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1.
Klin Lab Diagn ; 66(3): 172-176, 2021 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-33793117

RESUMO

Adenine nucleotides (ATP, ADP and AMP) play a central role in the regulation of metabolism and energy: they provide the energy balance of the cell, determine its redox state, act as allosteric effectors of a number of enzymes, modulate signaling and transcription factors and activate oxidation or biosynthesis substrates. A large number of methods have been developed to determine the level of ATP, ADP and AMP, but the most universal and effective method for the separation and analysis of complex mixtures is the reversed-phase high-performance liquid chromatography method (RP-HPLC). The aim of this study is to determine the optimal conditions for the qualitative separation and quantitative determination of standard solutions of ATP (1 mmol/l), ADP (0,5 mmol/l) and AMP (0,1 mmol/l) by RP-HPLC. The degree of separation of adenine nucleotides was estimated by the time of peak output in the chromatogram. To achieve the goal, the following tasks were set: assess the effect of the temperature of the analysis on the separation and change of the release time of the analytes in the chromatogram; determine the most optimal composition of the mobile phase for the separation of ATP, ADP and AMP in the chromatogram (the content of the organic solvent in the solution); to identify the effect of pH of the mobile phase on the separation of standard solutions of adenine nucleotides; set the optimal molarity of the mobile phase for the separation of ATP, ADP and AMP in the chromatogram. It was found that the temperature of the analysis does not affect the quality of peak separation, while the composition and pH of the mobile phase have a significant effect on the complete and clear separation of the studied nucleotides in the chromatogram. It was determined that the analysis temperature of 37°C and the mobile phase of 0.05 M KH2PO4 (pH 6.0) are optimal for separating the peaks of adenine nucleotides.


Assuntos
Nucleotídeos de Adenina , Trifosfato de Adenosina , Cromatografia Líquida de Alta Pressão , Indicadores e Reagentes
2.
Medicine (Baltimore) ; 100(10): e25100, 2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33725905

RESUMO

BACKGROUND: Mitochondrial diabetes mellitus (MDM) is characterized by maternal inheritance, progressive neurosensory deafness, insulin secretory disorder, and progressive microvascular complications. Mitochondria are critical organelles that provide energy in the form of adenosine triphosphate (ATP). An impairment of ATP production in pancreatic ß cells is regarded as the main cause of the insulin secretory disorder in patients with MDM, and these patients require insulin replacement therapy early after the diagnosis. The amino acid 5-aminolevulinic acid (5-ALA), a precursor of heme metabolites, is a non-proteinogenic δ amino acid synthesized in mitochondria. An addition of ferrous iron to 5-ALA enhances heme biosynthesis and increases ATP production through an upregulation of the respiratory complex. Several studies have reported that the administration of 5-ALA and ferrous iron to existing treatment improved the glycemic control in both patients with prediabetes and those with type 2 diabetes mellitus. The additional administration of 5-ALA and ferrous iron to MDM patients on insulin therapy may improve their insulin secretory capacity and glycemic control by improving their mitochondrial function. The findings of this study are expected to provide new treatment options for MDM and improve the patients' glycemic control and prognosis. METHODS/DESIGN: This study is a single-arm, open-label pilot intervention study using clinical endpoints to investigate the effects of treatment with 5-ALA plus sodium ferrous citrate (SFC) to patients with MDM on their glucose tolerance. A total of 5 patients with MDM will be administered 5-ALA/SFC (200 mg/d) for 24 weeks. We will perform a 75-g oral glucose tolerance test before and at 24 weeks after the start of this 5-ALA/SFC treatment to evaluate glucose-dependent insulin responses. DISCUSSION: To the best of our knowledge, this study will be the first assessment of the effects of 5-ALA/SFC in patients with MDM. This study will obtain an evidence regarding the effectiveness and safety of 5-ALA/SFC for patients with MDM. TRIAL REGISTRATION: This study was registered with the University Hospital Medical Information Network (UMIN000040581) on July 1, 2020 and with the Japan Registry of Clinical Trials (jRCTs071200025) on August 3, 2020.


Assuntos
Surdez/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Compostos Ferrosos/administração & dosagem , Intolerância à Glucose/tratamento farmacológico , Insulina/administração & dosagem , Ácidos Levulínicos/administração & dosagem , Doenças Mitocondriais/tratamento farmacológico , Trifosfato de Adenosina/metabolismo , Adulto , Glicemia/análise , Surdez/sangue , Surdez/diagnóstico , Surdez/patologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/patologia , Quimioterapia Combinada/efeitos adversos , Quimioterapia Combinada/métodos , Feminino , Compostos Ferrosos/efeitos adversos , Intolerância à Glucose/sangue , Intolerância à Glucose/diagnóstico , Intolerância à Glucose/patologia , Teste de Tolerância a Glucose , Humanos , Japão , Ácidos Levulínicos/efeitos adversos , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Doenças Mitocondriais/sangue , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/patologia , Projetos Piloto , Resultado do Tratamento
3.
Nat Commun ; 12(1): 1463, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33674590

RESUMO

Kinesin-1 is a processive motor protein that uses ATP-derived energy to transport a variety of intracellular cargoes toward the cell periphery. The ability to visualize and monitor kinesin transport in live cells is critical to study the myriad of functions associated with cargo trafficking. Herein we report the discovery of a fluorogenic small molecule substrate (QPD-OTf) for kinesin-1 that yields a precipitating dye along its walking path on microtubules (MTs). QPD-OTf enables to monitor native kinesin-1 transport activity in cellulo without external modifications. In vitro assays show that kinesin-1 and MTs are sufficient to yield fluorescent crystals; in cells, kinesin-1 specific transport of cargo from the Golgi appears as trails of fluorescence over time. These findings are further supported by docking studies, which suggest the binding of the activity-based substrate in the nucleotide binding site of kinesin-1.


Assuntos
Cinesina/química , Cinesina/metabolismo , Microtúbulos/metabolismo , Trifosfato de Adenosina , Animais , Sítios de Ligação , Complexo de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos , Cinesina/genética , Camundongos , Paclitaxel , Transporte Proteico , Células RAW 264.7
4.
Food Chem ; 351: 129336, 2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-33662909

RESUMO

To investigate the effect of relative humidity (RH) on chilling injury (CI), zucchini fruit were stored in cold rooms (4 ± 0.4 ℃) with different RHs (near-saturated RH [NSH] with 96-100% and normal RH with 72-76% served as control). Storage in NSH delayed weight loss and CI, maintained firmness and skin color. Higher antioxidant enzyme activities and greater scavenging capacities of free radicals were found in NSH-fruit than in the control fruit. The decrease of the unsaturated fatty acids was delayed in NSH-fruit due to lower activities of related membrane lipid degrading enzymes as compared to the control fruit. NSH-fruit also maintained higher activities of energy metabolism-associated enzymes than control fruit, leading to high levels of adenosine triphosphate (ATP). Taken together, we attributed the alleviation of CI by NSH storage to its enhancement of antioxidant capacities and its effect on maintaining higher energy status in zucchini fruit.


Assuntos
Antioxidantes/metabolismo , Temperatura Baixa , Cucurbita/metabolismo , Metabolismo Energético , Frutas/metabolismo , Umidade , Trifosfato de Adenosina/metabolismo , Armazenamento de Alimentos
5.
Molecules ; 26(4)2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33671368

RESUMO

Multidrug resistance protein-4 (MRP4) belongs to the ABC transporter superfamily and promotes the transport of xenobiotics including drugs. A non-synonymous single nucleotide polymorphisms (nsSNPs) in the ABCC4 gene can promote changes in the structure and function of MRP4. In this work, the interaction of certain endogen substrates, drug substrates, and inhibitors with wild type-MRP4 (WT-MRP4) and its variants G187W and Y556C were studied to determine differences in the intermolecular interactions and affinity related to SNPs using protein threading modeling, molecular docking, all-atom, coarse grained, and umbrella sampling molecular dynamics simulations (AA-MDS and CG-MDS, respectively). The results showed that the three MRP4 structures had significantly different conformations at given sites, leading to differences in the docking scores (DS) and binding sites of three different groups of molecules. Folic acid (FA) had the highest variation in DS on G187W concerning WT-MRP4. WT-MRP4, G187W, Y556C, and FA had different conformations through 25 ns AA-MD. Umbrella sampling simulations indicated that the Y556C-FA complex was the most stable one with or without ATP. In Y556C, the cyclic adenosine monophosphate (cAMP) and ceefourin-1 binding sites are located out of the entrance of the inner cavity, which suggests that both cAMP and ceefourin-1 may not be transported. The binding site for cAMP and ceefourin-1 is quite similar and the affinity (binding energy) of ceefourin-1 to WT-MRP4, G187W, and Y556C is greater than the affinity of cAMP, which may suggest that ceefourin-1 works as a competitive inhibitor. In conclusion, the nsSNPs G187W and Y556C lead to changes in protein conformation, which modifies the ligand binding site, DS, and binding energy.


Assuntos
Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Mutantes/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Benzotiazóis/química , Benzotiazóis/metabolismo , Sítios de Ligação , AMP Cíclico/química , AMP Cíclico/metabolismo , Ácido Fólico/química , Ácido Fólico/metabolismo , Ligantes , Domínios Proteicos , Homologia Estrutural de Proteína , Termodinâmica , Triazóis/química , Triazóis/metabolismo
6.
Molecules ; 26(4)2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673272

RESUMO

Selective recognition of nucleotides with synthetic receptors is an emerging direction to solve a series of nucleic acid-related challenges in biochemistry. Towards this goal, a new aza-cyclophane with two different dyes, naphthalimide and pyrene, connected through a triamine linker has been synthesized and studied for the ability to bind and detect nucleoside triphosphates in an aqueous solution. The receptor shows Foerster resonance energy transfer (FRET) in fluorescence spectra upon excitation in DMSO, which is diminished dramatically in the presence of water. According to binding studies, the receptor has a preference to bind ATP (adenosine triphosphate) and CTP (cytidine triphosphate) with a "turn-on" fluorescence response. Two separate emission bands of dyes allow one to detect nucleotides in a ratiometric manner in a broad concentration range of 10-5-10-3 M. Spectroscopic measurements and quantum chemical calculations suggest the formation of receptor-nucleotide complexes, which are stabilized by dispersion interactions between a nucleobase and dyes, while hydrogen bonding interactions of nucleobases with the amine linkers are responsible for selectivity.


Assuntos
Éteres Cíclicos/química , Naftalimidas/química , Nucleotídeos/química , Piperidinas/química , Pirenos/química , Trifosfato de Adenosina , Corantes Fluorescentes/química , Ligação de Hidrogênio , Estrutura Molecular , Soluções/química , Espectrometria de Fluorescência , Água/química
7.
PLoS Biol ; 19(2): e3001109, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33596198

RESUMO

Coronavirus Disease 2019 (COVID-19), caused by the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has affected over 30 million globally to date. Although high rates of venous thromboembolism and evidence of COVID-19-induced endothelial dysfunction have been reported, the precise aetiology of the increased thrombotic risk associated with COVID-19 infection remains to be fully elucidated. Therefore, we assessed clinical platelet parameters and circulating platelet activity in patients with severe and nonsevere COVID-19. An assessment of clinical blood parameters in patients with severe COVID-19 disease (requiring intensive care), patients with nonsevere disease (not requiring intensive care), general medical in-patients without COVID-19, and healthy donors was undertaken. Platelet function and activity were also assessed by secretion and specific marker analysis. We demonstrated that routine clinical blood parameters including increased mean platelet volume (MPV) and decreased platelet:neutrophil ratio are associated with disease severity in COVID-19 upon hospitalisation and intensive care unit (ICU) admission. Strikingly, agonist-induced ADP release was 30- to 90-fold higher in COVID-19 patients compared with hospitalised controls and circulating levels of platelet factor 4 (PF4), soluble P-selectin (sP-selectin), and thrombopoietin (TPO) were also significantly elevated in COVID-19. This study shows that distinct differences exist in routine full blood count and other clinical laboratory parameters between patients with severe and nonsevere COVID-19. Moreover, we have determined all COVID-19 patients possess hyperactive circulating platelets. These data suggest abnormal platelet reactivity may contribute to hypercoagulability in COVID-19 and confirms the role that platelets/clotting has in determining the severity of the disease and the complexity of the recovery path.


Assuntos
Plaquetas/virologia , /sangue , Trifosfato de Adenosina/metabolismo , Idoso , Coagulação Sanguínea , Plaquetas/citologia , Ensaio de Imunoadsorção Enzimática , Feminino , Hemostasia , Humanos , Inflamação , Unidades de Terapia Intensiva , Masculino , Volume Plaquetário Médio , Pessoa de Meia-Idade , Selectina-P/sangue , Fenótipo , Fator Plaquetário 4/sangue , Testes de Função Plaquetária , Trombopoetina/sangue
8.
Clin Transl Gastroenterol ; 12(2): e00314, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33620880

RESUMO

INTRODUCTION: During the coronavirus disease 2019 (COVID-19) pandemic, endoscopists have high risks of exposure to exhaled air from patients during gastroscopy. To minimize this risk, we transformed the oxygen mask into a fully closed negative-pressure gastroscope isolation mask. This study aimed to evaluate the effectiveness, safety, and feasibility of use of this mask during gastroscopy. METHODS: From February 28, 2020, to March 10, 2020, 320 patients undergoing gastroscopy were randomly assigned into the mask group (n = 160) or conventional group (n = 160). Patients in the mask group wore the isolation mask during gastroscopy, whereas patients in the conventional group did not wear the mask. The adenosine triphosphate fluorescence and carbon dioxide (CO2) concentration in patients' exhaled air were measured to reflect the degree of environmental pollution by exhaled air. Patients' vital signs, operation time, and adverse events during endoscopy were also evaluated. RESULTS: Four patients were excluded because of noncooperation or incomplete data. A total of 316 patients were included in the final analysis. The difference between the highest CO2 concentration around patients' mouth and CO2 concentration in the environment was significantly decreased in the mask group compared with the conventional group. There was no significant difference in the adenosine triphosphate fluorescence, vital signs, and operation time between the 2 groups. No severe adverse events related to the isolation mask, endoscopy failure, or new coronavirus infection during follow-up were recorded. DISCUSSION: This new isolation mask showed excellent feasibility of use and safety compared with routine gastroscopy during the COVID-19 pandemic.


Assuntos
/transmissão , Gastroscopia/efeitos adversos , Máscaras/virologia , Isoladores de Pacientes/virologia , Trifosfato de Adenosina/metabolismo , Adulto , /epidemiologia , Dióxido de Carbono/análise , Estudos de Casos e Controles , Desenho de Equipamento/métodos , Expiração/fisiologia , Estudos de Viabilidade , Feminino , Fluorescência , Gastroscopia/métodos , Humanos , Masculino , Máscaras/efeitos adversos , Máscaras/estatística & dados numéricos , Pessoa de Meia-Idade , Duração da Cirurgia , Estudos Prospectivos , Segurança , Resultado do Tratamento
9.
Science ; 371(6530)2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33574186

RESUMO

Kinesin motors are essential for the transport of cellular cargo along microtubules. How the motors step, detach, and cooperate with each other is still unclear. To dissect the molecular motion of kinesin-1, we developed germanium nanospheres as ultraresolution optical trapping probes. We found that single motors took 4-nanometer center-of-mass steps. Furthermore, kinesin-1 never detached from microtubules under hindering load conditions. Instead, it slipped on microtubules in microsecond-long, 8-nanometer steps and remained in this slip state before detaching or reengaging in directed motion. Unexpectedly, reengagement and thus rescue of directed motion was more frequent. Our observations broaden our knowledge on the mechanochemical cycle and slip state of kinesin. This state and rescue need to be accounted for to understand long-range transport by teams of motors.


Assuntos
Germânio , Cinesina/química , Cinesina/metabolismo , Nanosferas , Pinças Ópticas , Trifosfato de Adenosina/metabolismo , Transporte Biológico , Cinética , Bicamadas Lipídicas , Microtúbulos/metabolismo , Modelos Biológicos , Imagem Individual de Molécula
10.
Mol Cell ; 81(4): 642-644, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33606971

RESUMO

Luengo et al. (2020) demonstrate that pyruvate dehydrogenase (PDH) overactivation blunts NAD+ regeneration by overcharging the mitochondrial membrane potential and driving ATP synthesis beyond demand. Under these conditions, some cells prioritize aerobic glycolysis to meet the need for oxidized cofactors in biosynthetic metabolism.


Assuntos
NAD , Complexo Piruvato Desidrogenase , Trifosfato de Adenosina , Glucose , Glicólise , NAD/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Estações do Ano
11.
Nat Commun ; 12(1): 851, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33558474

RESUMO

ATP-independent chaperones are usually considered to be holdases that rapidly bind to non-native states of substrate proteins and prevent their aggregation. These chaperones are thought to release their substrate proteins prior to their folding. Spy is an ATP-independent chaperone that acts as an aggregation inhibiting holdase but does so by allowing its substrate proteins to fold while they remain continuously chaperone bound, thus acting as a foldase as well. The attributes that allow such dual chaperoning behavior are unclear. Here, we used the topologically complex protein apoflavodoxin to show that the outcome of Spy's action is substrate specific and depends on its relative affinity for different folding states. Tighter binding of Spy to partially unfolded states of apoflavodoxin limits the possibility of folding while bound, converting Spy to a holdase chaperone. Our results highlight the central role of the substrate in determining the mechanism of chaperone action.


Assuntos
Trifosfato de Adenosina/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Periplásmicas/metabolismo , Anabaena/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Azotobacter/metabolismo , Escherichia coli/metabolismo , Flavodoxina/química , Flavodoxina/metabolismo , Cinética , Espectroscopia de Ressonância Magnética , Conformação Molecular , Proteínas Mutantes/metabolismo , Proteínas Periplásmicas/química , Ligação Proteica , Dobramento de Proteína , Especificidade por Substrato
12.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33540748

RESUMO

Tuberculosis (TB) remains one of the major health concerns worldwide. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can flexibly change its metabolic processes during different life stages. Regulation of key metabolic enzyme activities by intracellular conditions, allosteric inhibition or feedback control can effectively contribute to Mtb survival under different conditions. Phosphofructokinase (Pfk) is one of the key enzymes regulating glycolysis. Mtb encodes two Pfk isoenzymes, Pfk A/Rv3010c and Pfk B/Rv2029c, which are differently expressed upon transition to the hypoxia-induced non-replicating state of the bacteria. While pfkB gene and protein expression are upregulated under hypoxic conditions, Pfk A levels decrease. Here, we present biochemical characterization of both Pfk isoenzymes, revealing that Pfk A and Pfk B display different kinetic properties. Although the glycolytic activity of Pfk A is higher than that of Pfk B, it is markedly inhibited by an excess of both substrates (fructose-6-phosphate and ATP), reaction products (fructose-1,6-bisphosphate and ADP) and common metabolic allosteric regulators. In contrast, synthesis of fructose-1,6-bisphosphatase catalyzed by Pfk B is not regulated by higher levels of substrates, and metabolites. Importantly, we found that only Pfk B can catalyze the reverse gluconeogenic reaction. Pfk B thus can support glycolysis under conditions inhibiting Pfk A function.


Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/enzimologia , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Regulação Alostérica , Proteínas de Bactérias/antagonistas & inibidores , Catálise , Indução Enzimática , Retroalimentação Fisiológica , Frutosedifosfatos/biossíntese , Frutosedifosfatos/farmacologia , Frutosefosfatos/metabolismo , Frutosefosfatos/farmacologia , Gluconeogênese , Glicólise , Hexosefosfatos/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Cinética , L-Lactato Desidrogenase/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Oxigênio/farmacologia , Piruvato Quinase/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
13.
Nat Commun ; 12(1): 1138, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33602916

RESUMO

DNA nanostructures have been demonstrated as promising carriers for gene delivery. In the carrier design, spatiotemporally programmable assembly of DNA under nanoconfinement is important but has proven highly challenging due to the complexity-scalability-error of DNA. Herein, a DNA nanotechnology-based strategy via the cascade hybridization chain reaction (HCR) of DNA hairpins in polymeric nanoframework has been developed to achieve spatiotemporally programmable assembly of DNA under nanoconfinement for precise siRNA delivery. The nanoframework is prepared via precipitation polymerization with Acrydite-DNA as cross-linker. The potential energy stored in the loops of DNA hairpins can overcome the steric effect in the nanoframework, which can help initiate cascade HCR of DNA hairpins and achieve efficient siRNA loading. The designer tethering sequence between DNA and RNA guarantees a triphosadenine triggered siRNA release specifically in cellular cytoplasm. Nanoframework provides stability and ease of functionalization, which helps address the complexity-scalability-error of DNA. It is exemplified that the phenylboronate installation on nanoframework enhanced cellular uptake and smoothed the lysosomal escape. Cellular results show that the siRNA loaded nanoframework down-regulated the levels of relevant mRNA and protein. In vivo experiments show significant therapeutic efficacy of using siPLK1 loaded nanoframework to suppress tumor growth.


Assuntos
DNA/química , Técnicas de Transferência de Genes , Nanoestruturas/química , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Polímeros/química , RNA Interferente Pequeno/administração & dosagem , Trifosfato de Adenosina/metabolismo , Animais , Antineoplásicos/farmacologia , Materiais Biocompatíveis/química , Ácidos Borônicos/química , Linhagem Celular Tumoral , Feminino , Inativação Gênica , Lisossomos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Fatores de Tempo
14.
Am J Physiol Heart Circ Physiol ; 320(3): H1055-H1065, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33449849

RESUMO

Pannexin 1 (Panx1) channels export ATP and may contribute to increased concentration of the vasodilator ATP in plasma during hypoxia in vivo. We hypothesized that Panx1 channels and associated ATP export contribute to hypoxic vasodilation, a mechanism that facilitates the matching of oxygen delivery to metabolic demand of tissue. Male and female mice devoid of Panx1 (Panx1-/-) and wild-type controls (WT) were anesthetized, mechanically ventilated, and instrumented with a carotid artery catheter or femoral artery flow transducer for hemodynamic and plasma ATP monitoring during inhalation of 21% (normoxia) or 10% oxygen (hypoxia). ATP export from WT vs. Panx1-/-erythrocytes (RBC) was determined ex vivo via tonometer experimentation across progressive deoxygenation. Mean arterial pressure (MAP) was similar in Panx1-/- (n = 6) and WT (n = 6) mice in normoxia, but the decrease in MAP in hypoxia seen in WT was attenuated in Panx1-/- mice (-16 ± 9% vs. -2 ± 8%; P < 0.05). Hindlimb blood flow (HBF) was significantly lower in Panx1-/- (n = 6) vs. WT (n = 6) basally, and increased in WT but not Panx1-/- mice during hypoxia (8 ± 6% vs. -10 ± 13%; P < 0.05). Estimation of hindlimb vascular conductance using data from the MAP and HBF experiments showed an average response of 28% for WT vs. -9% for Panx1-/- mice. Mean venous plasma ATP during hypoxia was 57% lower in Panx1-/- (n = 6) vs. WT mice (n = 6; P < 0.05). Mean hypoxia-induced ATP export from RBCs from Panx1-/- mice (n = 8) was 82% lower than that from WT (n = 8; P < 0.05). Panx1 channels participate in hemodynamic responses consistent with hypoxic vasodilation by regulating hypoxia-sensitive extracellular ATP levels in blood.NEW & NOTEWORTHY Export of vasodilator ATP from red blood cells requires pannexin 1. Blood plasma ATP elevations in response to hypoxia in mice require pannexin 1. Hemodynamic responses to hypoxia are accompanied by increased plasma ATP in mice in vivo and require pannexin 1.


Assuntos
Trifosfato de Adenosina/sangue , Conexinas/sangue , Eritrócitos/metabolismo , Hemodinâmica , Membro Posterior/irrigação sanguínea , Hipóxia/sangue , Proteínas do Tecido Nervoso/sangue , Oxigênio/sangue , Animais , Pressão Arterial , Conexinas/deficiência , Conexinas/genética , Modelos Animais de Doenças , Feminino , Frequência Cardíaca , Hiperemia/sangue , Hiperemia/genética , Hiperemia/fisiopatologia , Hipotensão/sangue , Hipotensão/genética , Hipotensão/fisiopatologia , Hipóxia/genética , Hipóxia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Fluxo Sanguíneo Regional , Vasodilatação
15.
Nature ; 590(7847): 624-629, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33461211

RESUMO

In the type III CRISPR-Cas immune response of prokaryotes, infection triggers the production of cyclic oligoadenylates that bind and activate proteins that contain a CARF domain1,2. Many type III loci are associated with proteins in which the CRISPR-associated Rossman fold (CARF) domain is fused to a restriction  endonuclease-like domain3,4. However, with the exception of the well-characterized Csm6 and Csx1 ribonucleases5,6, whether and how these inducible effectors provide defence is not known. Here we investigated a type III CRISPR accessory protein, which we name cyclic-oligoadenylate-activated single-stranded ribonuclease and single-stranded deoxyribonuclease 1 (Card1). Card1 forms a symmetrical dimer that has a large central cavity between its CRISPR-associated Rossmann fold and restriction endonuclease domains that binds cyclic tetra-adenylate. The binding of ligand results in a conformational change comprising the rotation of individual monomers relative to each other to form a more compact dimeric scaffold, in which a manganese cation coordinates the catalytic residues and activates the cleavage of single-stranded-but not double-stranded-nucleic acids (both DNA and RNA). In vivo, activation of Card1 induces dormancy of the infected hosts to provide immunity against phage infection and plasmids. Our results highlight the diversity of strategies used in CRISPR systems to provide immunity.


Assuntos
Nucleotídeos de Adenina/metabolismo , Sistemas CRISPR-Cas/imunologia , DNA de Cadeia Simples/metabolismo , Desoxirribonucleases/metabolismo , Endorribonucleases/metabolismo , Oligorribonucleotídeos/metabolismo , RNA/metabolismo , Staphylococcus/enzimologia , Staphylococcus/imunologia , Nucleotídeos de Adenina/imunologia , Trifosfato de Adenosina/metabolismo , Bacteriófagos/imunologia , Bacteriófagos/fisiologia , Biocatálise , Domínio Catalítico , Desoxirribonucleases/química , Desoxirribonucleases/genética , Endorribonucleases/química , Endorribonucleases/genética , Ativação Enzimática , Ligantes , Manganês/química , Manganês/metabolismo , Modelos Moleculares , Oligorribonucleotídeos/imunologia , Plasmídeos/genética , Plasmídeos/metabolismo , Multimerização Proteica , Rotação , Staphylococcus/crescimento & desenvolvimento , Staphylococcus/virologia , Especificidade por Substrato
16.
J Med Chem ; 64(2): 1197-1219, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33417773

RESUMO

Significant inhibition of Aurora B was achieved by the synthesis of simplified fragments of benzosceptrins and oroidin belonging to the marine pyrrole-2-aminoimidazoles metabolites isolated from sponges. Evaluation of kinase inhibition enabled the discovery of a synthetically accessible rigid acetylenic structural analogue EL-228 (1), whose structure could be optimized into the potent CJ2-150 (37). Here we present the synthesis of new inhibitors of Aurora B kinase, which is an important target for cancer therapy through mitosis regulation. The biologically oriented synthesis yielded several nanomolar inhibitors. The optimized compound CJ2-150 (37) showed a non-ATP competitive allosteric mode of action in a mixed-type inhibition for Aurora B kinase. Molecular docking identified a probable binding mode in the allosteric site "F" and highlighted the key interactions with the protein. We describe the improvement of the inhibitory potency and specificity of the novel scaffold as well as the characterization of the mechanism of action.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Aurora Quinase B/antagonistas & inibidores , Poríferos/química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Mitose/efeitos dos fármacos , Modelos Moleculares , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade
17.
Food Chem ; 348: 129113, 2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-33508609

RESUMO

The effect of chitosan coating exposure on juice sac granulation and energy metabolism in harvested pummelo fruit was investigated. Pummelo fruits were exposed to 1.5% chitosan coating, and then stored at 20 ± 2 °C for about 150 days. Postharvest chitosan coating treatment apparently alleviated the development of juice sac granulation as well as the increases in weight loss, pulp firmness, cell membrane permeability and cellulose content. The levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and energy charge (EC) in the chitosan-coated fruit showed significantly higher levels than those of the respective controls. Meanwhile, the enzymses actively engaged in energy metabolism such as H+-ATPase, Ca2+-ATPase, Mg2+-ATPase, cytochrome C oxidase (CCO), succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) were markedly maintained by chitosan coating. Besides, notably high contents of acetyl-CoA, cis-aconitate, succinate, fumarate and oxaloacetate were observed in the chitosan-coated fruit. The results highlighted that chitosan coating could delay postharvest senescence of pummelo fruit by reducing the rate of energy depletion while maintaining higher levels of key metabolites taking part in tricarboxylic acid (TCA) cycle at room temperature storage.


Assuntos
Quitosana/farmacologia , Citrus/metabolismo , Metabolismo Energético/efeitos dos fármacos , Acetilcoenzima A/metabolismo , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Celulose/metabolismo , Citrus/química , Frutas/química , Frutas/metabolismo
18.
Methods Mol Biol ; 2240: 207-230, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33423236

RESUMO

Depletion of oxygen (O2) levels and reduction in the ATP synthesis (or even its complete blockage) are important characteristics of mitochondrial dysfunction; features that are often correlated with neurodegeneration. The measurement of oxygen consumption rate (OCR) is thus essential to evaluate cellular metabolism, survival, and neuroprotective strategies. In the present chapter, we describe the oxygen consumption assay using a Clark-type oxygen electrode in different types of samples named cells suspension (from primary and established cell culture), brain slices (ex vivo), and fresh brain tissues. In addition, we demonstrate herein how the program Oxygraph can be used in order to analyze the data and different approaches to normalize it.


Assuntos
Trifosfato de Adenosina/metabolismo , Bioensaio , Encéfalo/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Fosforilação Oxidativa , Consumo de Oxigênio , Animais , Encéfalo/efeitos dos fármacos , Linhagem Celular , Humanos , Técnicas In Vitro , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Cultura Primária de Células , Ratos , Fatores de Tempo
19.
J Neuroimmune Pharmacol ; 16(1): 48-58, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33462776

RESUMO

Declared as a global public health emergency, coronavirus disease 2019 (COVID-19) is presented as a disease of the respiratory tract, although severe cases can affect the entire organism. Several studies have shown neurological symptoms, ranging from dizziness and loss of consciousness to cerebrovascular and neurodegenerative diseases. In this context, Guillain-Barré syndrome, an immune-mediated inflammatory neuropathy, has been closely associated with critical cases of infection with "severe acute respiratory syndrome of coronavirus 2" (SARS-CoV-2), the etiological agent of COVID-19. Its pathophysiology is related to a generalized inflammation that affects the nervous system, but neurotropism was also revealed by the new coronavirus, which may increase the risk of neurological sequel, as well as the mortality of the disease. Thus, considering the comorbidities that SARS-CoV-2 infection can promote, the modulation of purinergic signaling can be applied as a potential therapy. In this perspective, given the role of adenosine triphosphate (ATP) in neural intercommunication, the P2X7 receptor (P2X7R) acts on microglia cells and its inhibition may be able to reduce the inflammatory condition of neurodegenerative diseases. Finally, alternative measures to circumvent the reality of the COVID-19 pandemic need to be considered, given the severity of critical cases and the viral involvement of multiple organs.


Assuntos
Trifosfato de Adenosina , Síndrome de Guillain-Barré/etiologia , Síndrome de Guillain-Barré/fisiopatologia , Receptores Purinérgicos , Transdução de Sinais , Humanos , Receptores Purinérgicos P2X7
20.
Mol Cell ; 81(5): 1074-1083.e5, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33453169

RESUMO

The RAD51 recombinase forms nucleoprotein filaments to promote double-strand break repair, replication fork reversal, and fork stabilization. The stability of these filaments is highly regulated, as both too little and too much RAD51 activity can cause genome instability. RADX is a single-strand DNA (ssDNA) binding protein that regulates DNA replication. Here, we define its mechanism of action. We find that RADX inhibits RAD51 strand exchange and D-loop formation activities. RADX directly and selectively interacts with ATP-bound RAD51, stimulates ATP hydrolysis, and destabilizes RAD51 nucleofilaments. The RADX interaction with RAD51, in addition to its ssDNA binding capability, is required to maintain replication fork elongation rates and fork stability. Furthermore, BRCA2 can overcome the RADX-dependent RAD51 inhibition. Thus, RADX functions in opposition to BRCA2 in regulating RAD51 nucleofilament stability to ensure the right level of RAD51 function during DNA replication.


Assuntos
Proteína BRCA2/genética , Replicação do DNA , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a RNA/genética , Rad51 Recombinase/genética , Trifosfato de Adenosina/metabolismo , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Hidrólise , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Ligação a RNA/metabolismo , Rad51 Recombinase/metabolismo , Transdução de Sinais , Imagem Individual de Molécula
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