Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 67
Filtrar
1.
Sci Rep ; 11(1): 69, 2021 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420156

RESUMO

Nucleation is generally viewed as a structural fluctuation that passes a critical size to eventually become a stable emerging new phase. However, this concept leaves out many details, such as changes in cluster composition and competing pathways to the new phase. In this work, both experimental and computer modeling studies are used to understand the cluster composition and pathways. Monte Carlo and molecular dynamics approaches are used to analyze the thermodynamic and kinetic contributions to the nucleation landscape in barium silicate glasses. Experimental techniques examine the resulting polycrystals that form. Both the modeling and experimental data indicate that a silica rich core plays a dominant role in the nucleation process.

2.
Trends Ecol Evol ; 2021 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-33436278

RESUMO

Biologists have long appreciated the critical role that energy turnover plays in understanding variation in performance and fitness among individuals. Whole-organism metabolic studies have provided key insights into fundamental ecological and evolutionary processes. However, constraints operating at subcellular levels, such as those operating within the mitochondria, can also play important roles in optimizing metabolism over different energetic demands and time scales. Herein, we explore how mitochondrial aerobic metabolism influences different aspects of organismal performance, such as through changing adenosine triphosphate (ATP) and reactive oxygen species (ROS) production. We consider how such insights have advanced our understanding of the mechanisms underpinning key ecological and evolutionary processes, from variation in life-history traits to adaptation to changing thermal conditions, and we highlight key areas for future research.

3.
PLoS One ; 15(10): e0239804, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33031404

RESUMO

Mitochondrial OXPHOS generates most of the energy required for cellular function. OXPHOS biogenesis requires the coordinated expression of the nuclear and mitochondrial genomes. This represents a unique challenge that highlights the importance of nuclear-mitochondrial genetic communication to cellular function. Here we investigated the transcriptomic and functional consequences of nuclear-mitochondrial genetic divergence in vitro and in vivo. We utilized xenomitochondrial cybrid cell lines containing nuclear DNA from the common laboratory mouse Mus musculus domesticus and mitochondrial DNA (mtDNA) from Mus musculus domesticus, or exogenous mtDNA from progressively divergent mouse species Mus spretus, Mus terricolor, Mus caroli and Mus pahari. These cybrids model a wide range of nuclear-mitochondrial genetic divergence that cannot be achieved with other research models. Furthermore, we used a xenomitochondrial mouse model generated in our laboratory that harbors wild-type, C57BL/6J Mus musculus domesticus nuclear DNA and homoplasmic mtDNA from Mus terricolor. RNA sequencing analysis of xenomitochondrial cybrids revealed an activation of interferon signaling pathways even in the absence of OXPHOS dysfunction or immune challenge. In contrast, xenomitochondrial mice displayed lower baseline interferon gene expression and an impairment in the interferon-dependent innate immune response upon immune challenge with herpes simplex virus, which resulted in decreased viral control. Our work demonstrates that nuclear-mitochondrial genetic divergence caused by the introduction of exogenous mtDNA can modulate the interferon immune response both in vitro and in vivo, even when OXPHOS function is not compromised. This work may lead to future insights into the role of mitochondrial genetic variation and the immune function in humans, as patients affected by mitochondrial disease are known to be more susceptible to immune challenges.


Assuntos
Núcleo Celular/genética , DNA Mitocondrial , Interferons/imunologia , Mitocôndrias/genética , Animais , Linhagem Celular , Feminino , Genótipo , Imunidade Inata , Masculino , Camundongos/classificação , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação Oxidativa
4.
Int J Mol Sci ; 21(6)2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-32244971

RESUMO

The lack of effective treatments for mitochondrial disease has seen the development of new approaches, including those that aim to stimulate mitochondrial biogenesis to boost ATP generation above a critical disease threshold. Here, we examine the effects of the peroxisome proliferator-activated receptor γ (PPARγ) activator pioglitazone (PioG), in combination with deoxyribonucleosides (dNs), on mitochondrial biogenesis in cybrid cells containing >90% of the m.3243A>G mutation associated with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). PioG + dNs combination treatment increased mtDNA copy number and mitochondrial mass in both control (CON) and m.3243A>G (MUT) cybrids, with no adverse effects on cell proliferation. PioG + dNs also increased mtDNA-encoded transcripts in CON cybrids, but had the opposite effect in MUT cybrids, reducing the already elevated transcript levels. Steady-state levels of mature oxidative phosphorylation (OXPHOS) protein complexes were increased by PioG + dNs treatment in CON cybrids, but were unchanged in MUT cybrids. However, treatment was able to significantly increase maximal mitochondrial oxygen consumption rates and cell respiratory control ratios in both CON and MUT cybrids. Overall, these findings highlight the ability of PioG + dNs to improve mitochondrial respiratory function in cybrid cells containing the m.3243A>G MELAS mutation, as well as their potential for development into novel therapies to treat mitochondrial disease.

5.
FEBS Lett ; 594(4): 590-610, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31944285

RESUMO

Mitochondria provide the main source of energy for eukaryotic cells, oxidizing fatty acids and sugars to generate ATP. Mitochondrial fatty acid ß-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are two key pathways involved in this process. Disruption of FAO can cause human disease, with patients commonly presenting with liver failure, hypoketotic glycaemia and rhabdomyolysis. However, patients with deficiencies in the FAO enzyme short-chain enoyl-CoA hydratase 1 (ECHS1) are typically diagnosed with Leigh syndrome, a lethal form of subacute necrotizing encephalomyelopathy that is normally associated with OXPHOS dysfunction. Furthermore, some ECHS1-deficient patients also exhibit secondary OXPHOS defects. This sequela of FAO disorders has long been thought to be caused by the accumulation of inhibitory fatty acid intermediates. However, new evidence suggests that the mechanisms involved are more complex, and that disruption of OXPHOS protein complex biogenesis and/or stability is also involved. In this review, we examine the clinical, biochemical and genetic features of all ECHS1-deficient patients described to date. In particular, we consider the secondary OXPHOS defects associated with ECHS1 deficiency and discuss their possible contribution to disease pathogenesis.


Assuntos
Enoil-CoA Hidratase/deficiência , Fosforilação Oxidativa , Animais , Ácidos Graxos/metabolismo , Humanos , Mitocôndrias/metabolismo , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia
6.
Child Psychiatry Hum Dev ; 51(4): 552-562, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-31664631

RESUMO

Cognitive-behavioural models of obsessive-compulsive disorder (OCD) propose that inflated responsibility beliefs are central to the maintenance of the disorder and are proposed to originate during early childhood via experiences of harsh and/or controlling parenting. The current study aimed to examine the associations between perceived parental rearing behaviours, inflated responsibility/threat beliefs, and OCD severity and impairment in children (aged 7-12 years) and adolescents (aged 13-17 years) with OCD (n = 136). Results indicated that for younger children, greater child perceptions of overprotection and anxious rearing were each associated with increased inflated responsibility beliefs. For older children, these positive associations remained, and furthermore, inflated responsibility beliefs mediated the association between perceived maternal anxious rearing and OCD impairment. Results highlight the role of the family in the development of inflated responsibility bias and OCD-related impairment.

7.
J Am Acad Orthop Surg ; 27(23): e1059-e1067, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-30889040

RESUMO

BACKGROUND: It is unknown whether more expensive total knee prostheses provide better improvements in patient-determined outcomes compared with less expensive prostheses. A physician-owned distributorship (POD) was created with a goal to provide lower cost implants to hospitals as an alternative to higher cost prostheses sold by the large orthopaedic implant companies. The hypothesis was that lower cost total knee prostheses would have equivalent outcomes, while resulting in lower costs to the hospitals purchasing them compared with higher cost industry-supplied knee prostheses. METHODS: From May 2013 until January 2015, a POD existed which included five surgeons that performed total knee arthroplasties and were willing to follow the outcomes to ensure quality. The POD sold two knee arthroplasty systems at a cost that was lower than that of the large industry companies. Surgeons were allowed to use either POD knees or industry knees at their own discretion. Patients were followed up prospectively to determine The Knee Injury and Osteoarthritis Outcome Score (KOOS) outcomes at 2 years and any incidence of knee complications that required surgery. RESULTS: Two hundred-nine knees (35.2%) had a POD knee implanted, and 385 knees had an industry knee implanted. Both POD knees and industry knees showed statistically significant improvements (P < 0.0001) for all subgroups of the KOOS. No statistically significant difference was observed in improvement in any subgroup of the KOOS between the groups. Knee complications requiring surgical intervention were similar (2.9% POD knees versus 3.6% industry knees; P = 0.58). Using lower cost POD knees saved $209,875.71. CONCLUSIONS: No difference was observed in improvements in outcomes or complications in the lower cost POD-supplied knees compared with the higher cost industry-supplied knees. Hospitals and surgeons may consider using lower cost prostheses because the increased cost of the prosthesis has not been correlated to improved outcomes. LEVEL OF EVIDENCE: Level II therapeutic prospective cohort study.


Assuntos
Artroplastia do Joelho/instrumentação , Prótese do Joelho/economia , Propriedade/economia , Médicos/economia , Idoso , Artroplastia do Joelho/economia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento
8.
Methods Mol Biol ; 1928: 69-76, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30725451

RESUMO

The mitochondrial membrane potential (Δψm) drives the generation of ATP by mitochondria. Interestingly, Δψm is higher in many cancer cells comparted to healthy noncancerous cell types, providing a unique metabolic marker. This feature has also been exploited for therapeutic use by utilizing drugs that specifically accumulate in the mitochondria of cancer cells with high Δψm. As such, the assessment of Δψm can provide very useful information as to the metabolic state of a cancer cell, as well as its potential for malignancy. In addition, the measurement of Δψm can also be used to test the ability of novel anticancer therapies to disrupt mitochondrial metabolism and cause cell death.Here, we outline two methods for assessing Δψm in cancer cells using confocal microscopy and the potentiometric fluorescent dye tetramethylrhodamine methyl ester (TMRM). In the first protocol, we describe a technique to quantitatively measure Δψm, which can be used to compare Δψm between different cell types. In the second protocol, we describe a technique for assessing changes to Δψm over time, which can be used to determine the effectiveness of different therapeutic compounds or drugs in modulating mitochondrial function.


Assuntos
Corantes Fluorescentes , Potencial da Membrana Mitocondrial , Microscopia de Fluorescência , Rodaminas , Corantes Fluorescentes/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Confocal , Microscopia de Fluorescência/métodos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Rodaminas/metabolismo
9.
Cells ; 7(6)2018 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-29882869

RESUMO

Mitochondrial fatty acid β-oxidation (FAO) is the primary pathway for fatty acid metabolism in humans, performing a key role in liver, heart and skeletal muscle energy homeostasis. FAO is particularly important during times of fasting when glucose supply is limited, providing energy for many organs and tissues, including the heart, liver and brain. Deficiencies in FAO can cause life-threatening metabolic disorders in early childhood that present with liver dysfunction, hypoglycemia, dilated hypertrophic cardiomyopathy and Reye-like Syndrome. Alternatively, FAO defects can also cause ‘milder’ adult-onset disease with exercise-induced myopathy and rhabdomyolysis. Short-chain enoyl-CoA hydratase (ECHS1) is a key FAO enzyme involved in the metabolism of fatty acyl-CoA esters. ECHS1 deficiency (ECHS1D) also causes human disease; however, the clinical manifestation is unlike most other FAO disorders. ECHS1D patients commonly present with Leigh syndrome, a lethal form of subacute necrotizing encephalomyelopathy traditionally associated with defects in oxidative phosphorylation (OXPHOS). In this article, we review the clinical, biochemical and genetic features of the ESHS1D patients described to date, and discuss the significance of the secondary OXPHOS defects associated with ECHS1D and their contribution to overall disease pathogenesis.

10.
Sci Rep ; 8(1): 153, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29317722

RESUMO

Medium-chain acyl-Coenzyme A dehydrogenase (MCAD) is involved in the initial step of mitochondrial fatty acid ß-oxidation (FAO). Loss of function results in MCAD deficiency, a disorder that usually presents in childhood with hypoketotic hypoglycemia, vomiting and lethargy. While the disruption of mitochondrial fatty acid metabolism is the primary metabolic defect, secondary defects in mitochondrial oxidative phosphorylation (OXPHOS) may also contribute to disease pathogenesis. Therefore, we examined OXPHOS activity and stability in MCAD-deficient patient fibroblasts that have no detectable MCAD protein. We found a deficit in mitochondrial oxygen consumption, with reduced steady-state levels of OXPHOS complexes I, III and IV, as well as the OXPHOS supercomplex. To examine the mechanisms involved, we generated an MCAD knockout (KO) using human 143B osteosarcoma cells. These cells also exhibited defects in OXPHOS complex function and steady-state levels, as well as disrupted biogenesis of newly-translated OXPHOS subunits. Overall, our findings suggest that the loss of MCAD is associated with a reduction in steady-state OXPHOS complex levels, resulting in secondary defects in OXPHOS function which may contribute to the pathology of MCAD deficiency.


Assuntos
Acil-CoA Desidrogenase/deficiência , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Ácidos Graxos/metabolismo , Erros Inatos do Metabolismo Lipídico/genética , Erros Inatos do Metabolismo Lipídico/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Acil-CoA Desidrogenase/genética , Acil-CoA Desidrogenase/metabolismo , Respiração Celular , Células Cultivadas , DNA Mitocondrial , Fibroblastos/metabolismo , Deleção de Genes , Técnicas de Inativação de Genes , Marcação de Genes , Humanos , Fosforilação Oxidativa , Estabilidade Proteica , Espécies Reativas de Oxigênio
11.
Cell Death Discov ; 3: 17062, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28900542

RESUMO

Mitochondrial DNA copy number is strictly regulated during development as naive cells differentiate into mature cells to ensure that specific cell types have sufficient copies of mitochondrial DNA to perform their specialised functions. Mitochondrial DNA haplotypes are defined as specific regions of mitochondrial DNA that cluster with other mitochondrial sequences to show the phylogenetic origins of maternal lineages. Mitochondrial DNA haplotypes are associated with a range of phenotypes and disease. To understand how mitochondrial DNA haplotypes induce these characteristics, we used four embryonic stem cell lines that have the same set of chromosomes but possess different mitochondrial DNA haplotypes. We show that mitochondrial DNA haplotypes influence changes in chromosomal gene expression and affinity for nuclear-encoded mitochondrial DNA replication factors to modulate mitochondrial DNA copy number, two events that act synchronously during differentiation. Global DNA methylation analysis showed that each haplotype induces distinct DNA methylation patterns, which, when modulated by DNA demethylation agents, resulted in skewed gene expression patterns that highlight the effectiveness of the new DNA methylation patterns established by each haplotype. The haplotypes differentially regulate α-ketoglutarate, a metabolite from the TCA cycle that modulates the TET family of proteins, which catalyse the transition from 5-methylcytosine, indicative of DNA methylation, to 5-hydroxymethylcytosine, indicative of DNA demethylation. Our outcomes show that mitochondrial DNA haplotypes differentially modulate chromosomal gene expression patterns of naive and differentiating cells by establishing mitochondrial DNA haplotype-specific DNA methylation patterns.

12.
J Vis Exp ; (119)2017 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-28190045

RESUMO

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (ΔΨm) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains ΔΨm, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix. We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and ΔΨm in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of ΔΨm using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and ΔΨm simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of ΔΨm determined.


Assuntos
Cálcio/análise , Potencial da Membrana Mitocondrial , Microscopia de Fluorescência/métodos , Mitocôndrias/metabolismo , Compostos de Anilina/metabolismo , Cálcio/metabolismo , Linhagem Celular , Corantes Fluorescentes/metabolismo , Humanos , Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal , Membranas Mitocondriais/metabolismo , Rodaminas/metabolismo , Xantenos/metabolismo
13.
J Leukoc Biol ; 101(4): 901-911, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27881605

RESUMO

The interleukin (IL)-1 family member IL-37 is one of few anti-inflammatory cytokines, and it is capable of countering a broad spectrum of proinflammatory assaults. Although it is known that leukocytes are a major source of IL-37, knowledge on IL-37 production and secretion in specific immune cell types remains limited. Thus, we investigated IL-37 mRNA expression as well as protein production and secretion in human PBMCs. In PBMCs stimulated with agonists of Toll-like receptors (TLRs) 1-6 and 9, IL1F7 (the IL-37-encoding gene) was induced up to 9-fold, peaked at 6-8 h and returned to steady-state at 72 h. LPS-induced IL1F7 expression comprised isoforms b and c but not a and e Flow cytometry revealed that among IL-37+ PBMCs, monocytes predominated (81-91%), but T cells (6-8%) and myeloid dendritic cells (mDCs, 1-2%) also contributed to the IL-37+ leukocyte pool. Monocytes and mDCs, but not T cells, were capable of secreting IL-37. Whereas monocytes and mDCs secreted IL-37 upon LPS stimulation, only mDCs also released IL-37 at steady-state. Among monocyte subsets, IL-37 was LPS inducible and secreted only in classical and, although less pronounced, in intermediate monocytes; secretion was observed as early as 3 h after stimulation. Overall, our data suggest that constitutive IL-37 secretion by mDCs may serve to maintain an anti-inflammatory milieu at steady state, whereas IL-37 is stored in monocytes to be available for rapid release upon inflammatory encounters, thus acting as a novel anti-inflammatory alarmin. These insights may prove important to advancing towards clinical use the protective functions of one of the most powerful anti-inflammatory mediators so far discovered.


Assuntos
Células Dendríticas/metabolismo , Interleucina-1/metabolismo , Monócitos/metabolismo , Adulto , Células Dendríticas/efeitos dos fármacos , Citometria de Fluxo , Humanos , Cinética , Ligantes , Lipopolissacarídeos/farmacologia , Monócitos/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Receptores Toll-Like/metabolismo
14.
Elife ; 52016 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-27554484

RESUMO

The TIM22 complex mediates the import of hydrophobic carrier proteins into the mitochondrial inner membrane. While the TIM22 machinery has been well characterised in yeast, the human complex remains poorly characterised. Here, we identify Tim29 (C19orf52) as a novel, metazoan-specific subunit of the human TIM22 complex. The protein is integrated into the mitochondrial inner membrane with it's C-terminus exposed to the intermembrane space. Tim29 is required for the stability of the TIM22 complex and functions in the assembly of hTim22. Furthermore, Tim29 contacts the Translocase of the Outer Mitochondrial Membrane, TOM complex, enabling a mechanism for transport of hydrophobic carrier substrates across the aqueous intermembrane space. Identification of Tim29 highlights the significance of analysing mitochondrial import systems across phylogenetic boundaries, which can reveal novel components and mechanisms in higher organisms.


Assuntos
Proteínas de Transporte da Membrana Mitocondrial/análise , Membranas Mitocondriais/enzimologia , Subunidades Proteicas/análise , Linhagem Celular , Humanos , Multimerização Proteica
16.
Pharm Res ; 33(9): 2298-306, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27283829

RESUMO

PURPOSE: The purpose of this study is to investigate a sol-gel transition property and content release profiles for thermosensitive poly-(D,L-lactide-co-glycolide)-block-poly-(ethylene glycol)-block-poly-(D,L-lactide-co-glycolide) (PLGA-b-PEG-b-PLGA) hydrogels carrying paclitaxel, rapamycin, and LS301, and to present a proof-of-concept that PLGA-b-PEG-b-PLGA hydrogels carrying paclitaxel, rapamycin, and LS301, called TheranoGel, exhibit excellent theranostic activity in peritoneal ES-2-luc ovarian cancer xenograft mice. METHODS: Thermosensitive PLGA-b-PEG-b-PLGA hydrogels carrying paclitaxel, rapamycin, and LS301, individually or in combination, were prepared via a lyophilization method, characterized with content release kinetics, and assessed with theranostic activity in ES-2-luc xenograft mice. RESULTS: A thermosensitive PLGA-b-PEG-b-PLGA sol-gel system was able to entrain 3 poorly water-soluble payloads, paclitaxel, rapamycin, and LS301 (TheranoGel). TheranoGel made a sol-to-gel transition at 37°C and slowly released 3 drugs at a simultaneous release rate in response to the physical dissociation of hydrogels in vitro. TheranoGel enabled loco-regional delivery of multi-drugs by forming a gel-depot in the peritoneal cavity of ES-2-luc xenograft mice. An intraperitoneal (IP) administration of TheranoGel resulted in excellent therapeutic and diagnostic activities, leading to the improved peritoneal surgery in ES-2-luc xenograft mice. CONCLUSIONS: TheranoGel prepared via a facile lyophiliation method enabled successful IP delivery of multi-drugs and exhibited excellent theranostic activity in vivo.


Assuntos
Hidrogéis/química , Neoplasias Ovarianas/dietoterapia , Paclitaxel/química , Neoplasias Peritoneais/tratamento farmacológico , Poliésteres/química , Polietilenoglicóis/química , Polímeros/química , Animais , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Feminino , Injeções Intraperitoneais/métodos , Camundongos , Camundongos Nus , Paclitaxel/administração & dosagem , Poliésteres/administração & dosagem , Polietilenoglicóis/administração & dosagem , Sirolimo/administração & dosagem , Sirolimo/química , Cirurgia Assistida por Computador/métodos
17.
PLoS One ; 11(4): e0154371, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27110715

RESUMO

Mutations in mitochondrial DNA (mtDNA) can cause mitochondrial disease, a group of metabolic disorders that affect both children and adults. Interestingly, individual mtDNA mutations can cause very different clinical symptoms, however the factors that determine these phenotypes remain obscure. Defects in mitochondrial oxidative phosphorylation can disrupt cell signaling pathways, which may shape these disease phenotypes. In particular, mitochondria participate closely in cellular calcium signaling, with profound impact on cell function. Here, we examined the effects of a homoplasmic m.13565C>T mutation in MT-ND5 on cellular calcium handling using transmitochondrial cybrids (ND5 mutant cybrids). We found that the oxidation of NADH and mitochondrial membrane potential (Δψm) were significantly reduced in ND5 mutant cybrids. These metabolic defects were associated with a significant decrease in calcium uptake by ND5 mutant mitochondria in response to a calcium transient. Inhibition of glycolysis with 2-deoxy-D-glucose did not affect cytosolic calcium levels in control cybrids, but caused an increase in cytosolic calcium in ND5 mutant cybrids. This suggests that glycolytically-generated ATP is required not only to maintain Δψm in ND5 mutant mitochondria but is also critical for regulating cellular calcium homeostasis. We conclude that the m.13565C>T mutation in MT-ND5 causes defects in both mitochondrial oxidative metabolism and mitochondrial calcium sequestration. This disruption of mitochondrial calcium handling, which leads to defects in cellular calcium homeostasis, may be an important contributor to mitochondrial disease pathogenesis.


Assuntos
Cálcio/metabolismo , Complexo I de Transporte de Elétrons/genética , Fibroblastos/metabolismo , Células Híbridas/metabolismo , Síndrome MELAS/genética , Proteínas Mitocondriais/genética , Trifosfato de Adenosina/biossíntese , Linhagem Celular Tumoral , Desoxiglucose/farmacologia , Complexo I de Transporte de Elétrons/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Regulação da Expressão Gênica , Glicólise/efeitos dos fármacos , Glicólise/genética , Humanos , Células Híbridas/efeitos dos fármacos , Células Híbridas/patologia , Síndrome MELAS/metabolismo , Síndrome MELAS/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Mutação , NAD/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Cultura Primária de Células , Transdução de Sinais
19.
Sci Rep ; 6: 23229, 2016 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-26987907

RESUMO

An increasing number of women fail to achieve pregnancy due to either failed fertilization or embryo arrest during preimplantation development. This often results from decreased oocyte quality. Indeed, reduced mitochondrial DNA copy number (mitochondrial DNA deficiency) may disrupt oocyte quality in some women. To overcome mitochondrial DNA deficiency, whilst maintaining genetic identity, we supplemented pig oocytes selected for mitochondrial DNA deficiency, reduced cytoplasmic maturation and lower developmental competence, with autologous populations of mitochondrial isolate at fertilization. Supplementation increased development to blastocyst, the final stage of preimplantation development, and promoted mitochondrial DNA replication prior to embryonic genome activation in mitochondrial DNA deficient oocytes but not in oocytes with normal levels of mitochondrial DNA. Blastocysts exhibited transcriptome profiles more closely resembling those of blastocysts from developmentally competent oocytes. Furthermore, mitochondrial supplementation reduced gene expression patterns associated with metabolic disorders that were identified in blastocysts from mitochondrial DNA deficient oocytes. These results demonstrate the importance of the oocyte's mitochondrial DNA investment in fertilization outcome and subsequent embryo development to mitochondrial DNA deficient oocytes.


Assuntos
DNA Mitocondrial/genética , Desenvolvimento Embrionário , Mitocôndrias/metabolismo , Oócitos/metabolismo , Sus scrofa/genética , Animais , Blastocisto/metabolismo , Meios de Cultura/química , Variações do Número de Cópias de DNA , Técnicas de Cultura Embrionária , Feminino , Fertilização In Vitro , Perfilação da Expressão Gênica , Mitocôndrias/genética , Gravidez , Sus scrofa/embriologia , Suínos
20.
FASEB J ; 30(6): 2236-48, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26929434

RESUMO

Mitochondrial complex I (NADH:ubiquinone oxidoreductase) must be assembled precisely from 45 protein subunits for it to function correctly. One of its mitochondrial DNA (mtDNA) encoded subunits, ND1, is incorporated during the early stages of complex I assembly. However, little is known about how mutations in ND1 affect this assembly process. We found that in human 143B cybrid cells carrying a homoplasmic MT-ND1 mutation, ND1 protein could not be translated. As a result, the early stages of complex I assembly were disrupted, with mature complex I undetectable and complex I-linked respiration severely reduced to 2.0% of control levels. Interestingly, complex IV (ferrocytochrome c:oxygen oxidoreductase) steady-state levels were also reduced to 40.3%, possibly due to its diminished stability in the absence of respiratory supercomplex formation. This was in comparison with 143B cybrid controls (that contained wild-type mtDNA on the same nuclear background), which exhibited normal complex I, complex IV, and supercomplex assembly. We conclude that the loss of ND1 stalls complex I assembly during the early stages of its biogenesis, which not only results in the loss of mature complex I but also disrupts the stability of complex IV and the respiratory supercomplex to cause mitochondrial dysfunction.-Lim, S. C., Hroudová, J., Van Bergen, N. J., Lopez Sanchez, M. I. G., Trounce, I. A., McKenzie, M. Loss of mitochondrial DNA-encoded protein ND1 results in disruption of complex I biogenesis during early stages of assembly.


Assuntos
DNA Mitocondrial/metabolismo , Regulação da Expressão Gênica/fisiologia , NADH Desidrogenase/metabolismo , Linhagem Celular Tumoral , DNA Mitocondrial/genética , Humanos , Mutação , NADH Desidrogenase/genética , Transcriptoma
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...