytoSorb® (CytoSorbents Inc., Princeton, NJ, USA) has been shown to efficiently remove ticagrelor from whole blood in vitro. A promising clinical experience was made with the integration of the hemoadsorption cartridge on the cardiopulmonary bypass (CPB) circuit during cardiac surgery to reduce adverse events. Materials and Methods: In this report, we describe a novel approach using a new apheresis platform, PUR-01 (Nikkisio Co., Ltd., Tokyo, Japan), which was used as the extracorporeal circuit where CytoSorb® could be installed for the removal of ticagrelor during off-pump coronary artery bypass (OPCAB) procedures. Results: In a 74-year-old male (index case) with coronary artery disease and dual antiplatelet therapy, hemoadsorption was initiated with a skin incision for OPCAB surgery and was continued for 221 min to eliminate ticagrelor. The blood volume that had circulated through the CytoSorb® was 39.04 L in total. Thus far, this treatment strategy has been used in four cases with CHD and DAPT who needed OPCAB surgery. The intraoperative and postoperative courses were uneventful in all patients. No device-related adverse events occurred. Conclusions: The combination of the PUR-01 apheresis pump and hemoadsorption with the CytoSorb® column during OPCAB procedures appears to be safe and effective in eliminating antiplatelet drugs.
Assuntos
Remoção de Componentes Sanguíneos , Ponte de Artéria Coronária sem Circulação Extracorpórea , Masculino , Humanos , Idoso , Ponte de Artéria Coronária sem Circulação Extracorpórea/métodos , Inibidores da Agregação Plaquetária/uso terapêutico , Fibrinolíticos , Ticagrelor/uso terapêutico , Ponte Cardiopulmonar/efeitos adversosRESUMO
BACKGROUND: Patients requiring coronary artery bypass grafting (CABG) are often loaded with antithrombotic drugs (AT) and are at an increased risk for perioperative bleeding complications. Active AT removal by a hemoadsorption cartridge integrated in the cardiopulmonary bypass circuit is increasingly used in this setting to reduce bleeding, and herein we describe the extension of this application in patients on AT undergoing off-pump coronary artery bypass (OPCAB). METHODS: Ten patients (80% male; mean age: 67.4 ± 9.2years) were treated with ticagrelor (eight patients), rivaroxaban and ticagrelor (one patient), and rivaroxaban (one patient) prior to OPCAB surgery. AT's were discontinued one day before surgery in nine patients and on the day of surgery in one patient, and all patients were also on aspirin. The cohort mean EuroSCORE-II was 2.9 ± 1.5%. A hemoadsorption cartridge was integrated into a dialysis device (n=4) or a stand-alone apheresis pump (n=6) periprocedural, for a treatment time of 145 ± 33 min. Outcome measures included bleeding according to Bleeding Academic Research Consortium (BARC)-4 and 24-hour chest-tube-drainage (CTD). RESULTS: Mean operation time was 184 ± 35 min. All patients received a left internal thoracic artery with a mean of 2.3 ± 0.9 total grafts. One patient had a BARC-4 bleeding event and there were no surgical re-explorations for bleeding. Mean 24-hours CTD was 680 ± 307mL. During follow-up of 19.5 ± 17.0 months, none of the patients died or required further reinterventions. No device-related adverse events were reported. CONCLUSIONS: Hemoadsorption via a stand-alone apheresis pump during OPCAB surgery was feasible and safe. This innovative and new approach showed favorable bleeding rates in patients on antithrombotic drugs requiring bypass surgery.
Assuntos
Ponte de Artéria Coronária sem Circulação Extracorpórea , Humanos , Masculino , Pessoa de Meia-Idade , Idoso , Feminino , Ponte de Artéria Coronária sem Circulação Extracorpórea/efeitos adversos , Fibrinolíticos , Ticagrelor , Rivaroxabana , Ponte de Artéria Coronária , Resultado do TratamentoRESUMO
PURPOSE: To advance a transition towards an indication-based chest radiograph (CXR) ordering in intensive care units (ICUs) without compromising patient safety. MATERIALS AND METHODS: Single-center prospective cohort study with a retrospective reference group including 857 ICU patients. The routine group (n = 415) received CXRs at the discretion of the ICU physician, the restrictive group (n = 442) if specified by an indication catalogue. Documented data include number of CXRs per day and CXR radiation dose as primary outcomes, re-intubation and re-admission rates, hours of mechanical ventilation and ICU length of stay. RESULTS: CXR numbers were reduced in the restrictive group (964 CXRs in 2479 days vs. 1281 CXRs in 2318 days) and median radiation attributed to CXR per patient was significantly lowered in the restrictive group (0.068 vs. 0.076 Gy x cm2, P = 0.003). For patients staying ≥24 h, median number of CXRs per day was significantly reduced in the restrictive group (0.41 (IQR 0.21-0.61) vs. 0.55 (IQR 0.34-0.83), P < 0.001). Survival analysis proved non-inferiority. Secondary outcome parameters were not significantly different between the groups. CXR reduction was significant even for patients in most critical conditions. CONCLUSIONS: A substantial reduction of the number of CXRs on ICUs was feasible and safe using an indication catalogue thereby improving resource management. TRIAL REGISTRATION: DRKS00015621, German Clinical Trials Register.
Assuntos
Unidades de Terapia Intensiva , Radiografia Torácica , Humanos , Estudos Prospectivos , Radiografia , Estudos RetrospectivosRESUMO
The inner membrane of mitochondria is organized in two morphologically distinct domains, the inner boundary membrane (IBM) and the cristae membrane (CM), which are connected by narrow, tubular cristae junctions. The protein composition of these domains, their dynamics, and their biogenesis and maintenance are poorly understood at the molecular level. We have used quantitative immunoelectron microscopy to determine the distribution of a collection of representative proteins in yeast mitochondria belonging to seven major processes: oxidative phosphorylation, protein translocation, metabolite exchange, mitochondrial morphology, protein translation, iron-sulfur biogenesis, and protein degradation. We show that proteins are distributed in an uneven, yet not exclusive, manner between IBM and CM. The individual distributions reflect the physiological functions of proteins. Moreover, proteins can redistribute between the domains upon changes of the physiological state of the cell. Impairing assembly of complex III affects the distribution of partially assembled subunits. We propose a model for the generation of this dynamic subcompartmentalization of the mitochondrial inner membrane.
Assuntos
Membranas Intracelulares/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Compartimento Celular , Técnicas Imunoenzimáticas , Proteínas Mitocondriais/genética , Fosforilação Oxidativa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Loss-of-function mutations in the parkin gene (PARK2) and PINK1 gene (PARK6) are associated with autosomal recessive parkinsonism. PINK1 deficiency was recently linked to mitochondrial pathology in human cells and Drosophila melanogaster, which can be rescued by parkin, suggesting that both genes play a role in maintaining mitochondrial integrity. Here we demonstrate that an acute down-regulation of parkin in human SH-SY5Y cells severely affects mitochondrial morphology and function, a phenotype comparable with that induced by PINK1 deficiency. Alterations in both mitochondrial morphology and ATP production caused by either parkin or PINK1 loss of function could be rescued by the mitochondrial fusion proteins Mfn2 and OPA1 or by a dominant negative mutant of the fission protein Drp1. Both parkin and PINK1 were able to suppress mitochondrial fragmentation induced by Drp1. Moreover, in Drp1-deficient cells the parkin/PINK1 knockdown phenotype did not occur, indicating that mitochondrial alterations observed in parkin- or PINK1-deficient cells are associated with an increase in mitochondrial fission. Notably, mitochondrial fragmentation is an early phenomenon upon PINK1/parkin silencing that also occurs in primary mouse neurons and Drosophila S2 cells. We propose that the discrepant findings in adult flies can be explained by the time of phenotype analysis and suggest that in mammals different strategies may have evolved to cope with dysfunctional mitochondria.
Assuntos
Proteínas de Drosophila/fisiologia , Mitocôndrias/metabolismo , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Apoptose , Linhagem Celular , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitina-Proteína Ligases/genéticaRESUMO
The MDM31 and MDM32 genes are required for normal distribution and morphology of mitochondria in the yeast Saccharomyces cerevisiae. They encode two related proteins located in distinct protein complexes in the mitochondrial inner membrane. Cells lacking Mdm31 and Mdm32 harbor giant spherical mitochondria with highly aberrant internal structure. Mitochondrial DNA (mtDNA) is instable in the mutants, mtDNA nucleoids are disorganized, and their association with Mmm1-containing complexes in the outer membrane is abolished. Mutant mitochondria are largely immotile, resulting in a mitochondrial inheritance defect. Deletion of either one of the MDM31 and MDM32 genes is synthetically lethal with deletion of either one of the MMM1, MMM2, MDM10, and MDM12 genes, which encode outer membrane proteins involved in mitochondrial morphogenesis and mtDNA inheritance. We propose that Mdm31 and Mdm32 cooperate with Mmm1, Mmm2, Mdm10, and Mdm12 in maintenance of mitochondrial morphology and mtDNA.
Assuntos
DNA Mitocondrial/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Actinas/metabolismo , Epistasia Genética , Instabilidade Genômica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas de Membrana/classificação , Proteínas de Membrana/genética , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/classificação , Proteínas Mitocondriais/genética , Complexos Multiproteicos , Fenótipo , Filogenia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/classificação , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Mitochondrial distribution and morphology depend on MDM33, a Saccharomyces cerevisiae gene encoding a novel protein of the mitochondrial inner membrane. Cells lacking Mdm33 contain ring-shaped, mostly interconnected mitochondria, which are able to form large hollow spheres. On the ultrastructural level, these aberrant organelles display extremely elongated stretches of outer and inner membranes enclosing a very narrow matrix space. Dilated parts of Delta mdm33 mitochondria contain well-developed cristae. Overexpression of Mdm33 leads to growth arrest, aggregation of mitochondria, and generation of aberrant inner membrane structures, including septa, inner membrane fragments, and loss of inner membrane cristae. The MDM33 gene is required for the formation of net-like mitochondria in mutants lacking components of the outer membrane fission machinery, and mitochondrial fusion is required for the formation of extended ring-like mitochondria in cells lacking the MDM33 gene. The Mdm33 protein assembles into an oligomeric complex in the inner membrane where it performs homotypic protein-protein interactions. Our results indicate that Mdm33 plays a distinct role in the mitochondrial inner membrane to control mitochondrial morphology. We propose that Mdm33 is involved in fission of the mitochondrial inner membrane.
Assuntos
Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Candida albicans/genética , GTP Fosfo-Hidrolases/metabolismo , Genes Fúngicos , Membranas Intracelulares/metabolismo , Proteínas Luminescentes/metabolismo , Substâncias Macromoleculares , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Peso Molecular , Conformação Proteica , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de SequênciaRESUMO
Degeneration of dopaminergic neurons in the substantia nigra is characteristic for Parkinson's disease (PD), the second most common neurodegenerative disorder. Mitochondrial dysfunction is believed to contribute to the etiology of PD. Although most cases are sporadic, recent evidence points to a number of genes involved in familial variants of PD. Among them, a loss-of-function of phosphatase and tensin homolog-induced kinase 1 (PINK1; PARK6) is associated with rare cases of autosomal recessive parkinsonism. In HeLa cells, RNA interference-mediated downregulation of PINK1 results in abnormal mitochondrial morphology and altered membrane potential. Morphological changes of mitochondria can be rescued by expression of wild-type PINK1 but not by PD-associated PINK1 mutants. Moreover, primary cells derived from patients with two different PINK1 mutants showed a similar defect in mitochondrial morphology. Human parkin but not PD-associated mutants could rescue mitochondrial pathology in human cells like wild-type PINK1. Our results may therefore suggest that PINK1 deficiency in humans results in mitochondrial abnormalities associated with cellular stress, a pathological phenotype, which can be ameliorated by enhanced expression of parkin.
Assuntos
Mitocôndrias/enzimologia , Mitocôndrias/patologia , Proteínas Quinases/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Células Cultivadas , Células HeLa , Humanos , Mitocôndrias/genética , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/patologia , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , Fenótipo , Proteínas Quinases/deficiência , Proteínas Quinases/genéticaRESUMO
We report a case of a 19-year-old female who presented with right flank pain and microscopic hematuria. Three years earlier, she sustained a stab wound to the right flank and was managed conservatively. After being diagnosed with an enhancing renal mass using computed tomography (CT) scan, duplex ultrasound and angiography were performed revealing an intrarenal pseudoaneurysm. Endovascular coils were successfully employed to selectively embolize the pseudoaneurysm.
Assuntos
Falso Aneurisma/diagnóstico , Falso Aneurisma/terapia , Embolização Terapêutica , Artéria Renal/lesões , Adulto , Falso Aneurisma/complicações , Angiografia , Meios de Contraste , Diagnóstico Diferencial , Feminino , Dor no Flanco/etiologia , Hematúria/etiologia , Humanos , Tomografia Computadorizada por Raios X , Ultrassonografia Doppler DuplaRESUMO
Protein quality control is an essential function of the endoplasmic reticulum. Misfolded proteins unable to acquire their native conformation are retained in the endoplasmic reticulum, retro-translocated back into the cytosol, and degraded via the ubiquitin-proteasome system. We show that efficient degradation of soluble malfolded proteins in yeast requires a fully competent early secretory pathway. Mutations in proteins essential for ER-Golgi protein traffic severely inhibit ER degradation of the model substrate CPY*. We found ER localization of CPY* in WT cells, but no other specific organelle for ER degradation could be identified by electron microscopy studies. Because CPY* is degraded in COPI coat mutants, only a minor fraction of CPY* or of a proteinaceous factor required for degradation seems to enter the recycling pathway between ER and Golgi. Therefore, we propose that the disorganized structure of the ER and/or the mislocalization of Kar2p, observed in early secretory mutants, is responsible for the reduction in CPY* degradation. Further, we observed that mutations in proteins directly involved in degradation of malfolded proteins (Der1p, Der3/Hrd1p, and Hrd3p) lead to morphological changes of the endoplasmic reticulum and the Golgi, escape of CPY* into the secretory pathway and a slower maturation rate of wild-type CPY.
Assuntos
Retículo Endoplasmático/fisiologia , Complexo de Golgi/fisiologia , Saccharomyces cerevisiae/fisiologia , Cicloeximida/farmacologia , Genótipo , Cinética , Plasmídeos , Dobramento de Proteína , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sistemas do Segundo Mensageiro/fisiologiaRESUMO
Remodeling of crista junctions (CJs) is observed in numerous human disorders and during apoptosis. The functional interplay of OPA1 and MIC60, two key players in this context, is unclear. We show that OPA1 modulates cristae morphology but is dispensable for CJ formation. MIC60 is strongly enriched at CJs, whereas OPA1 is distributed evenly across the inner membrane. MIC60 levels are increased in OPA1-/- cells which show increased cellular resistance to apoptosis induction. Endogenous OPA1 and MIC60 show a physical interaction. Overall, we suggest that the regulation of CJ remodeling during apoptosis is mediated via an interplay between OPA1 and MIC60.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Musculares/metabolismo , Animais , Apoptose , Linhagem Celular , GTP Fosfo-Hidrolases/genética , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas Musculares/genética , Ligação ProteicaRESUMO
Transcranial direct current stimulation (tDCS) continues to demonstrate success as a medical intervention for neurodegenerative diseases, psychological conditions, and traumatic brain injury recovery. One aspect of tDCS still not fully comprehended is the influence of the tDCS electric field on neural functionality. To address this issue, we present a mathematical, multiscale model that couples tDCS administration to neuron electrodynamics. We demonstrate the model's validity and medical applicability with computational simulations using an idealized two-dimensional domain and then an MRI-derived, three-dimensional human head geometry possessing inhomogeneous and anisotropic tissue conductivities. We exemplify the capabilities of these simulations with real-world tDCS electrode configurations and treatment parameters and compare the model's predictions to those attained from medical research studies. The model is implemented using efficient numerical strategies and solution techniques to allow the use of fine computational grids needed by the medical community.
Assuntos
Imageamento por Ressonância Magnética/métodos , Processamento de Sinais Assistido por Computador , Estimulação Transcraniana por Corrente Contínua/métodos , Potenciais de Ação , Anisotropia , Encéfalo/patologia , Encéfalo/fisiologia , Simulação por Computador , Estimulação Elétrica , Eletrodos , Análise de Elementos Finitos , Cabeça , Humanos , Cinética , Neurônios/fisiologiaRESUMO
Crista junctions (CJs) are tubular invaginations of the inner membrane of mitochondria that connect the inner boundary with the cristae membrane. These architectural elements are critical for mitochondrial function. The yeast inner membrane protein Fcj1, called mitofilin in mammals, was reported to be preferentially located at CJs and crucial for their formation. Here we investigate the functional roles of individual domains of Fcj1. The most conserved part of Fcj1, the C-terminal domain, is essential for Fcj1 function. In its absence, formation of CJ is strongly impaired and irregular, and stacked cristae are present. This domain interacts with full-length Fcj1, suggesting a role in oligomer formation. It also interacts with Tob55 of the translocase of outer membrane ß-barrel proteins (TOB)/sorting and assembly machinery (SAM) complex, which is required for the insertion of ß-barrel proteins into the outer membrane. The association of the TOB/SAM complex with contact sites depends on the presence of Fcj1. The biogenesis of ß-barrel proteins is not significantly affected in the absence of Fcj1. However, down-regulation of the TOB/SAM complex leads to altered cristae morphology and a moderate reduction in the number of CJs. We propose that the C-terminal domain of Fcj1 is critical for the interaction of Fcj1 with the TOB/SAM complex and thereby for stabilizing CJs in close proximity to the outer membrane. These results assign novel functions to both the C-terminal domain of Fcj1 and the TOB/SAM complex.
Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência Conservada , Regulação para Baixo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/ultraestrutura , Relação Estrutura-AtividadeRESUMO
OBJECTIVES: Delayed urinary anastomotic leak after transperitoneal robot-assisted radical prostatectomy (RALP) is an uncommon complication. After failure of conventional measures, we successfully managed this problem using a nephroureteral stent placed on intermittent suction. METHODS: A 62-year-old man with clinical stage T1c prostate cancer (Gleason 3 + 3) developed a persistent urinary anastomotic leak after RALP. Conventional measures, including catheter traction, passive drainage, and needle vented Foley catheter suction, failed. On postoperative day 6 a unilateral nephroureteral stent was placed on intermittent suction. RESULTS: Placement of one nephroureteral stent on suction device immediately stopped the urinary anastomotic leakage into the peritoneal cavity. CONCLUSIONS: In case of a persistent urinary leak after RALP that fails conservative management, a nephroureteral stent on suction may aid to stop the anastomotic leak.
Assuntos
Laparoscopia/métodos , Prostatectomia/métodos , Robótica , Stents , Uretra/cirurgia , Bexiga Urinária/cirurgia , Anastomose Cirúrgica/efeitos adversos , Anastomose Cirúrgica/métodos , Desenho de Equipamento , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/cirurgia , Desenho de Prótese , Sucção/instrumentação , Procedimentos Cirúrgicos Urológicos/efeitos adversos , Procedimentos Cirúrgicos Urológicos/métodosRESUMO
The complexes of the respiratory chain represent mosaics of nuclear and mitochondrially encoded components. The processes by which synthesis and assembly of the various subunits are coordinated remain largely elusive. During evolution, many proteins of the mitochondrial ribosome acquired additional domains pointing at specific properties or functions of the translation machinery in mitochondria. Here, we analyzed the function of Mrpl36, a protein associated with the large subunit of the mitochondrial ribosome. This protein, homologous to the ribosomal protein L31 from bacteria, contains a mitochondria-specific C-terminal domain that is not required for protein synthesis per se; however, its absence decreases stability of Mrpl36. Cells lacking this C-terminal domain can still synthesize proteins, but these translation products fail to be properly assembled into respiratory chain complexes and are rapidly degraded. Surprisingly, overexpression of Mrpl36 seems to even increase the efficiency of mitochondrial translation. Our data suggest that Mrpl36 plays a critical role during translation that determines the rate of respiratory chain assembly. This important function seems to be carried out by a stabilizing activity of Mrpl36 on the interaction between large and small ribosomal subunits, which could influence accuracy of protein synthesis.
Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/biossíntese , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Modelos Biológicos , Mutação/genética , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Ribossômicas/química , Ribossomos/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , SolubilidadeRESUMO
The mitochondrial dynamin-like GTPase Mgm1 exists as a long (l-Mgm1) and a short isoform (s-Mgm1). They both are essential for mitochondrial fusion. Here we show that the isoforms interact in a homotypic and heterotypic manner. Their submitochondrial distribution between inner boundary membrane and cristae was markedly different. Overexpression of l-Mgm1 exerts a dominant negative effect on mitochondrial fusion. A functional GTPase domain is required only in s-Mgm1 but not in l-Mgm1. We propose that l-Mgm1 acts primarily as an anchor in the inner membrane that in concert with the GTPase activity of s-Mgm1 mediates the fusion of inner membranes.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Fusão de Membrana/fisiologia , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Dinaminas/química , Dinaminas/metabolismo , Microscopia Imunoeletrônica , Proteínas Mitocondriais/genética , Isoformas de Proteínas/metabolismoRESUMO
Crista junctions (CJs) are important for mitochondrial organization and function, but the molecular basis of their formation and architecture is obscure. We have identified and characterized a mitochondrial membrane protein in yeast, Fcj1 (formation of CJ protein 1), which is specifically enriched in CJs. Cells lacking Fcj1 lack CJs, exhibit concentric stacks of inner membrane in the mitochondrial matrix, and show increased levels of F(1)F(O)-ATP synthase (F(1)F(O)) supercomplexes. Overexpression of Fcj1 leads to increased CJ formation, branching of cristae, enlargement of CJ diameter, and reduced levels of F(1)F(O) supercomplexes. Impairment of F(1)F(O) oligomer formation by deletion of its subunits e/g (Su e/g) causes CJ diameter enlargement and reduction of cristae tip numbers and promotes cristae branching. Fcj1 and Su e/g genetically interact. We propose a model in which the antagonism between Fcj1 and Su e/g locally modulates the F(1)F(O) oligomeric state, thereby controlling membrane curvature of cristae to generate CJs and cristae tips.
Assuntos
Membranas Intracelulares , Mitocôndrias , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Modelos Anatômicos , Estrutura Quaternária de Proteína , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The F1F0-ATP synthase in mitochondria, in addition to its function in energy transduction, has a structural role in determining cristae morphology. This depends on its ability to form dimeric and higher oligomeric supracomplexes. Here we show that mutants of the dimer-specific subunits e and g, which destabilize dimeric and oligomeric F1F0-ATP synthase supracomplexes, have a decreased mitochondrial membrane potential delta psi. The degree of destabilization correlated with the reduction of the membrane potential. The enzymatic activities of F1F0-ATP synthase and cytochrome c oxidase, maximal respiration rate, coupling of oxidative phosphorylation, and tubular mitochondrial morphology were not affected or only to a minor extent. In mutants lacking one or two coiled-coil domains of subunit e, the reduction of the mitochondrial membrane potential was not due to loss of mitochondrial DNA, a reduced capacity of oxidative phosphorylation, or to altered cristae morphology. We propose a role for the supracomplexes of the F1F0-ATP synthase in organizing microdomains within the inner membrane, ensuring optimal bioenergetic competence of mitochondria.
Assuntos
Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/química , Citocromos/metabolismo , DNA/química , Dimerização , Potenciais da Membrana , Oxigênio/metabolismo , Fenótipo , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Oxygen radicals are formed by the endothelium and blood cells and have specific functions in various organs systems. On the level of the microcirculation, oxygen radicals take part in the regulation of the leukocyte-endothelial interaction. The involvement of oxygen radicals has previously been found in conditions such as sepsis, ischemia-reperfusion, and inflammation. Indomethacin is a clinically applied nonsteroidal antiphlogistic, and in previous studies in the rat, it has been found to induce an inflammatory reaction in the small intestine characterized by edema and reddening of the intestinal epithelium, ulceration, and dysregulation in the intestinal-epithelial barrier function. In the present study, we investigated the effect of N-acetylcysteine on erythrocyte velocity and the arteriolar diameter of the main arteriole in single villi, thus providing insight in the perfusion of the mucosa in indomethacin-induced intestinal inflammation. N-Acetylcysteine is known to inactivate superoxide and its precursors. Therefore, we used N-acetylcysteine to investigate whether superoxide and its precursors participate in the regulation of blood supply to single villi in this animal model. We found that indomethacin induced an increase in villous perfusion that was significantly reduced by N-acetylcysteine, indicating that superoxide and its precursors may participate in the regulation of blood supply to the mucosa in this animal model of intestinal inflammation.
Assuntos
Acetilcisteína/farmacologia , Inflamação/patologia , Mucosa Intestinal/irrigação sanguínea , Mucosa Intestinal/efeitos dos fármacos , Microcirculação/fisiologia , Animais , Modelos Animais de Doenças , Sequestradores de Radicais Livres/análise , Indometacina , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/fisiopatologia , Masculino , Microcirculação/efeitos dos fármacos , Probabilidade , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Valores de Referência , Fluxo Sanguíneo Regional , Estatísticas não ParamétricasRESUMO
Mitochondrial cytochrome P450 enzymes play a crucial role in the steroid biosynthesis in human adrenals, catalyzing regio- and stereospecific hydroxylations. In search of a new model system for the study of these enzymes, we expressed the human CYP11B2 (aldosterone synthase, P450(aldo)) in fission yeast Schizosaccharomyces pombe. Analysis of the subcellular localization of the P450 enzyme by Western blot analysis, fluorescence microscopy, and electron microscopy demonstrated that the mitochondrial localization signal of the human protein is functional in S. pombe. The transformed yeasts show the inducible ability to convert in vivo considerable amounts of 11-deoxycortisol to cortisol and 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone, and aldosterone, respectively. Although in mammalian cells, mitochondrial steroid hydroxylases depend for their activity on an electron transport chain that consists of two proteins, adrenodoxin and adrenodoxin reductase, no coexpression of these proteins is needed for efficient substrate conversion by intact fission yeast cells. Searching the fission yeast genome for adrenodoxin homologues, a gene was identified that codes for a protein with an amino terminal domain homologous to COX15 of Saccharomyces cerevisiae and a carboxy terminal ferredoxin domain. It was found that overexpression of this gene significantly enhances steroid hydroxylase activity of CYP11B2 expressing fission yeast cells. Moreover, the bacterially expressed ferredoxin domain of this protein can replace adrenodoxin in a reconstituted steroid hydroxylation assay and transfer electrons from adrenodoxin reductase to a mammalian or a bacterial cytochrome P450. Therefore, we suggest to name this protein etp1 (electron-transfer protein 1).