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1.
Biophys Chem ; 257: 106278, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31767207

RESUMO

The acclaimed explanation for mitochondrial oxidative phosphorylation (mOxPhos) is a proton or cation centric scheme. Such ideas were recently disclaimed and in lieu, an evidence-based oxygen-centric explanation, murburn concept, was proposed. The new understanding vouches for catalytic roles of diffusible reactive oxygen species (DROS). The involvement of DROS explains the "non-discoverability of an enzyme-linked high-energy phosphorylating intermediate", a historical predicament, which had fueled several trans-membrane potential (TMP) based mechano-electrical explanations like the Nath model. This communication aims to briefly apprise the readers some lacunae and inadmissible aspects of the Nath model and project the appeal of murburn scheme of mOxPhos.


Assuntos
Trifosfato de Adenosina , Fosforilação Oxidativa , Mitocôndrias , Prótons
2.
Biophys Chem ; 257: 106279, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31757522

RESUMO

In a recent publication, Manoj raises criticisms against consensus views on the ATP synthase. The radical statements and assertions are shown to contradict a vast body of available knowledge that includes i) pioneering single-molecule biochemical and biophysical studies from the respected experimental groups of Kinosita, Yoshida, Noji, Börsch, Dunn, Gräber, Frasch, and Dimroth etc., ii) state-of-the-art X-ray and EM/cryo-EM structural information garnered over the decades by the expert groups of Leslie-Walker, Kühlbrandt, Mueller, Meier, Rubinstein, Sazanov, Duncan, and Pedersen on ATP synthase, iii) the pioneering energy-based computer simulations of Warshel, and iv) the novel theoretical and experimental works of Nath. Valid objections against Mitchell's chemiosmotic theory and Boyer's binding change mechanism put forth by Manoj have been addressed satisfactorily by Nath's torsional mechanism of ATP synthesis and two-ion theory of energy coupling and published 10 to 20 years ago, but these papers are not cited by him. This communication shows conclusively and in great detail that none of his objections apply to Nath's mechanism/theory. Nath's theory is further consolidated based on its previous predictive record, its consistency with biochemical evidence, its unified nature, its application to other related energy transductions and to disease, and finally its ability to guide the design of new experiments. Some constructive suggestions for high-resolution structural experiments that have the power to delve into the heart of the matter and throw unprecedented light on the nature of coupled ion translocation in the membrane-bound FO portion of F1FO-ATP synthase are made.


Assuntos
Fosforilação Oxidativa , Fotofosforilação , Trifosfato de Adenosina , Termodinâmica
3.
Cancer Sci ; 111(1): 36-46, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31705593

RESUMO

Osteosarcoma (OS) is a highly malignant bone tumor and the prognosis for non-responders to chemotherapy remains poor. Previous studies have shown that human sarcomas contain sarcoma-initiating cells (SIC), which have the characteristics of high tumorigenesis and resistance to chemotherapy. In the present study, we characterized SIC of a novel OS cell line, screened for SIC-related genes, and tried to regulate the proliferation of OS by metabolic interference. Initially, we established a new human OS cell line (OS13) and isolated clones showing higher tumorigenesis as SIC (OSHIGH ) and counterpart clones. OSHIGH cells showed chemoresistance and their metabolism highly depended on aerobic glycolysis and suppressed oxidative phosphorylation. Using RNA-sequencing, we identified LIN28B as a SIC-related gene highly expressed in OSHIGH cells. mRNA of LIN28B was expressed in sarcoma cell lines including OS13, but its expression was not detectable in normal organs other than the testis and placenta. LIN28B protein was also detected in various sarcoma tissues. Knockdown of LIN28B in OS13 cells reduced tumorigenesis, decreased chemoresistance, and reversed oxidative phosphorylation function. Combination therapy consisting of a glycolysis inhibitor and low-dose chemotherapy had antitumor effects. In conclusion, manipulation of glycolysis combined with chemotherapy might be a good adjuvant treatment for OS. Development of immunotherapy targeting LIN28B, a so-called cancer/testis antigen, might be a good approach.


Assuntos
Neoplasias Ósseas/genética , Glicólise/genética , Osteossarcoma/genética , Proteínas de Ligação a RNA/genética , Animais , Neoplasias Ósseas/patologia , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Osteossarcoma/patologia , Fosforilação Oxidativa , Placenta/patologia , Gravidez , Prognóstico , RNA Mensageiro/genética , Testículo/patologia
4.
Int J Cancer ; 146(1): 10-17, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31396957

RESUMO

In contrast to prior belief, cancer cells require oxidative phosphorylation (OXPHOS) to strive, and exacerbated OXPHOS dependency frequently characterizes cancer stem cells, as well as primary or acquired resistance against chemotherapy or tyrosine kinase inhibitors. A growing arsenal of therapeutic agents is being designed to suppress the transfer of mitochondria from stromal to malignant cells, to interfere with mitochondrial biogenesis, to directly inhibit respiratory chain complexes, or to disrupt mitochondrial function in other ways. For the experimental treatment of cancers, OXPHOS inhibitors can be advantageously combined with tyrosine kinase inhibitors, as well as with other strategies to inhibit glycolysis, thereby causing a lethal energy crisis. Unfortunately, most of the preclinical data arguing in favor of OXPHOS inhibition have been obtained in xenograft models, in which human cancer cells are implanted in immunodeficient mice. Future studies on OXPHOS inhibitors should elaborate optimal treatment schedules and combination regimens that stimulate-or at least are compatible with-anticancer immune responses for long-term tumor control.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Fosforilação Oxidativa , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Antineoplásicos/farmacologia , Carcinogênese , Glicólise , Humanos , Camundongos , Neoplasias/enzimologia , Neoplasias/metabolismo , Biogênese de Organelas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Bioelectrochemistry ; 131: 107350, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31518962

RESUMO

Curcumin (Cur), the yellow pigment of well-known turmeric (Curcuma longa L.) is effective in multiple cancers including triple negative breast cancer (TNBC). In combination with electrical pulses (EP), enhanced effects of curcumin (Cur + EP) are observed in TNBC cells. To gain insights into the mechanisms of enhanced anticancer effects of Cur + EP, we studied the proteins involved in the anticancer activity of Cur + EP in MDA-MB-231, human TNBC cells using high-throughput global proteomics. A curcumin dose of 50 µM was applied with eight, 1200 V/cm, 100 µs pulses, the most commonly used electrochemotherapy (ECT) parameter in clinics. Results show that the Cur + EP treatment reduced the clonogenic ability in MDA-MB-231 cells, with the induction of apoptosis. Proteomic analysis identified a total of 1456 proteins, of which 453 proteins were differentially regulated, including kinases, heat shock proteins, transcription factors, structural proteins, and metabolic enzymes. Eight key glycolysis proteins (ALDOA, ENO2, LDHA, LDHB, PFKP, PGM1, PGAM1 and PGK1) were downregulated in Cur + EP from Cur. There was a switch in the metabolism with upregulation of 10 oxidative phosphorylation pathway proteins and 8 tricarboxylic acid (TCA) cycle proteins in the Cur + EP sample, compared to curcumin. These results provide novel systematic insights into the mechanisms of ECT with curcumin.


Assuntos
Antineoplásicos/uso terapêutico , Curcumina/uso terapêutico , Eletroquimioterapia/métodos , Proteínas de Neoplasias/metabolismo , Proteômica , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Curcumina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Feminino , Glicólise , Humanos , Fosforilação Oxidativa , Via de Pentose Fosfato/efeitos dos fármacos , Reprodutibilidade dos Testes , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
6.
Adv Exp Med Biol ; 1206: 329-357, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31776993

RESUMO

Autophagy is a lysosome-dependent catabolic process. Both extra- and intra-cellular components are engulfed in autophagic vacuoles and degraded to simple molecules, such as monosaccharides, fatty acids and amino acids. Then, these molecules can be further used to produce ATP through catabolic reactions and/or provide building blocks for the synthesis of essential proteins. Therefore, we consider autophagy a critical and fine-tuned process in maintaining energy homeostasis. The complicated relationships between autophagy and energy metabolism have raised broad interest and have been extensively studied. In this chapter, we summarize the relationships enabling autophagy to control or modulate energy metabolism and allowing metabolic pathways to regulate autophagy. Specifically, we review the correlations between autophagy and energy homeostasis in terms of oxidative phosphorylation, reactive oxygen species in mitochondria, glycolysis, metabolism of glycogen and protein, and so on. An understanding of the role of autophagy in energy homeostasis could help us better appreciate how autophagy determines cell fate under stressful conditions or pathological processes.


Assuntos
Autofagia , Metabolismo Energético , Mitocôndrias , Fosforilação Oxidativa , Espécies Reativas de Oxigênio , Estresse Fisiológico
7.
J Microbiol ; 57(12): 1095-1104, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31758395

RESUMO

Subglacial ecosystems harbor diverse chemoautotrophic microbial communities in areas with limited organic carbon, and lithological H2 produced during glacial erosion has been considered an important energy source in these ecosystems. To verify the H2-utilizing potential there and to identify the related energy-converting metabolic mechanisms of these communities, we performed metagenomic analysis on subglacial sediment samples from East Antarctica with and without H2 supplementation. Genes coding for several [NiFe]-hydrogenases were identified in raw sediment and were enriched after H2 incubation. All genes in the dissimilatory nitrate reduction and denitrification pathways were detected in the subglacial community, and the genes coding for these pathways became enriched after H2 was supplied. Similarly, genes transcribing key enzymes in the Calvin cycle were detected in raw sediment and were also enriched. Moreover, key genes involved in H2 oxidization, nitrate reduction, oxidative phosphorylation, and the Calvin cycle were identified within one metagenome-assembled genome belonging to a Polaromonas sp. As suggested by our results, the microbial community in the subglacial environment we investigated consisted of chemoautotrophic populations supported by H2 oxidation. These results further confirm the importance of H2 in the cryosphere.


Assuntos
Sedimentos Geológicos/microbiologia , Hidrogênio/metabolismo , Metagenoma , Microbiota/fisiologia , Regiões Antárticas , Archaea/classificação , Archaea/enzimologia , Archaea/genética , Archaea/metabolismo , Bactérias/classificação , Bactérias/enzimologia , Bactérias/genética , Bactérias/metabolismo , Ciclo do Carbono , Crescimento Quimioautotrófico , Comamonadaceae/enzimologia , Comamonadaceae/metabolismo , Genes Arqueais/genética , Genes Bacterianos/genética , Hidrogenase/classificação , Hidrogenase/genética , Hidrogenase/isolamento & purificação , Redes e Vias Metabólicas , Microbiota/genética , Nitratos/metabolismo , Fosforilação Oxidativa , Fotossíntese , Análise de Sequência de DNA
8.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 31(9): 1167-1169, 2019 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-31657347

RESUMO

OBJECTIVE: Metabolic reprogramming is the response of cells to environmental changes, such as cell activation, proliferation and differentiation, which involves changes in metabolism-related enzymes, metabolites and metabolic pathways. Sepsis-associated immune cells undergo metabolic reprogramming in response to inflammatory signals, which not only provides biological energy and biosynthesis requirements, but also determines cell fate and function in a highly specific way. In this paper, the changes in glycolysis, tricarboxylic acid cycle, oxidative phosphorylation and other glucose metabolism pathways of macrophages, T lymphocytes, dendritic cells, neutrophils and other sepsis related immune cells are described, so as to provide feasibility for future research and metabolic therapy.


Assuntos
Glicólise , Sepse , Células Dendríticas , Glicogênio , Humanos , Fosforilação Oxidativa
9.
Nucleic Acids Res ; 47(19): 10267-10281, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31665743

RESUMO

Post-transcriptional RNA modifications, the epitranscriptome, play important roles in modulating the functions of RNA species. Modifications of rRNA are key for ribosome production and function. Identification and characterization of enzymes involved in epitranscriptome shaping is instrumental for the elucidation of the functional roles of specific RNA modifications. Ten modified sites have been thus far identified in the mammalian mitochondrial rRNA. Enzymes responsible for two of these modifications have not been characterized. Here, we identify METTL15, show that it is the main N4-methylcytidine (m4C) methyltransferase in human cells and demonstrate that it is responsible for the methylation of position C839 in mitochondrial 12S rRNA. We show that the lack of METTL15 results in a reduction of the mitochondrial de novo protein synthesis and decreased steady-state levels of protein components of the oxidative phosphorylation system. Without functional METTL15, the assembly of the mitochondrial ribosome is decreased, with the late assembly components being unable to be incorporated efficiently into the small subunit. We speculate that m4C839 is involved in the stabilization of 12S rRNA folding, therefore facilitating the assembly of the mitochondrial small ribosomal subunits. Taken together our data show that METTL15 is a novel protein necessary for efficient translation in human mitochondria.


Assuntos
Metiltransferases/genética , Mitocôndrias/genética , Ribossomos Mitocondriais/química , RNA Ribossômico/genética , Citidina/genética , Humanos , Metilação , Mitocôndrias/química , Fosforilação Oxidativa , Biossíntese de Proteínas/genética , Dobramento de RNA/genética , Processamento Pós-Transcricional do RNA/genética , RNA Ribossômico/química
10.
Invest Ophthalmol Vis Sci ; 60(13): 4187-4195, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31596927

RESUMO

Purpose: Metastatic uveal melanoma (UM) has a very poor prognosis and no effective therapy. Despite remarkable advances in treatment of cutaneous melanoma, UM remains recalcitrant to chemotherapy, small-molecule kinase inhibitors, and immune-based therapy. Methods: We assessed two sets of oxidative phosphorylation (OxPhos) genes within 9858 tumors across 31 cancer types. An OxPhos inhibitor was used to characterize differential metabolic programming of highly metastatic monosomy 3 (M3) UM. Seahorse analysis and global metabolomics profiling were done to identify metabolic vulnerabilities. Analyses of UM TCGA data set were performed to determine expressions of key OxPhos effectors in M3 and non-M3 UM. We used targeted knockdown of succinate dehydrogenase A (SDHA) to determine the role of SDHA in M3 UM in conferring resistance to OxPhos inhibition. Results: We identified UM to have among the highest median OxPhos levels and showed that M3 UM exhibits a distinct metabolic profile. M3 UM shows markedly low succinate levels and has highly increased levels of SDHA, the enzyme that couples the tricarboxylic acid cycle with OxPhos by oxidizing (lowering) succinate. We showed that SDHA-high M3 UM have elevated expression of key OxPhos molecules, exhibit abundant mitochondrial reserve respiratory capacity, and are resistant to OxPhos antagonism, which can be reversed by SDHA knockdown. Conclusions: Our study has identified a critical metabolic program within poor prognostic M3 UM. In addition to the heightened mitochondrial functional capacity due to elevated SDHA, M3 UM SDHA-high mediate resistance to therapy that is reversible with targeted treatment.


Assuntos
Melanoma/metabolismo , Succinato Desidrogenase/fisiologia , Neoplasias Uveais/metabolismo , Humanos , Fosforilação Oxidativa , Succinato Desidrogenase/metabolismo , Ácido Succínico/metabolismo , Células Tumorais Cultivadas
11.
PLoS Genet ; 15(10): e1008410, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31584940

RESUMO

Mitochondria have been increasingly recognized as a central regulatory nexus for multiple metabolic pathways, in addition to ATP production via oxidative phosphorylation (OXPHOS). Here we show that inducing mitochondrial DNA (mtDNA) stress in Drosophila using a mitochondrially-targeted Type I restriction endonuclease (mtEcoBI) results in unexpected metabolic reprogramming in adult flies, distinct from effects on OXPHOS. Carbohydrate utilization was repressed, with catabolism shifted towards lipid oxidation, accompanied by elevated serine synthesis. Cleavage and translocation, the two modes of mtEcoBI action, repressed carbohydrate rmetabolism via two different mechanisms. DNA cleavage activity induced a type II diabetes-like phenotype involving deactivation of Akt kinase and inhibition of pyruvate dehydrogenase, whilst translocation decreased post-translational protein acetylation by cytonuclear depletion of acetyl-CoA (AcCoA). The associated decrease in the concentrations of ketogenic amino acids also produced downstream effects on physiology and behavior, attributable to decreased neurotransmitter levels. We thus provide evidence for novel signaling pathways connecting mtDNA to metabolism, distinct from its role in supporting OXPHOS.


Assuntos
Reprogramação Celular/genética , DNA Mitocondrial/genética , Diabetes Mellitus Tipo 2/genética , Mitocôndrias/genética , Trifosfato de Adenosina/genética , Animais , Metabolismo dos Carboidratos/genética , Carboidratos/genética , Enzimas de Restrição do DNA/genética , Diabetes Mellitus Tipo 2/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Redes e Vias Metabólicas/genética , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Estresse Oxidativo/genética
13.
EMBO J ; 38(22): e101056, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31609012

RESUMO

The mitochondrial membrane potential (ΔΨm ) is the main driver of oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane (IMM), consisting of cristae and inner boundary membranes (IBM), is considered to carry a uniform ΔΨm . However, sequestration of OXPHOS components in cristae membranes necessitates a re-examination of the equipotential representation of the IMM. We developed an approach to monitor ΔΨm at the resolution of individual cristae. We found that the IMM was divided into segments with distinct ΔΨm , corresponding to cristae and IBM. ΔΨm was higher at cristae compared to IBM. Treatment with oligomycin increased, whereas FCCP decreased, ΔΨm heterogeneity along the IMM. Impairment of cristae structure through deletion of MICOS-complex components or Opa1 diminished this intramitochondrial heterogeneity of ΔΨm . Lastly, we determined that different cristae within the individual mitochondrion can have disparate membrane potentials and that interventions causing acute depolarization may affect some cristae while sparing others. Altogether, our data support a new model in which cristae within the same mitochondrion behave as independent bioenergetic units, preventing the failure of specific cristae from spreading dysfunction to the rest.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/fisiologia , Membranas Mitocondriais/metabolismo , Mioblastos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Células Cultivadas , Feminino , Células HeLa , Humanos , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/metabolismo , Mioblastos/citologia , Fosforilação Oxidativa
14.
Anticancer Res ; 39(9): 4865-4876, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519589

RESUMO

BACKGROUND/AIM: Hypoxia promotes tumor proliferation and metastasis in colorectal cancer (CRC). Since the tumor microenvironment is generally characterized by hypoxia, its understanding is important for cancer therapy. We hypothesized that hypoxia promotes the mitochondrial function, mobility, and proliferation of CRC by up-regulating peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). MATERIALS AND METHODS: To assess the effects of PGC-1α under hypoxia, we investigated the mitochondrial function, cell motility, and sphere formation as well as proliferation and apoptosis of CRC. RESULTS: Under hypoxia, we confirmed the increased expression of PGC-1α and reduced production of reactive oxygen species (ROS) by activating anti-oxidant enzymes. Also, up-regulation of PGC-1α enhanced the motility, sphere formation, and proliferation of CRC. Under the presence of the anti-cancer drug 5-fluorouracil (5FU), up-regulation of PGC-1α under hypoxia promoted resistance of CRC against 5FU-induced apoptosis. CONCLUSION: Targeting PGC-1α could to be a powerful strategy for CRC therapy.


Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Hipóxia/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Apoptose , Catalase/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Modelos Biológicos , Fosforilação Oxidativa , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo
15.
Reprod Domest Anim ; 54 Suppl 3: 22-28, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31512320

RESUMO

The biological nature of age-related declines in fertility in males of any species, including stallions, has been elusive. In horses, the economic costs to the breeding industry are frequently extensive. Mitochondrial function in ejaculated sperm, which is essential for sperm motility, is reflected by adenosine triphosphate production, mitochondrial oxidative efficiency and production of reactive oxygen species, and that this balance may become compromised in ageing stallions and during the process of cryopreservation. This presentation will focus on mitochondrial integrity and function as an avenue for understanding the pathophysiology of sperm when undergoing cryopreservation and male ageing. We discuss the importance of understanding the differences and similarities of sperm mitochondria to that of somatic cells regarding structure and mitochondrial biochemistry relating to sperm function. The roles of oxidative phosphorylation and glycolysis in sperm mitochondria are outlined as is the method of determining oxygen consumption and calcium homoeostasis in sperm mitochondria. Further, we outline the role of oxidative stress and reactive oxygen species.


Assuntos
Cavalos/fisiologia , Mitocôndrias/fisiologia , Espermatozoides/fisiologia , Envelhecimento/fisiologia , Animais , Criopreservação/veterinária , Fertilidade/fisiologia , Glicólise , Masculino , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Técnicas de Reprodução Assistida/veterinária , Motilidade Espermática/fisiologia , Espermatozoides/citologia , Espermatozoides/metabolismo
16.
J Dairy Sci ; 102(11): 9767-9780, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31495615

RESUMO

Our objective was to determine the effects of uncouplers of oxidative phosphorylation on feeding behavior of lactating dairy cows. We hypothesized that uncouplers of oxidative phosphorylation would increase meal size and meal length and performed 2 experiments to test our hypothesis. In experiment 1, 4 late-lactation cows (345 ± 48.4 d in milk; mean ± SD) were administered a daily intrajugular injection of either 10 mg/kg of BW0.75 of 2,4-dinitrophenol methyl ether (DNPME) and propylene carbonate or propylene carbonate (control; CON) in a crossover design with 2-d periods. In experiment 2, 8 early-lactation cows (11.3 ± 0.89 d in milk) were administered a daily intrajugular injection via jugular catheter of either 50 mg/kg of BW of sodium salicylate (SAL) and saline or saline (control; CON) in a crossover design with 1-d periods. Feeding behavior was recorded by a computerized data acquisition system and analyzed for the first 4 h after access to feed within 15 min of treatment for both experiments. Neither DNPME nor SAL affected meal size over the first 4 h after access to feed. However, DNPME increased meal length by 6.4 min (26.3 vs. 19.9 min) and tended to decrease the number of meals (2.55 vs. 2.78 meals/4 h) over the first 4 h after access to feed compared with CON. Both DNPME and SAL decreased eating rate over the first 4 h after access to feed compared with their respective controls (0.10 vs. 0.12 kg/min for DNPME vs. CON; 0.06 vs. 0.07 kg/min for SAL vs. CON). Lack of treatment effects on meal size may have been caused by increased rate of oxidation of fuels compensating for the disruption of oxidative phosphorylation.


Assuntos
Bovinos/fisiologia , Comportamento Alimentar/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Salicilato de Sódio/farmacologia , Desacopladores/farmacologia , Ração Animal/análise , Animais , Aleitamento Materno , Estudos Cross-Over , Dieta/veterinária , Feminino , Lactação/efeitos dos fármacos , Fígado/química , Leite , Salicilato de Sódio/administração & dosagem , Desacopladores/administração & dosagem
17.
Int J Med Microbiol ; 309(6): 151330, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31471070

RESUMO

OBJECTIVE: Alcohol dehydrogenase I is encoded by ADH1 in Candida albicans, and is one of the key enzymes in fungal metabolism by which it catalyzes the conversion from acetaldehyde to ethanol. The role of the associated protein Adh1p, encoded by ADH1 in fungal pathogenicity has not been thoroughly studied despite its near ubiquity in the fungal kingdom. Using C. albicans as a model, this study proposes to determine the possible pathogenic roles for ADH1 and its possible underlying mechanisms. METHODS: The SAT1 flipper strategy was used to construct the ADH1 deletion mutant. Growth curves and spot assay were used to compare growth and cell viability of the mutant to wild type C. albicans. Three host model systems (infected mice, C. elegans, and G. mellonella) were used to investigate the effects of ADH1 deletion in vivo on C. albicans pathogenicity. Then, adhesion, hyphal formation, biofilm formation, cell surface hydrophobicity (CSH) and RT-qPCR were performed to investigate the effects of ADH1 deletion in vitro on C. albicans virulence. Finally, Xfe 96 seahorse assay, ROS level, mitochondrial membrane potential, and intracellular ATP content were used to determine the effects of ADH1 deletion on bioenergetics. RESULTS: ADH1 deletion has no effects on the growth and cell viability of C. albicans, but significantly prolongs survival time in each of the three host models, decreases fungal burden in kidney and liver, and lessens pathological tissue damage (P <  0.05). In addition, ADH1 deletion significantly increases CSH and reduces C. albicans virulence in terms of adhesion, hyphal formation and biofilm formation in accord with the downregulation of virulence-related genes such as ALS1, ALS3, HWP1, and CSH1 (P <  0.05). For bioenergetics, ADH1 deletion has no obvious effect on glycolysis, but a lack of ADH1 significantly increases ROS levels and decreases mitochondrial membrane potential and intracellular ATP content even through the mitochondrial oxygen consumption rate and NADH/NAD+ ratio are elevated (P <  0.05). CONCLUSION: Our results suggest that the fermentative enzyme ADH1 is required for the pathogenicity of C. albicans under one of the presumed mechanisms viaits effects on oxidative phosphorylation activities in mitochondria.


Assuntos
Álcool Desidrogenase/metabolismo , Candida albicans/patogenicidade , Candidíase/metabolismo , Proteínas Fúngicas/metabolismo , Fosforilação Oxidativa , Virulência/genética , Álcool Desidrogenase/genética , Animais , Biofilmes/crescimento & desenvolvimento , Caenorhabditis elegans , Candida albicans/genética , Candidíase/microbiologia , Adesão Celular , Modelos Animais de Doenças , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Hifas/crescimento & desenvolvimento , Camundongos Endogâmicos ICR , Mitocôndrias/metabolismo , Mariposas , Espécies Reativas de Oxigênio/metabolismo , Deleção de Sequência
18.
Ann Clin Lab Sci ; 49(4): 425-438, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31471331

RESUMO

The purpose of this review is to elucidate how low blood cholesterol promotes mitochondrial dysfunction and mortality by the loss of thioretinaco ozonide from opening of the mitochondrial permeability transition pore (mPTP). Mortality from infections and cancer are both inversely associated with blood cholesterol, as determined by multiple cohort studies from 10 to 30 years earlier. Moreover, low-density lipoprotein (LDL) is inversely related to all-cause and/or cardiovascular mortality, as determined by followup study of elderly cohorts. LDL adheres to and inactivates most microorganisms and their toxins, causing aggregation of LDL and homocysteinylated autoantibodies which obstruct vasa vasorum and produce intimal microabscesses, the vulnerable atherosclerotic plaques. The active site of mitochondrial oxidative phosphorylation and adenosine triphosphate (ATP) biosynthesis is proposed to consist of thioretinaco, a complex of two molecules of thioretinamide with cobalamin, oxidized to the disulfonium thioretinaco ozonide and complexed with oxygen, nicotinamide adenine dinucleotide (NAD+), phosphate, and ATP. Loss of the active site complex from mitochondria results from the opening of the mPTP and from decomposition of the disulfonium active site by electrophilic carcinogens, oncogenic viruses, microbes, and by reactive oxygen radicals from ionizing and non-ionizing radiation. Suppression of innate immunity is caused by the depletion of adenosyl methionine because of increased polyamine biosynthesis, resulting in inhibition of nitric oxide and peroxynitrite biosynthesis. Opening of the mPTP produces a loss of thioretinaco ozonide from mitochondria. This loss impairs ATP biosynthesis and causes the mitochondrial dysfunction observed in carcinogenesis, atherosclerosis, aging and dementia. Cholesterol inhibits the opening of the mPTP by preventing integration of the pro-apoptotic Bcl-2-associated X protein (BAX) in the outer mitochondrial membrane. This inhibition explains how elevated LDL reduces mitochondrial dysfunction by preventing loss of the active site of oxidative phosphorylation from mitochondria.


Assuntos
Colesterol/toxicidade , Homocisteína/análogos & derivados , Homocisteína/toxicidade , Mitocôndrias/patologia , Mortalidade , Vitamina B 12/análogos & derivados , Humanos , Mitocôndrias/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Vitamina B 12/toxicidade
19.
J Exp Clin Cancer Res ; 38(1): 337, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31383001

RESUMO

BACKGROUND: Metabolic rewiring is a common feature of many cancer types, including prostate cancer (PCa). Alterations in master genes lead to mitochondrial metabolic rewiring and provide an appealing target to inhibit cancer progression and improve survival. Phospholipase C (PLC)ε is a regulator of tumor generation and progression. However, its role in mitochondrial metabolism remains unclear. METHODS: The GEO, The Cancer Genome Atlas, and the GTEx databases were used to determine Twist1 mRNA levels in tumors and their non-tumor counterparts. Fifty-five PCa and 48 benign prostatic hypertrophy tissue samples were tested for the presence of PLCε and Twist1 immunohistochemically. An association between PLCε and Twist1 was determined by Pearson's correlation analysis. PLCε was knocked down with a lentiviral short hairpin RNA. Mitochondrial activity was assessed by measuring the oxygen consumption rate. Western blotting analyses were used to measure levels of PPARß, Twist1, phosphorylated (p)-Twist1, p-MEK, p-ERK, p-P38, and p-c-Jun N-terminal kinase (JNK). Cells were treated with inhibitors of MEK, JNK, and P38 MAPK, and an agonist and inhibitor of peroxisome proliferator activated receptor (PPAR) ß, to evaluate which signaling pathways were involved in PLCε-mediated Twist1 expression. The stability of Twist1 was determined after blocking protein synthesis with cycloheximide. Reporter assays utilized E-cadherin or N-cadherin luciferase reporters under depletion of PLCε or Twist1. Transwell assays assessed cell migration. Finally, a nude mouse tumor xenograft assay was conducted to verify the role of PLCε in tumor formation. RESULTS: Our findings revealed that the expression of PLCε was positively associated with Twist1 in clinical PCa samples. PLCε knockdown promoted mitochondrial oxidative metabolism in PCa cells. Mechanistically, PLCε increased phosphorylation of Twist1 and stabilized the Twist1 protein through MAPK signaling. The transcriptional activity of Twist1, and the Twist1-mediated epithelial-to-mesenchymal transition, cell migration, and transcription regulation, were suppressed by PLCε knockdown and by blocking PPARß nuclear translocation. The tumor xenograft assay demonstrated that PLCε depletion diminished PCa cell tumorigenesis. CONCLUSIONS: These findings reveal an undiscovered physiological role for PLCε in the suppression of mitochondrial oxidative metabolism that has significant implications for understanding PCa occurrence and migration.


Assuntos
Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação Oxidativa , Fosfoinositídeo Fosfolipase C/metabolismo , Neoplasias da Próstata/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/genética , Modelos Biológicos , Gradação de Tumores , Estadiamento de Neoplasias , Proteínas Nucleares/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Transporte Proteico , Proteína 1 Relacionada a Twist/genética
20.
EBioMedicine ; 46: 356-367, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31383553

RESUMO

BACKGROUND: TK2 is a nuclear gene encoding the mitochondrial matrix protein thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial nucleotide salvage pathway. Deficiency of TK2 activity causes mitochondrial DNA (mtDNA) depletion, which in humans manifests predominantly as a mitochondrial myopathy with onset typically in infancy and childhood. We previously showed that oral treatment of the Tk2 H126N knock-in mouse model (Tk2-/-) with the TK2 substrates, deoxycytidine (dCtd) and thymidine (dThd), delayed disease onset and prolonged median survival by 3-fold. Nevertheless, dCtd + dThd treated Tk2-/- mice showed mtDNA depletion in brain as early as postnatal day 13 and in virtually all other tissues at age 29 days. METHODS: To enhance mechanistic understanding and efficacy of dCtd + dThd therapy, we studied the bioavailability of dCtd and dThd in various tissues as well as levels of the cytosolic enzymes, TK1 and dCK that convert the deoxynucleosides into dCMP and dTMP. FINDINGS: Parenteral treatment relative to oral treatment produced higher levels of dCtd and dThd and improved mtDNA levels in liver and heart, but did not ameliorate molecular defects in brain or prolong survival. Down-regulation of TK1 correlated with temporal- and tissue-specificity of response to dCtd + dThd. Finally, we observed in human infant and adult muscle expression of TK1 and dCK, which account for the long-term efficacy to dCtd + dThd therapy in TK2 deficient patients. INTERPRETATIONS: These data indicate that the cytosolic pyrimidine salvage pathway enzymes TK1 and dCK are critical for therapeutic efficacy of deoxynucleoside therapy for Tk2 deficiency. FUND: National Institutes of Health P01HD32062.


Assuntos
Desoxirribonucleosídeos/farmacologia , Timidina Quinase/deficiência , Animais , Disponibilidade Biológica , Barreira Hematoencefálica/metabolismo , DNA Mitocondrial , Desoxirribonucleosídeos/farmacocinética , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Especificidade de Órgãos , Fosforilação Oxidativa , Fenótipo , Timidina Quinase/genética , Timidina Quinase/metabolismo
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