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1.
Cell Physiol Biochem ; 54(1): 15-26, 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31916734

RESUMO

BACKGROUND/AIMS: The primary cilium is a nanoscale membrane protrusion believed to act as a mechano-chemical sensor in a range of different cell types. Disruptions in its structure and signalling have been linked to a number of medical conditions, referred to as ciliopathies, but remain poorly understood due to lack of techniques capable of investigating signal transduction in cilia at nanoscale. Here we set out to use latest advances in nanopipette technology to address the question of ion channel distribution along the structure of primary cilium. METHODS: We used glass nanopipettes and Scanning Ion Conductance Microscopy (SICM) to image 3D topography of intact primary cilia in inner medullary collecting duct (IMCD) cells with nanoscale resolution. The high-resolution topographical images were then used to navigate the nanopipette along the structure of each cilium and perform spatially resolved single-channel recordings under precisely controlled mechanical and chemical stimulation. RESULTS: We have successfully obtained first single-channel recordings at specific locations of intact primary cilia. Our experiments revealed significant differences between the populations of channels present at the ciliary base, tip and within extra-ciliary regions in terms of mean conductance and sensitivity to membrane displacement as small as 100 nm. Ion channels at the base of cilium, where mechanical strain is expected to be the highest, appeared particularly sensitive to the mechanical displacement. CONCLUSION: Our results suggest the distribution of ion channels in the membrane of primary cilia is non-homogeneous. The relationship between the location and function of ciliary ion channels could be key to understanding signal transduction in primary cilia.


Assuntos
Membrana Celular/metabolismo , Cílios/metabolismo , Canais Iônicos/metabolismo , Nanotecnologia/métodos , Potenciais de Ação/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Mecanotransdução Celular , Camundongos
2.
Anticancer Res ; 40(1): 323-333, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31892583

RESUMO

BACKGROUND/AIM: Despite the Warburg effect, mitochondria play an essential role in the survival and maintenance of cancer cells. Thus, mitochondria have been considered a target for anticancer agents. Here, we identified a mitochondria-targeting anticancer agent from natural products. MATERIALS AND METHODS: Morphological and functional changes in mitochondria were determined by a fluorescence-based High Content Imaging System. Using human non-small cell lung cancer (NSCLC) cell lines (H1299, H226B, and A549), cell viability and colony formation assays, cell cycle analysis, and immunoblotting were performed to determine cytotoxic and proapoptotic effects of papuamine. RESULTS: Using a natural product chemical library, we identified papuamine as an active compound to inhibit viability and ATP production of NSCLC cells. Papuamine depleted intracellular ATP by causing mitochondrial dysfunction, as indicated by the loss of the mitochondrial membrane potential and increased mitochondrial superoxide generation. Papuamine significantly inhibited viability and colony formation of NSCLC cells by inducing apoptosis. CONCLUSION: Papuamine has a potential as a novel mitochondria-targeting anticancer agent.


Assuntos
Alcaloides/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Mitocôndrias/patologia , Células A549 , Trifosfato de Adenosina/metabolismo , Adenilato Quinase/metabolismo , Alcaloides/química , Alcaloides/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Sobrevivência Celular/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/patologia , Mitocôndrias/efeitos dos fármacos , Ensaio Tumoral de Célula-Tronco , Regulação para Cima/efeitos dos fármacos
3.
Phys Chem Chem Phys ; 22(3): 1534-1542, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31872818

RESUMO

Dynein, a large and complex motor protein, harnesses energy from adenosine triphosphate (ATP) hydrolysis to regulate essential cellular activities. The ATP hydrolysis mechanism for the dynein motor is still shrouded in mystery. Herein, molecular dynamics simulations of a dynein motor disclosed that two water molecules are present close to the γ-phosphate of ATP and Glu1742 at the AAA1 site of dynein. We have proposed three possible mechanisms for the ATP hydrolysis. We divulge by using a quantum mechanics/molecular mechanics (QM/MM) study that two water molecules and Glu1742 are crucial for facilitating the ATP hydrolysis reaction in dynein. Moreover, the ATP hydrolysis step is initiated by the activation of lytic water (W1) by Glu1742 through relay proton transfers with the help of auxiliary water (W2) yielding HPO42- and ADP, as a product. In the next step, a proton is shifted back from Glu1742 to generate inorganic phosphate (H2PO4-) via another relay proton transfer event. The overall activation barrier for the Glu1742 assisted ATP hydrolysis is found to be the most favourable pathway compared to other plausible pathways. We also unearthed that ATP hydrolysis in dynein follows a so-called associative-like pathway in its rate-limiting step. Our study ascertained the important indirect roles of the two amino acids (such as Arg2109, Asn1792) and Mg2+ ion in the ATP hydrolysis of dynein. Additionally, multiple sequence alignment of the different organisms of dynein motors has conveyed the evolutionary importance of the Glu1742, Asn1742, and Arg2109 residues, respectively. As similar mechanisms are also prevalent in other motors, and GTPase and ATPase enzymes, the present finding spells out the definitive requirement of a proton relay process through an extended water-chain as one of the key components in an enzymatic ATP hydrolysis reaction.


Assuntos
Trifosfato de Adenosina/metabolismo , Dineínas/química , Hidrólise , Trifosfato de Adenosina/química , Teoria Quântica
4.
Biochemistry (Mosc) ; 84(11): 1247-1255, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31760915

RESUMO

Fo×F1-ATPases of mitochondria, chloroplasts, and microorganisms catalyze transformation of proton motive force (the difference between the electrochemical potentials of hydrogen ion across a coupling membrane) to the free energy of ATP phosphoryl potential. It is often stated that Fo×F1-ATPases operate as reversible chemo-mechano-electrical molecular machines that provide either ATP synthesis or hydrolysis depending on particular physiological demands of an organism; the microreversibility principle of the enzyme catalysis is usually taken as a dogma. Since 1980, the author has upheld the view that the mechanisms of ATP synthesis and hydrolysis by the Fo×F1 complex are different (Vinogradov, A. D. (2000) J. Exp. Biol., 203, 41-49). In this paper, the author proposes a new model considering the existence in coupling membranes of two non-equilibrium isoforms of Fo×F1 unidirectionally catalyzing synthesis and/or hydrolysis of ATP.


Assuntos
Trifosfato de Adenosina/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Biocatálise , Cloroplastos/enzimologia , Hidrólise , Cinética , Mitocôndrias/enzimologia , Paracoccus denitrificans/enzimologia , Força Próton-Motriz
5.
Biomed Khim ; 65(5): 398-402, 2019 Aug.
Artigo em Russo | MEDLINE | ID: mdl-31666412

RESUMO

The experimental study of the cardioprotective effect of uridine, the metabolic precursor of the endogenous activator of mitochondrial ATP-dependent K+-channels (mitoKATP-channels), was performed using the model of myocardial ischemia/reperfusion (I/RP) in rats. Ischemia for 30 min followed by reperfusion for 120 min resulted in a significant decrease in ATP and phosphocreatine (PC) content, intensification of lipid peroxidation (LPO), and inhibition of the antioxidant system (AOS) in cardiomyocytes. Uridine in a dose of 30 mg/kg, administered intravenously prior to reperfusion, had a protective effect on myocardial metabolism in the I/RP zone. It prevented the decrease of ATP and PC, limited the LPO processes, evaluated by the content of lipid hydroperoxides and conjugated dienes, and improved the AOS state by, preventing the decrease of superoxide dismutase (SOD) activity and increasing the content of reduced glutathione (GSH). The mitoKATP-channel blocker 5-hydroxydecanoate (5-HD, 5 mg/kg) eliminated the ability of uridine to maintain the ATP level and to exhibit its positive effect on the intensity of the LPO and activity of AOS. The obtained data allow us to conclude that activation of mitoKATP-channels play an important role in the mechanism of the cardioprotective effect of uridine in I/RP damage of myocardium.


Assuntos
Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Uridina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Antioxidantes/metabolismo , Ácidos Decanoicos , Glutationa/metabolismo , Hidroxiácidos , Peroxidação de Lipídeos , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio , Substâncias Protetoras/farmacologia , Ratos , Superóxido Dismutase/metabolismo
6.
Chem Commun (Camb) ; 55(85): 12857-12860, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31598611

RESUMO

5-Hydroxy-2-phenyl-7-(thiiran-2-ylmethoxy)-4H-chromen-4-one (compound 52) was found as a DNA non-intercalative topo II specific catalytic inhibitor by targeting its ATP-binding domain. Showing changes in interaction with Mg2+, it exhibited highly selective properties against the α-isoform with less toxicity, unlike other topo II poisons, such as etoposide.


Assuntos
Trifosfato de Adenosina/química , DNA Topoisomerases Tipo II/química , Proteínas de Ligação a DNA/química , Inibidores da Topoisomerase II/química , Trifosfato de Adenosina/metabolismo , Biocatálise , DNA/química , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Etoposídeo/química , Humanos , Domínios Proteicos , Isoformas de Proteínas , Inibidores da Topoisomerase II/metabolismo
7.
J Agric Food Chem ; 67(38): 10637-10645, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31513389

RESUMO

Previous studies have shown that selenite, a representative of inorganic form selenium, exerts its anticancer effect by inducing apoptosis in androgen-dependent LNCaP prostate cancer cells, but few studies have determined the nature of cell death induced by selenite in metastatic androgen-refractory PC-3 cells. Our study showed that necrosis-like cell death rather than apoptosis, pyroptosis, or autophagic cell death was caused by selenite in PC-3 cells. Mechanistically, this type of cell death was caused by ATP depletion (26.28 ± 3.39 nmol/mg of control versus 9.12 ± 2.44 nmol/mg of 10 µM selenite treatment) that resulted from phosphofructokinase activity reduction (100.17 ± 0.17% of control versus 21.74 ± 6.65% of 10 µM selenite treatment). Our study also showed that ROS production is necessary for the decrease in cellular ATP levels and in phosphofructokinase activity. To our knowledge, this is the first study showing that selenite can induce necrosis-like cell death in PC-3 cells. Our findings support selenite as an effective compound for the therapy of apoptosis-resistant prostate cancer.


Assuntos
Morte Celular/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Neoplasias da Próstata/fisiopatologia , Ácido Selenioso/farmacologia , Trifosfato de Adenosina/metabolismo , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Masculino , Fosfofrutoquinases/metabolismo , Neoplasias da Próstata/metabolismo
8.
Ultrason Sonochem ; 59: 104751, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31473421

RESUMO

Ultrasonic sterilization (US), as a promising non-thermal sterilization method, exhibits unique superiorities than traditional sterilization methods. In this study, the inactivation mechanism of E. coli O157:H7 under US was investigated in cucumber and bitter gourd vegetable juices. Results revealed that the US treatment showed good antibacterial ability in countering E. coli O157:H7. Through determinations of conductivity and ß-galactosidase activity, significant augmentation in membrane permeability of the bacteria was confirmed after the US treatment. The morphologies of the US treated E. coli O157:H7 demonstrated that the integrity of the cell membrane was disrupted by US treatment. SDS-PAGE and LSCM data further proved the disruptive action of US, leading to the leakage of proteins and DNA through the breakage on cell membrane. The decrease of metabolic-related enzyme activity was verified through investigation of bacterial metabolism. The antibacterial mechanism analysis indicated that the US can generate free radicals which resulted in the rise of intracellular oxidative stress, attenuation of energy metabolism and inhibition of hexose monophosphate pathway. As the application verification, the US treatment can cause the deprivation of E. coli O157:H7 cell viability in vegetable juices without obvious impact on the sensory quality.


Assuntos
Escherichia coli O157/fisiologia , Viabilidade Microbiana , Sonicação , Esterilização/métodos , Trifosfato de Adenosina/metabolismo , Permeabilidade da Membrana Celular , Cor , DNA Bacteriano/metabolismo , Escherichia coli O157/citologia , Escherichia coli O157/metabolismo , Sucos de Frutas e Vegetais/microbiologia , Espaço Intracelular/metabolismo , Oxirredução , Paladar , beta-Galactosidase/metabolismo
9.
J Chem Theory Comput ; 15(11): 6368-6381, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31538783

RESUMO

The molecular chaperone HSP90 oversees the functional activation of a large number of client proteins. Because of its role in multiple pathways linked to cancer and neurodegeneration, drug discovery targeting HSP90 has been actively pursued. Yet, a number of inhibitors failed to meet expectations due to induced toxicity problems. In this context, allosteric perturbation has emerged as an alternative strategy for the pharmacological modulation of HSP90 functions. Specifically, novel allosteric stimulators showed the interesting capability of accelerating HSP90 closure dynamics and ATPase activities while inducing tumor cell death. Here, we gain atomistic insight into the mechanisms of allosteric ligand recognition and their consequences on the functional dynamics of HSP90, starting from the fully unbound state. We integrate advanced computational sampling methods based on FunnelMetadynamics, with the analysis of internal dynamics of the structural ensembles visited during the simulations. We observe several binding/unbinding events, and from these, we derive an accurate estimation of the absolute binding free energy. Importantly, we show that different binding poses induce different dynamics states. Our work for the first time explicitly correlates HSP90 responses to binding/unbinding of an allosteric ligand to the modulation of functionally oriented protein motions.


Assuntos
Proteínas de Choque Térmico HSP90/química , Ligantes , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sítios de Ligação , Proteínas de Choque Térmico HSP90/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Termodinâmica
10.
J Agric Food Chem ; 67(38): 10678-10684, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31475535

RESUMO

γ-Hydroxyvalerate (4HV) is an important monomer used to produce various valuable polymers and products. In this study, an engineered 3-hydroxybutyrate dehydrogenase that can convert levulinic acid (LA) into 4HV was co-expressed with a cofactor (NADH) regeneration system mediated by an NAD+-dependent formate dehydrogenase (CbFDH) in the Escherichia coli strain, MG1655. The resulting strain produced 23-fold more 4HV in a shake flask. The 4HV production was not dependent on ATP and required low aeration; all of these are considered beneficial characteristics for the production of target compounds, especially at an industrial scale. Under optimized conditions in a 5 L fermenter, the titer, productivity, and molar conversion efficiency for 4HV reached 100 g/L, 4.2 g/L/h, and 92%, respectively. Our system could prove to be a promising method for the large-scale production of 4HV from LA at low-cost and using a renewable biomass source.


Assuntos
Escherichia coli/metabolismo , Ácidos Levulínicos/metabolismo , Valeratos/metabolismo , Trifosfato de Adenosina/metabolismo , Biotransformação , Escherichia coli/genética , Fermentação , Engenharia Metabólica
11.
Expert Rev Cardiovasc Ther ; 17(9): 683-697, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31483166

RESUMO

Introduction: A critical mechanism of how hypoxia/ischemia causes irreversible myocardial injury is through the exhaustion of adenosine triphosphate (ATP). Cyclocreatine (CCr) and its water-soluble salt Cyclocreatine-Phosphate (CCrP) are potent bioenergetic agents that preserve high levels of ATP during ischemia. Areas covered: CCr and CCrP treatment prior to the onset of ischemia, preserved high levels of ATP in ischemic myocardium, reduced myocardial cell injury, exerted anti-inflammatory and anti-apoptotic activities, and restored contractile function during reperfusion in animal models of acute myocardial infarction (AMI), global cardiac arrest, cardiopulmonary bypass, and heart transplantation. Medline and Embase (1970 - Feb 2019), the WIPO databank (up to Feb 2019); no language restriction. Expert opinion: This review provides the basis for a number of clinical applications of CCrP and CCr to minimize ischemic injury and necrosis. One strategy is to administer CCrP to AMI patients in the pre-hospital phase, as well as during, or after Percutaneous Coronary Intervention (PCI) procedure to potentially achieve protection of the myocardium, reduce infarcted-size, and, thus, limit the progression to heart failure. Another clinical applications are in predictable myocardial ischemia where pretreatment with CCrP would likely improve outcome and quality of life of patients who will undergo cardiopulmonary bypass for coronary revascularization and end-stage heart failure patients scheduled for heart transplantation.


Assuntos
Creatinina/análogos & derivados , Infarto do Miocárdio/fisiopatologia , Isquemia Miocárdica/fisiopatologia , Trifosfato de Adenosina/metabolismo , Animais , Creatinina/metabolismo , Coração/fisiopatologia , Parada Cardíaca/fisiopatologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Intervenção Coronária Percutânea/métodos , Qualidade de Vida
12.
J Photochem Photobiol B ; 198: 111564, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31382090

RESUMO

Light irradiation has been used in clinical therapy for several decades. In this context, photobiomodulation (PBM) modulates signaling pathways via ROS, ATP, Ca2+, while photodynamic therapy (PDT) generates reactive oxygen species by excitation of a photosensitizer. NO generation could be an important tool when combined with both kinds of light therapy. By using a metal-based compound, we found that PBM combined with PDT could be a beneficial cancer treatment option. We used two types of ruthenium compounds, ([Ru(Pc)], Pc = phthalocyanine) and trans-[Ru(NO)(NO2)(Pc)]. The UV-vis spectra of both complexes displayed a band in the 660 nm region. In the case of 0.5 µM trans-[Ru(NO)(NO2)(Pc)], light irradiation at the Q-band reduced the percentage of viable human melanoma (A375) cells to around 50% as compared to [Ru(Pc)]. We hypothesized that these results were due to a synergistic effect between singlet oxygen and nitric oxide. Similar experiments performed with PDT (660 nm) combined with PBM (850 nm) induced more photocytotoxicity using both [Ru(Pc)] and trans-[Ru(NO)(NO2)(Pc)]. This was interpreted as PBM increasing cell metabolism (ATP production) and the consequent higher uptake of the ruthenium phthalocyanine compounds and more efficient apoptosis. The use of metal-based photosensitizers combined with light therapy may represent an advance in the field of photodynamic therapy.


Assuntos
Trifosfato de Adenosina/metabolismo , Complexos de Coordenação/química , Óxido Nítrico/metabolismo , Compostos Organometálicos/química , Fármacos Fotossensibilizantes/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/farmacologia , Complexos de Coordenação/uso terapêutico , Humanos , Luz , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Melanoma/patologia , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Oxigênio Singlete/metabolismo
13.
Anal Bioanal Chem ; 411(26): 6899-6911, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31407049

RESUMO

Adenosine triphosphate (ATP) is the main energy source in cells and an important biomolecule participating in cellular reactions in living organisms. Since the ATP level changes dynamically reflecting the development of a debilitating disease or carcinogenesis, we have focused in this work on monitoring of the oligomycin (OMC)-modulated ATP synthase inhibition using a fluorescent-switching DNA aptamer designed for the detection of ATP (Apt(ATP)), as the model for studies of dynamic ATP level variation. The behavior of the ATP aptamer has been characterized using fluorescence spectroscopy. The Intramolecular fluorescence resonance energy transfer (iFRET) operates in the proposed aptamer from the FAM dye moiety to guanines of the aptamer G-quadruplex when the target ATP is present and binds to the aptamer changing its conformation. The iFRET process enables the detection of ATP down to the limit of detection, LOD = 17 µM, without resorting to any extra chemi-amplification schemes. The selectivity coefficients for relevant interferent triphosphates (UTP, GTP, and CTP) are low for the same concentration as that of ATP. We have demonstrated an efficient transfection of intact cells and OMC-treated SW480 colon cancer cells with Apt(ATP), using microscopic imaging, iFRET measurements, and cell viability testing with MTT method. The applicability of the switching DNA aptamer for the analysis of real samples, obtained by lysis of SW480 cells, was also tested. The proposed Apt(ATP) may be considered as a viable candidate for utilization in measurements of dynamic ATP level modulation in cells in different stages of cancer development and testing of new drugs in pharmacological studies. Graphical abstract.


Assuntos
Trifosfato de Adenosina/análise , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Trifosfato de Adenosina/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/química , Quadruplex G , Humanos , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Neoplasias/metabolismo , Oligomicinas/farmacologia
14.
Bioelectromagnetics ; 40(7): 488-497, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31392747

RESUMO

Changes in impedance at 2 kHz, adenosine triphosphate (ATP) content, and muscle contraction were evaluated in yellowtail during 0 (ice), 5, 10, 15, and 20°C storage. Histological changes during ice storage were also measured. At any temperature, although impedance increased with both rigor mortis and ATP consumption during early storage, it began to decrease rapidly when ATP was almost depleted. Moreover, temporarily increasing impedance had a strong relationship with ATP content; decreasing impedance had a significant correlation with storage temperature after ATP depletion. Furthermore, impedance increased with narrowing of intercellular spaces when sarcolemma was intact and decreased with expansion of intercellular spaces when sarcolemma was leaky. Meanwhile, changes of sarcolemma and intercellular spaces were accompanied by ATP change. Thus, ATP is one significant physiological factor for impedance change, and temperature greatly influenced impedance after depletion of ATP. Results suggest that impedance analysis can be used as a convenient and nondestructive method to diagnose condition of tissue at different storage temperatures. Bioelectromagnetics. 2019;40:488-497. © 2019 Bioelectromagnetics Society.


Assuntos
Trifosfato de Adenosina/metabolismo , Impedância Elétrica , Peixes , Armazenamento de Alimentos , Músculos/metabolismo , Animais , Tecnologia de Alimentos/métodos , Temperatura Ambiente
15.
Life Sci ; 234: 116734, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31394126

RESUMO

AIMS: Acute myocardial insulin resistance is an independent risk factor for patients who undergo cardiac surgery with cardiopulmonary bypass (CPB). However, the underlying mechanism of insulin resistance during CPB has not been fully investigated. MATERIALS AND METHODS: To explore the role of myocardial insulin resistance on the cardiac function and its underlying mechanism, CPB operation and pharmacological intervention were applied in mini pigs, and myocardial insulin signaling, glucose uptake, ATP production and cardiac function were examined. KEY FINDINGS: Our data showed that CPB elicited not only hyperglycemia and hyperinsulinemia, but also inactivated Akt, and impaired the transposition of membrane glucose transporter-4 (GLUT-4), reduced glucose uptake and ATP production in the myocardium as well, which in turn was accompanied with cardiac dysfunction. Meanwhile, linear correlations were established among reduced myocardial glucose uptake, ATP production, and depressed cardiac systolic or diastolic function. Reactivation of Akt by SC79, an Akt agonist, partially alleviated myocardial insulin resistance and restored post CPB cardiac function via augmenting myocardial glucose uptake and ATP production. SIGNIFICANCE: These findings revealed that acute myocardial insulin resistance due to inactivation of Akt played a key role in cardiac dysfunction post CPB via suppressing glucose metabolism related energy supply.


Assuntos
Ponte Cardiopulmonar/efeitos adversos , Resistência à Insulina , Insulina/metabolismo , Miocárdio/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Coração/fisiopatologia , Masculino , Miocárdio/patologia , Suínos , Porco Miniatura
16.
J Microbiol Biotechnol ; 29(8): 1288-1298, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31370116

RESUMO

Bacterial ATP synthases drive ATP synthesis by a rotary mechanism, and play a vital role in physiology and cell metabolism. Corynebacterium glutamicum is well known as an industrial workhorse for amino acid production, and its ATP synthase operon contains eight structural genes and two adjacent genes, cg1360 and cg1361. So far, the physiological functions of Cg1360 (GenBank CAF19908) and Cg1361 (GenBank CAF19909) remain unclear. Here, we showed that Cg1360 was a hydrophobic protein with four transmembrane helices (TMHs), while no TMH was found in Cg1361. Deletion of cg1360, but not cg1361, led to significantly reduced cell growth using glucose and acetic acid as carbon sources, reduced F1 portions in the membrane, reduced ATP-driven proton-pumping activity and ATPase activity, suggesting that Cg1360 plays an important role in ATP synthase function. The intracellular ATP concentration in the Δcg1360 mutant was decreased to 72% of the wild type, while the NADH and NADPH levels in the Δcg1360 mutant were increased by 29% and 26%, respectively. However, the Δcg1361 mutant exhibited comparable intracellular ATP, NADH and NADPH levels with the wild-type strain. Moreover, the effect of cg1360 deletion on L-valine production was examined in the L-valine-producing V-10 strain. The final production of L-valine in the V-10-Δcg1360 mutant reached 9.2 ± 0.3 g/l in shake flasks, which was 14% higher than that of the V-10 strain. Thus, Cg1360 can be used as an effective engineering target by altering energy metabolism for the enhancement of amino acid production in C. glutamicum.


Assuntos
Trifosfato de Adenosina/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Deleção de Genes , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Valina/biossíntese , Ácido Acético/metabolismo , Adenosina Trifosfatases , Carbono/metabolismo , Corynebacterium glutamicum/crescimento & desenvolvimento , Metabolismo Energético , Fermentação , Ordem dos Genes , Glucose/metabolismo , NAD/metabolismo , NADP/metabolismo , Alinhamento de Sequência
17.
Nucleic Acids Res ; 47(15): 7798-7808, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31372639

RESUMO

Homologous recombination is a fundamental process in all living organisms that allows the faithful repair of DNA double strand breaks, through the exchange of DNA strands between homologous regions of the genome. Results of three decades of investigation and recent fruitful observations have unveiled key elements of the reaction mechanism, which proceeds along nucleofilaments of recombinase proteins of the RecA family. Yet, one essential aspect of homologous recombination has largely been overlooked when deciphering the mechanism: while ATP is hydrolyzed in large quantity during the process, how exactly hydrolysis influences the DNA strand exchange reaction at the structural level remains to be elucidated. In this study, we build on a previous geometrical approach that studied the RecA filament variability without bound DNA to examine the putative implication of ATP hydrolysis on the structure, position, and interactions of up to three DNA strands within the RecA nucleofilament. Simulation results on modeled intermediates in the ATP cycle bring important clues about how local distortions in the DNA strand geometries resulting from ATP hydrolysis can aid sequence recognition by promoting local melting of already formed DNA heteroduplex and transient reverse strand exchange in a weaving type of mechanism.


Assuntos
Trifosfato de Adenosina/química , DNA de Cadeia Simples/química , DNA/química , Recombinação Homóloga , Ácidos Nucleicos Heteroduplexes/química , Recombinases Rec A/química , Trifosfato de Adenosina/metabolismo , Bactérias/genética , Bactérias/metabolismo , Sítios de Ligação , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Hidrólise , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Ligação Proteica , Conformação Proteica , Recombinases Rec A/genética , Recombinases Rec A/metabolismo
18.
Adv Exp Med Biol ; 1155: 163-172, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468395

RESUMO

Taurine (2-aminoethanesulfonic acid), a sulfur-containing ß-amino acid, is a free amino acid present in high concentrations in mammalian tissues. Taurine has pivotal roles in anti-oxidation, membrane stabilization, osmoregulation, anti-inflammation, and other process. In a DNA microarray analysis, we previously found that taurine markedly increases the mRNA expression of thioredoxin interacting protein (TXNIP) in Caco-2 cells. In this study, we investigated the effect of these taurine-induced changes in TXNIP on the function of Caco-2 cells. We found that taurine decreases glucose uptake in a dose-dependent manner. The taurine-induced decrease in glucose uptake was completely abolished by transfection with siRNA against TXNIP, suggesting that TXNIP is involved in the taurine-induced down-regulation of glucose uptake. We also revealed that taurine induces AMPK activation and further increases the intracellular ATP content in Caco-2 cells. These results suggest that taurine could regulate the function of Caco-2 cells via TXNIP induction, leading to extend our understanding of the functions of taurine.


Assuntos
Proteínas de Transporte/metabolismo , Glucose/metabolismo , Taurina/farmacologia , Trifosfato de Adenosina/metabolismo , Adenilato Quinase/metabolismo , Transporte Biológico , Células CACO-2 , Regulação para Baixo , Humanos , RNA Interferente Pequeno
19.
Biochim Biophys Acta Rev Cancer ; 1872(2): 188304, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31348974

RESUMO

The concentration of sodium ions (Na+) is raised in solid tumours and can be measured at the cellular, tissue and patient levels. At the cellular level, the Na+ gradient across the membrane powers the transport of H+ ions and essential nutrients for normal activity. The maintenance of the Na+ gradient requires a large proportion of the cell's ATP. Na+ is a major contributor to the osmolarity of the tumour microenvironment, which affects cell volume and metabolism as well as immune function. Here, we review evidence indicating that Na+ handling is altered in tumours, explore our current understanding of the mechanisms that may underlie these alterations and consider the potential consequences for cancer progression. Dysregulated Na+ balance in tumours may open opportunities for new imaging biomarkers and re-purposing of drugs for treatment.


Assuntos
Neoplasias/metabolismo , Sódio/metabolismo , Trifosfato de Adenosina/metabolismo , Tamanho Celular , Homeostase , Humanos , Microambiente Tumoral
20.
Nature ; 571(7766): 515-520, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31341297

RESUMO

The mitochondrial ADP/ATP carrier (AAC) is a major transport protein of the inner mitochondrial membrane. It exchanges mitochondrial ATP for cytosolic ADP and controls cellular production of ATP. In addition, it has been proposed that AAC mediates mitochondrial uncoupling, but it has proven difficult to demonstrate this function or to elucidate its mechanisms. Here we record AAC currents directly from inner mitochondrial membranes from various mouse tissues and identify two distinct transport modes: ADP/ATP exchange and H+ transport. The AAC-mediated H+ current requires free fatty acids and resembles the H+ leak via the thermogenic uncoupling protein 1 found in brown fat. The ADP/ATP exchange via AAC negatively regulates the H+ leak, but does not completely inhibit it. This suggests that the H+ leak and mitochondrial uncoupling could be dynamically controlled by cellular ATP demand and the rate of ADP/ATP exchange. By mediating two distinct transport modes, ADP/ATP exchange and H+ leak, AAC connects coupled (ATP production) and uncoupled (thermogenesis) energy conversion in mitochondria.


Assuntos
Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Prótons , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Coenzimas/metabolismo , Ácidos Graxos/metabolismo , Transporte de Íons , Masculino , Camundongos , Consumo de Oxigênio
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