Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 547
Filtrar
Filtros adicionais











Intervalo de ano
1.
Mol Cell Biochem ; 458(1-2): 133-142, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31093850

RESUMO

Changes in the ecto-5'-nucleotidase activity-an extracellular nucleotide catabolic enzyme may lead to the inflammation and endothelial dysfunction. We investigated the effect of CD73 deletion on the endothelial function and L-arginine metabolism in various age groups of mice. 1-,3-,6-, and 12-month-old, male C57BL/6 J wild type (WT) and C57BL/6 J CD73-/- (CD73-/-) mice were used. Blood samples were used for the analysis of adenine nucleotide concentrations. Serum samples were analyzed for the concentration of amino acids, Interleukin 6 (IL-6), Intercellular Adhesion Molecule 1 (ICAM-1), Vascular Cell Adhesion Molecule 1 (VCAM-1), and endothelial nitric oxide synthase (eNOS) level. Serum and aortic nitrate/nitrite, as well as aortic arginase and NOS activity in endothelial cells (EC) were evaluated. CD73 deletion led to age-dependent increase in IL-6, ICAM-1, and VCAM-1 concentration compared to WT. All CD73-/- mice age groups were characterized by reduced L-Arginine concentration and eNOS level. Significantly lower NOS activity was noticed in EC isolated from CD73-/- mice lungs in comparison to EC isolated from WT lungs. The L-Arginine/ADMA ratio in the CD73-/- decreased in age-dependent manner in comparison to WT. The nitrate/nitrite ratio was reduced in serum and in aortas of 6-month-old CD73-/- mice as compared to WT. The ornithine/arginine and ornithine/citrulline ratios were increased in CD73-/- compared to controls. Blood (erythrocyte) Adenosine-5'-triphosphate and Adenosine-5'-diphosphate levels were reduced in favor to higher blood Adenosine-5'-monophosphate concentration in CD73-/- mice in comparison to WT. The CD73 deletion leads to the development of age-dependent endothelial dysfunction in mice, associated with impaired L-arginine metabolism. CD73 activity seems to protect endothelium.


Assuntos
5'-Nucleotidase/deficiência , Arginina/sangue , Endotélio Vascular/metabolismo , Difosfato de Adenosina/sangue , Difosfato de Adenosina/genética , Trifosfato de Adenosina/sangue , Trifosfato de Adenosina/genética , Animais , Arginina/genética , Endotélio Vascular/patologia , Molécula 1 de Adesão Intercelular/sangue , Molécula 1 de Adesão Intercelular/genética , Interleucina-6/sangue , Interleucina-6/genética , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/sangue , Óxido Nítrico Sintase Tipo III/genética , Molécula 1 de Adesão de Célula Vascular/sangue , Molécula 1 de Adesão de Célula Vascular/genética
2.
Nat Commun ; 10(1): 711, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30755613

RESUMO

Adenosine 5' triphosphate (ATP) is a universal intracellular energy source and an evolutionarily ancient, ubiquitous extracellular signal in diverse species. Here, we report the generation and characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of F0F1-ATPase from Bacillus PS3. On the cell surface and within the cytosol, iATPSnFR1.0 responds to relevant ATP concentrations (30 µM to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. After careful consideration of their modest pH sensitivity, iATPSnFRs represent promising reagents for imaging ATP in the extracellular space and within cells during a variety of settings, and for further application-specific refinements.


Assuntos
Trifosfato de Adenosina/química , Membrana Celular/química , Citosol/química , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas/metabolismo , Trifosfato de Adenosina/genética , Bacillus/citologia , Bacillus/genética , Bacillus/metabolismo , Proteínas de Bactérias/genética , Expressão Gênica , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Processamento de Imagem Assistida por Computador , Cinética , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica
3.
Proc Natl Acad Sci U S A ; 116(2): 566-574, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30587587

RESUMO

We report a patient who presented with congenital hypotonia, hypoventilation, and cerebellar histopathological alterations. Exome analysis revealed a homozygous mutation in the initiation codon of the NME3 gene, which encodes an NDP kinase. The initiation-codon mutation leads to deficiency in NME3 protein expression. NME3 is a mitochondrial outer-membrane protein capable of interacting with MFN1/2, and its depletion causes dysfunction in mitochondrial dynamics. Consistently, the patient's fibroblasts were characterized by a slow rate of mitochondrial dynamics, which was reversed by expression of wild-type or catalytic-dead NME3. Moreover, glucose starvation caused mitochondrial fragmentation and cell death in the patient's cells. The expression of wild-type and catalytic-dead but not oligomerization-attenuated NME3 restored mitochondrial elongation. However, only wild-type NME3 sustained ATP production and viability. Thus, the separate functions of NME3 in mitochondrial fusion and NDP kinase cooperate in metabolic adaptation for cell survival in response to glucose starvation. Given the critical role of mitochondrial dynamics and energy requirements in neuronal development, the homozygous mutation in NME3 is linked to a fatal mitochondrial neurodegenerative disorder.


Assuntos
Trifosfato de Adenosina , Metabolismo Energético/genética , Homozigoto , Dinâmica Mitocondrial/genética , Nucleosídeo NM23 Difosfato Quinases , Doenças Neurodegenerativas , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Linhagem Celular , Sobrevivência Celular , Feminino , Humanos , Masculino , Mitocôndrias/enzimologia , Mitocôndrias/genética , Mitocôndrias/patologia , Nucleosídeo NM23 Difosfato Quinases/genética , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Doenças Neurodegenerativas/enzimologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia
4.
Nucleic Acids Res ; 47(3): 1404-1415, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30541105

RESUMO

ASCE ATPases include ring-translocases such as cellular helicases and viral DNA packaging motors (terminases). These motors have conserved Walker A and B motifs that bind Mg2+-ATP and a catalytic carboxylate that activates water for hydrolysis. Here we demonstrate that Glu179 serves as the catalytic carboxylate in bacteriophage λ terminase and probe its mechanistic role. All changes of Glu179 are lethal: non-conservative changes abrogate ATP hydrolysis and DNA translocation, while the conservative E179D change attenuates ATP hydrolysis and alters single molecule translocation dynamics, consistent with a slowed chemical hydrolysis step. Molecular dynamics simulations of several homologous terminases suggest a novel mechanism, supported by experiments, wherein the conserved Walker A arginine 'toggles' between interacting with a glutamate residue in the 'lid' subdomain and the catalytic glutamate upon ATP binding; this switch helps mediate a transition from an 'open' state to a 'closed' state that tightly binds nucleotide and DNA, and also positions the catalytic glutamate next to the γ-phosphate to align the hydrolysis transition state. Concomitant reorientation of the lid subdomain may mediate mechanochemical coupling of ATP hydrolysis and DNA translocation. Given the strong conservation of these structural elements in terminase enzymes, this mechanism may be universal for viral packaging motors.


Assuntos
Empacotamento do DNA/genética , DNA Viral/genética , Genoma Viral/genética , Montagem de Vírus/genética , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Arginina/genética , Arginina/metabolismo , Bacteriófago lambda/enzimologia , Catálise , Endodesoxirribonucleases/genética , Ácido Glutâmico/genética , Hidrólise , Fosfatos/metabolismo
5.
Nucleic Acids Res ; 47(3): 1428-1439, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30590734

RESUMO

Fungal tRNA ligase (Trl1) is an essential enzyme that repairs RNA breaks with 2',3'-cyclic-PO4 and 5'-OH ends inflicted during tRNA splicing and non-canonical mRNA splicing in the fungal unfolded protein response. Trl1 is composed of C-terminal cyclic phosphodiesterase (CPD) and central GTP-dependent polynucleotide kinase (KIN) domains that heal the broken ends to generate the 3'-OH,2'-PO4 and 5'-PO4 termini required for sealing by an N-terminal ATP-dependent ligase domain (LIG). Here we report crystal structures of the Trl1-LIG domain from Chaetomium thermophilum at two discrete steps along the reaction pathway: the covalent LIG-(lysyl-Nζ)-AMP•Mn2+ intermediate and a LIG•ATP•(Mn2+)2 Michaelis complex. The structures highlight a two-metal mechanism whereby a penta-hydrated metal complex stabilizes the transition state of the ATP α phosphate and a second metal bridges the ß and γ phosphates to help orient the pyrophosphate leaving group. A LIG-bound sulfate anion is a plausible mimetic of the essential RNA terminal 2'-PO4. Trl1-LIG has a distinctive C-terminal domain that instates fungal Trl1 as the founder of an Rnl6 clade of ATP-dependent RNA ligase. We discuss how the Trl1-LIG structure rationalizes the large body of in vivo structure-function data for Saccharomyces cerevisiae Trl1.


Assuntos
Chaetomium/química , DNA Ligase Dependente de ATP/química , Diester Fosfórico Hidrolases/química , Polinucleotídeo 5'-Hidroxiquinase/química , Polinucleotídeo Ligases/química , Relação Estrutura-Atividade , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Sequência de Aminoácidos , Domínio Catalítico , Chaetomium/enzimologia , Cristalografia por Raios X , DNA Ligase Dependente de ATP/genética , Metais/química , Diester Fosfórico Hidrolases/genética , Polinucleotídeo 5'-Hidroxiquinase/genética , Polinucleotídeo Ligases/genética , Conformação Proteica , Domínios Proteicos , Processamento de RNA/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia
6.
Med Arch ; 72(2): 148-150, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29736106

RESUMO

Introduction: Arteriovenous Pearson syndrome is a very rare multisystemic mitochondrial disease characterized by sideroblastic anemia and exocrine pancreatic insufficiency. It is usually fatal in infancy. Case report: We reported a four-month-old infant presented with fever and pancytopenia. Bone marrow examination showed hypoplastic changes and sideroblastic features. Molecular Study showed a novel hetroplasmic mitochondrial deletions (m. 10760 -m. 15889+) in multiple genes (ND4,ND5,ND6, CYTB). In our patient the pathogenic mutation was 5.1 kb heteroplasmic deletions in multiple genes that are important and crucial for intact oxidative phosphorylation pathway and ATP production in the mitochondrial DNA. This mutation was not reported in literature including the mitomap.org website (which was last edited on Nov 30, 2017 and accessed on Jan 13, 2018).


Assuntos
Trifosfato de Adenosina/biossíntese , Trifosfato de Adenosina/genética , DNA Mitocondrial/genética , Insuficiência Pancreática Exócrina/genética , Doenças Mitocondriais/genética , Deleção de Sequência , Evolução Fatal , Feminino , Humanos , Lactente , Fosforilação Oxidativa , Tunísia
7.
Drug Metab Dispos ; 46(5): 628-635, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29437875

RESUMO

The identification of drug transporters expressed in human skin and interindividual differences in gene expression is important for understanding the role of drug transporters in human skin. In the present study, we evaluated the expression of ATP-binding cassette (ABC) and solute carrier (SLC) transporters using human skin tissues. In skin samples, ABCC3 was expressed at the highest levels, followed by SLCO3A1, SLC22A3, SLC16A7, ABCA2, ABCC1, and SLCO2B1. Among the quantitated transporters, ABCC3 accounted for 20.0% of the total mean transporter mRNA content. The expression of ABCC3 mRNA showed large interindividual variability (9.5-fold). None of the single nucleotide polymorphisms tested (-1767G>A, -1328G>A, -1213C>G, -897delC, -260T>A, and -211C>T) in the promoter region of the ABCC3 gene showed a significant change in ABCC3 mRNA levels. ABCC3 expression levels negatively correlated with the methylation status of the CpG island (CGI) located approximately 10 kilobase pairs upstream of ABCC3 (Rs: -0.323, P < 0.05). The reporter gene assay revealed a significant increase in transcriptional activity in the presence of CGI. ABCC3 mRNA was upregulated in HaCaT cells by the demethylating agent 5-aza-2'-deoxycytidine. Furthermore, the deletion of the region surrounding CGI using the clustered regularly interspaced short palindromic repeat/Cas9 system resulted in significantly lower ABCC3 mRNA levels than those in control clones in HaCaT cells. Herein, we demonstrated large interindividual differences in the expression of drug transporters in human skin. CGI may function as an enhancer of the transcription of ABCC3, and methylation levels in CGI contribute to the variability of ABCC3 expression in human skin.


Assuntos
Metilação de DNA/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Pele/metabolismo , Transcrição Genética/genética , Trifosfato de Adenosina/genética , Adulto , Transporte Biológico/genética , Ilhas de CpG/genética , Expressão Gênica/genética , Humanos , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Transportadores de Ácidos Monocarboxílicos/genética , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Adulto Jovem
8.
Biochim Biophys Acta Bioenerg ; 1859(5): 319-325, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29470949

RESUMO

F1 is a soluble part of FoF1-ATP synthase and performs a catalytic process of ATP hydrolysis and synthesis. The γ subunit, which is the rotary shaft of F1 motor, is composed of N-terminal and C-terminal helices domains, and a protruding Rossman-fold domain located between the two major helices parts. The N-terminal and C-terminal helices domains of γ assemble into an antiparallel coiled-coil structure, and are almost embedded into the stator ring composed of α3ß3 hexamer of the F1 molecule. Cyanobacterial and chloroplast γ subunits harbor an inserted sequence of 30 or 39 amino acids length within the Rossman-fold domain in comparison with bacterial or mitochondrial γ. To understand the structure-function relationship of the γ subunit, we prepared a mutant F1-ATP synthase of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, in which the γ subunit is split into N-terminal α-helix along with the inserted sequence and the remaining C-terminal part. The obtained mutant showed higher ATP-hydrolysis activities than those containing the wild-type γ. Contrary to our expectation, the complexes containing the split γ subunits were mostly devoid of the C-terminal helix. We further investigated the effect of post-assembly cleavage of the γ subunit. We demonstrate that insertion of the nick between two helices of the γ subunit imparts resistance to ADP inhibition, and the C-terminal α-helix is dispensable for ATP-hydrolysis activity and plays a crucial role in the assembly of F1-ATP synthase.


Assuntos
Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Cianobactérias/enzimologia , ATPases Translocadoras de Prótons/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cianobactérias/genética , Domínios Proteicos , Estrutura Secundária de Proteína , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Deleção de Sequência
9.
Sci Rep ; 8(1): 903, 2018 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-29343701

RESUMO

The enzyme Pantothenate synthetase (PS) represents a potential drug target in Mycobacterium tuberculosis. Its X-ray crystallographic structure has demonstrated the significance and importance of conserved active site residues including His44, His47, Asn69, Gln72, Lys160 and Gln164 in substrate binding and formation of pantoyl adenylate intermediate. In the current study, molecular mechanism of decreased affinity of the enzyme for ATP caused by alanine mutations was investigated using molecular dynamics (MD) simulations and free energy calculations. A total of seven systems including wild-type + ATP, H44A + ATP, H47A + ATP, N69A + ATP, Q72A + ATP, K160A + ATP and Q164A + ATP were subjected to 50 ns MD simulations. Docking score, MM-GBSA and interaction profile analysis showed weak interactions between ATP (substrate) and PS (enzyme) in H47A and H160A mutants as compared to wild-type, leading to reduced protein catalytic activity. However, principal component analysis (PCA) and free energy landscape (FEL) analysis revealed that ATP was strongly bound to the catalytic core of the wild-type, limiting its movement to form a stable complex as compared to mutants. The study will give insight about ATP binding to the PS at the atomic level and will facilitate in designing of non-reactive analogue of pantoyl adenylate which will act as a specific inhibitor for PS.


Assuntos
Trifosfato de Adenosina/metabolismo , Alanina/genética , Sítios de Ligação/genética , Domínio Catalítico/genética , Mutação/genética , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Monofosfato de Adenosina/genética , Trifosfato de Adenosina/genética , Catálise , Cristalografia por Raios X/métodos , Cinética , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/genética , Ligação Proteica/genética , Especificidade por Substrato
10.
Sci Rep ; 8(1): 949, 2018 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-29343702

RESUMO

We previously established human induced pluripotent stem (iPS) cells in two diabetic patients from different families with the mitochondrial A3243G mutation and isolated isogenic iPS cell clones with either undetectable or high levels of the mutation in both patients. In the present study, we analyzed the mitochondrial functions of two mutation-undetectable and two mutation-high clones in each patient through four methods to assess complex I activity, mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production. In the first patient, complex I activity, mitochondrial respiration, and mitochondrial ATP production were decreased in the mutation-high clones compared with the mutation-undetectable clones, and mitochondrial membrane potential was decreased in a mutation-high clone compared with a mutation-undetectable clone. In the second patient, complex I activity was decreased in one mutation-high clone compared with the other clones. The other parameters showed no differences in any clones. In addition, the complex I activity and mitochondrial respiration of the mutation-undetectable clones from both patients were located in the range of those of iPS cells from healthy subjects. The present study suggests that the mitochondrial function of the mutation-undetectable iPS cell clones obtained from two patients with the A3243G mutation is comparable to the control iPS cells.


Assuntos
DNA Mitocondrial/genética , Diabetes Mellitus/genética , Diabetes Mellitus/fisiopatologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Mitocôndrias/fisiologia , Mutação/genética , Trifosfato de Adenosina/genética , Adulto , Linhagem Celular , Feminino , Humanos , Masculino , Potencial da Membrana Mitocondrial/genética , Potencial da Membrana Mitocondrial/fisiologia , Pessoa de Meia-Idade
11.
Microb Pathog ; 115: 321-331, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29306008

RESUMO

Bacterial non-coding RNAs (ncRNAs), as important regulatory factors, are involved in many cellular processes, including virulence and protection against environmental stress. The 5' untranslated region (UTR) of malS (named malS-5'UTR), a regulatory ncRNA, increases the invasive capacity and influences histidine biosynthesis in Salmonella enterica serovar Typhi (S. Typhi). In this study, we found that overexpression of the malS-5'UTR decreased S. Typhi survival within macrophages. A microarray analysis of a strain overexpressing the malS-5'UTR revealed a significant increase in the mRNA levels of the atp operon. The intracellular ATP levels were elevated in the malS-5'UTR overexpression strain. Quantitative real-time polymerase chain reaction results showed that the malS-5'UTR downregulated the mRNA levels of phoP, phoQ, and mgtC. MgtC, its expression is regulated by PhoP/PhoQ two-component regulatory system, inhibits the F1F0 ATP synthase, thereby preventing the accumulation of ATP to non-physiological levels and the acidification of the cytoplasm within macrophages. Thus, we propose that the malS-5'UTR weakens the ability of S. Typhi to survive in macrophages, probably because of the accumulation of ATP within macrophages, by regulating the mRNA levels of mgtC and the atp operon in a phoP-dependent manner.


Assuntos
Regiões 5' não Traduzidas/genética , Trifosfato de Adenosina/metabolismo , Amilases/genética , Regulação Bacteriana da Expressão Gênica/genética , Macrófagos/microbiologia , Salmonella typhi/crescimento & desenvolvimento , Trifosfato de Adenosina/genética , Amilases/biossíntese , Proteínas de Bactérias/genética , Proteínas de Transporte de Cátions/genética , Linhagem Celular Tumoral , Regulação para Baixo/genética , Histidina/biossíntese , Humanos , Células THP-1
12.
Biochim Biophys Acta Bioenerg ; 1859(2): 99-109, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29097244

RESUMO

The physiological role of the mitochondrial ATP synthase complex is to generate ATP through oxidative phosphorylation. Indeed, the enzyme can reverse its activity and hydrolyze ATP under ischemic conditions, as shown in isolated mitochondria and in mammalian heart and liver. However, what occurs when cancer cells experience hypoxia or anoxia has not been well explored. In the present study, we investigated the bioenergetics of cancer cells under hypoxic/anoxic conditions with particular emphasis on ATP synthase, and the conditions driving it to work in reverse. In this context, we further examined the role exerted by its endogenous inhibitor factor, IF1, that it is overexpressed in cancer cells. Metabolic and bioenergetic analysis of cancer cells exposed to severe hypoxia (down to 0.1% O2) unexpectedly showed that Δψm is preserved independently of the presence of IF1 and that ATP synthase still phosphorylates ADP though at a much lower rate than in normoxia. However, when we induced an anoxia-mimicking condition by collapsing ΔµΗ+ with the FCCP uncoupler, the IF1-silenced clones only reversed the ATP synthase activity hydrolyzing ATP in order to reconstitute the electrochemical proton gradient. Notably, in cancer cells IF1 overexpression fully prevents ATP synthase hydrolytic activity activation under uncoupling conditions. Therefore, our results suggest that IF1 overexpression promotes cancer cells survival under temporary anoxic conditions by preserving cellular ATP despite mitochondria dysfunction.


Assuntos
Adaptação Fisiológica , Mitocôndrias/metabolismo , Membranas Mitocondriais , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteínas/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Mitocôndrias/genética , Mitocôndrias/patologia , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Proteínas/genética
13.
Balkan Med J ; 35(3): 272-274, 2018 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-29219112

RESUMO

Background: Ovotesticular disorder is characterized by the presence of testicular and ovarian tissues in the same individual. Single gene mutations in SRY, SOX9, DMRT1 and DAX1 can lead to ovotesticular disorder of sexual development. Case Report: Herein, we report a 3-month-old phenotypically female baby in whom differentiated tissues of both Müllerian and Wolffian ducts were detected on pathological analysis of laparoscopic biopsy material. Chromosomal analysis observed 46,XY, der(9)t(3;9)(p25;p24) with deletion of 9p24.3p23 including the DMRT gene cluster and duplication of 3p26.3p24.3 on array comparative genomic hybridisation. Conclusion: In support of previous literature, we found that haploinsufficiency of the DMRT gene cluster leads to ovotesticular disorder of sexual development. In addition, we emphasize that array comparative genomic hybridisation is an important technique in the molecular diagnosis of ovotesticular disorder of sexual.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Haploinsuficiência/genética , Transtornos Ovotesticulares do Desenvolvimento Sexual/genética , Tionucleosídeos/genética , Trifosfato de Adenosina/genética , Feminino , Disgenesia Gonadal 46 XY/diagnóstico , Disgenesia Gonadal 46 XY/genética , Humanos , Hibridização in Situ Fluorescente , Lactente , Masculino , Família Multigênica , Transtornos Ovotesticulares do Desenvolvimento Sexual/diagnóstico , Fatores de Transcrição SOX9 , Testículo
14.
Stroke ; 49(1): 165-174, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29212737

RESUMO

BACKGROUND AND PURPOSE: Lrp4 (low-density lipoprotein receptor-related protein 4) is predominantly expressed in astrocytes, where it regulates glutamatergic neurotransmission by suppressing ATP release. Here, we investigated Lrp4's function in ischemia/stroke-induced brain injury response, which includes glutamate-induced neuronal death and reactive astrogliosis. METHODS: The brain-specific Lrp4 conditional knockout mice (Lrp4GFAP-Cre), astrocytic-specific Lrp4 conditional knockout mice (Lrp4GFAP-creER), and their control mice (Lrp4f/f) were subjected to photothrombotic ischemia and the transient middle cerebral artery occlusion. After ischemia/stroke, mice or their brain samples were subjected to behavior tests, brain histology, immunofluorescence staining, Western blot, and quantitative real-time polymerase chain reaction. In addition, primary astrocytes and neurons were cocultured with or without oxygen and glucose deprivation and in the presence or absence of the antagonist for adenosine-A2AR (adenosine A2A receptor) or ATP-P2X7R (P2X purinoceptor 7) signaling. Gliotransmitters, such as glutamate, d-serine, ATP, and adenosine, in the condition medium of cultured astrocytes were also measured. RESULTS: Lrp4, largely expressed in astrocytes, was increased in response to ischemia/stroke. Both Lrp4GFAP-Cre and Lrp4GFAP-creER mice showed less brain injury, including reduced neuronal death, and impaired reactive astrogliosis. Mechanistically, Lrp4 conditional knockout in astrocytes increased ATP release and the production of ATP derivative, adenosine, which were further elevated by oxygen and glucose deprivation. Pharmacological inhibition of ATP-P2X7R or adenosine-A2AR signaling diminished Lrp4GFAP-creER's protective effect. CONCLUSIONS: The astrocytic Lrp4 plays an important role in ischemic brain injury response. Lrp4 deficiency in astrocytes seems to be protective in response to ischemic brain injury, likely because of the increased ATP release and adenosine-A2AR signaling.


Assuntos
Trifosfato de Adenosina/metabolismo , Astrócitos/metabolismo , Lesões Encefálicas/metabolismo , Isquemia Encefálica/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptores de LDL/metabolismo , Transdução de Sinais , Trifosfato de Adenosina/genética , Animais , Astrócitos/patologia , Lesões Encefálicas/genética , Lesões Encefálicas/patologia , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Camundongos , Camundongos Knockout , Receptor A2A de Adenosina/genética , Receptores de LDL/genética , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo
15.
Biochim Biophys Acta Biomembr ; 1860(1): 166-173, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28389204

RESUMO

Pannexins are a three-member family of vertebrate plasma membrane spanning molecules that have homology to the invertebrate gap junction forming proteins, the innexins. However, pannexins do not form gap junctions but operate as plasma membrane channels. The best-characterized member of these proteins, Pannexin1 (Panx1) was suggested to be functionally associated with purinergic P2X and N-methyl-D-aspartate (NMDA) receptor channels. Activation of these receptor channels by their endogenous ligands leads to cross-activation of Panx1 channels. This in turn potentiates P2X and NMDA receptor channel signaling. Two potentiation concepts have been suggested: enhancement of the current responses and/or sustained receptor channel activation by ATP released through Panx1 pore and adenosine generated by ectonucleotidase-dependent dephosphorylation of ATP. Here we summarize the current knowledge and hypotheses about interactions of Panx1 channels with P2X and NMDA receptor channels. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.


Assuntos
Trifosfato de Adenosina/metabolismo , Adenosina/metabolismo , Conexinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Purinérgicos P2X/metabolismo , Trifosfato de Adenosina/genética , Animais , Conexinas/genética , Humanos , Proteínas do Tecido Nervoso/genética , Receptores de N-Metil-D-Aspartato/genética , Receptores Purinérgicos P2X/genética
16.
Trends Cancer ; 3(12): 857-870, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29198441

RESUMO

Until recently, the dual roles of mitochondria in ATP production (bioenergetics) and apoptosis (cell life/death decision) were thought to be separate. New evidence points to a more intimate link between these two functions, mediated by the remodeling of the mitochondrial ultrastructure during apoptosis. While most of the key molecular players that regulate this process have been identified (primarily membrane proteins), the exact mechanisms by which they function are not yet understood. Because resistance to apoptosis is a hallmark of cancer, and because ultimately all chemotherapies are believed to result directly or indirectly in induction of apoptosis, a better understanding of the biophysical processes involved may lead to new avenues for therapy.


Assuntos
Apoptose/genética , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Metabolismo Energético/genética , Humanos , Mitocôndrias/patologia , Membranas Mitocondriais/metabolismo , Neoplasias/genética , Neoplasias/patologia
17.
Adv Exp Med Biol ; 1038: 149-171, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29178075

RESUMO

Mitochondria are generally considered as a powerhouse in a cell where the majority of the cellular ATP and metabolite productions occur. Metabolic rewiring and reprogramming may be initiated and regulated by mitochondrial enzymes. The hypothesis that cellular metabolic rewiring and reprogramming processes may occur as cellular microenvironment is disturbed, resulting in alteration of cell phenotype, such as cancer cells resistant to therapeutics seems to be now acceptable. Cancer metabolic reprogramming regulated by mitochondrial enzymes is now one of the hallmarks of cancer. This chapter provides an overview of cancer metabolism and summarizes progress made in mitochondria-mediated metabolic regulation in cancer drug resistance.


Assuntos
Trifosfato de Adenosina , Resistencia a Medicamentos Antineoplásicos/genética , Mitocôndrias , Neoplasias , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia
18.
Adv Exp Med Biol ; 1051: 107-122, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29134605

RESUMO

The P2Y11 receptor is a G protein-coupled receptor that is stimulated by endogenous purine nucleotides, particularly ATP. Amongst P2Y receptors it has several unique properties; (1) it is the only human P2Y receptor gene that contains an intron in the coding sequence; (2) the gene does not appear to be present in the rodent genome; (3) it couples to stimulation of both phospholipase C and adenylyl cyclase. Its absence in mice and rats, along with a limited range of selective pharmacological tools, has hampered the development of our knowledge and understanding of its properties and functions. Nonetheless, through a combination of careful use of the available tools, suppression of receptor expression using siRNA and genetic screening for SNPs, possible functions of native P2Y11 receptors have been identified in a variety of human cells and tissues. Many are in blood cells involved in inflammatory responses, consistent with extracellular ATP being a damage-associated signalling molecule in the immune system. Thus proposed potential therapeutic applications relate, in the main, to modulation of acute and chronic inflammatory responses.


Assuntos
Trifosfato de Adenosina/metabolismo , Fases de Leitura Aberta , Polimorfismo de Nucleotídeo Único , Receptores Purinérgicos P2 , Trifosfato de Adenosina/genética , Animais , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Íntrons , Camundongos , Especificidade de Órgãos , Ratos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Especificidade da Espécie
19.
Am J Hum Genet ; 101(5): 833-843, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29100093

RESUMO

Gorlin-Chaudhry-Moss syndrome (GCMS) is a dysmorphic syndrome characterized by coronal craniosynostosis and severe midface hypoplasia, body and facial hypertrichosis, microphthalmia, short stature, and short distal phalanges. Variable lipoatrophy and cutis laxa are the basis for a progeroid appearance. Using exome and genome sequencing, we identified the recurrent de novo mutations c.650G>A (p.Arg217His) and c.649C>T (p.Arg217Cys) in SLC25A24 in five unrelated girls diagnosed with GCMS. Two of the girls had pronounced neonatal progeroid features and were initially diagnosed with Wiedemann-Rautenstrauch syndrome. SLC25A24 encodes a mitochondrial inner membrane ATP-Mg/Pi carrier. In fibroblasts from affected individuals, the mutated SLC25A24 showed normal stability. In contrast to control cells, the probands' cells showed mitochondrial swelling, which was exacerbated upon treatment with hydrogen peroxide (H2O2). The same effect was observed after overexpression of the mutant cDNA. Under normal culture conditions, the mitochondrial membrane potential of the probands' fibroblasts was intact, whereas ATP content in the mitochondrial matrix was lower than that in control cells. However, upon H2O2 exposure, the membrane potential was significantly elevated in cells harboring the mutated SLC25A24. No reduction of mitochondrial DNA copy number was observed. These findings demonstrate that mitochondrial dysfunction with increased sensitivity to oxidative stress is due to the SLC25A24 mutations. Our results suggest that the SLC25A24 mutations induce a gain of pathological function and link mitochondrial ATP-Mg/Pi transport to the development of skeletal and connective tissue.


Assuntos
Anormalidades Múltiplas/genética , Antiporters/genética , Proteínas de Ligação ao Cálcio/genética , Anormalidades Craniofaciais/genética , Craniossinostoses/genética , Permeabilidade do Canal Arterial/genética , Hipertricose/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Mutação/genética , Trifosfato de Adenosina/genética , Adolescente , Criança , Pré-Escolar , Cútis Laxa/genética , DNA Mitocondrial/genética , Exoma/genética , Feminino , Retardo do Crescimento Fetal/genética , Fibroblastos/patologia , Humanos , Peróxido de Hidrogênio/farmacologia , Lactente , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/genética , Progéria/genética
20.
Adv Exp Med Biol ; 1051: 233-253, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29064066

RESUMO

Adenosine 5'-triphosphate (ATP), a ubiquitously dispersed biomolecule, is not only a major source of biochemical energy for living cells, but also acts as a critical signaling molecule through inter-cellular communication. Recent studies have clearly shown that extracellular ATP is involved in various physiological processes in plants, including root growth, stomata movement, pollen tube development, gravitropism, and abiotic/biotic stress responses. The first plant purinergic receptor for extracellular ATP, DORN1 (the founding member of the P2K family of purinergic receptors), was identified in Arabidopsis thaliana by a forward genetic screen. DORN1 consists of an extracellular lectin domain, transmembrane domain, and serine/threonine kinase, intracellular domain. The predicted structure of the DORN1 extracellular domain revealed putative key ATP binding residues but an apparent lack of sugar binding. In this chapter, we summarize recent studies on the molecular mechanism of plant recognition of extracellular ATP with specific reference to the role of DORN1.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Desenvolvimento Vegetal/fisiologia , Proteínas Quinases/metabolismo , Estresse Fisiológico , Trifosfato de Adenosina/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Domínios Proteicos , Proteínas Quinases/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA