Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 16 de 16
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Chem Phys ; 151(19): 195101, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31757137

RESUMO

The solid-state photochemically induced dynamic nuclear polarization (photo-CIDNP) effect has been studied in a quinone-depleted uniformly (u-)13C,15N-labeled photosynthetic reaction center (RC) protein from purple bacterium Rhodobacter (R.) sphaeroides wild type (WT). As a method for investigation, solid-state 15N NMR under magic-angle spinning (MAS) is applied under both continuous illumination (steady state) and nanosecond-laser flashes (time-resolved). While all previous 15N photo-CIDNP MAS NMR studies on the purple bacterial RC used the carotenoid-less mutant R26, this is the first using WT samples. The absence of further photo-CIDNP mechanisms (compared to R26) and various couplings (compared to 13C NMR experiments on 13C-labeled samples) allows the simplification of the spin-system. We report 15N signals of the three cofactors forming the spin-correlated radical pair (SCRP) and, based on density-functional theory calculations, their assignment. The simulation of photo-CIDNP intensities and time-resolved 15N photo-CIDNP MAS NMR data matches well to the frame of the mechanistic interpretation. Three spin-chemical processes, namely, radical pair mechanism, three spin mixing, and differential decay, generate emissive (negative) 15N polarization in the singlet decay channel and absorptive (positive) polarization in the triplet decay channel of the SCRP. The absorptive 15N polarization of the triplet decay channel is transiently obscured during the lifetime of the triplet state of the carotenoid (3Car); therefore, the observed 15N signals are strongly emissive. Upon decay of 3Car, the transiently obscured polarization becomes visible by reducing the excess of emissive polarization. After the decline of 3Car, the remaining nuclear hyperpolarization decays with nuclear T1 relaxation kinetics.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Complexo de Proteínas do Centro de Reação Fotossintética/química , Rhodobacter sphaeroides/metabolismo , Modelos Moleculares , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Conformação Proteica
2.
J Alzheimers Dis ; 69(2): 363-375, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30958376

RESUMO

In Alzheimer's disease (AD), disturbances in the circadian rhythm and sleep-wake cycle are frequently observed. Both are controlled by the master clock: the suprachiasmatic nucleus (SCN), which was reported in postmortem studies of AD subjects to be compromised. However, the influence of age and gender on the biophysical integrity and subtle microstructural changes of SCN and mechanistic connections between SCN dysfunction and AD progression in vivo remain to be explored. In the present study, we utilized state-of-the-art in vivo magnetic resonance relaxation measurements in combination with immunohistochemistry to follow microstructural changes in SCN of the Tg2576 mouse model of AD. Longitudinal monitoring of in vivo T2 relaxation with age shows significant shortening of T2 values in the SCN of transgenic mice and more substantially in female transgenic than aged-matched controls. Multiexponential T2 analysis detected a unique long T2 component in SCN of transgenic mice which was absent in wild-type mice. Immunohistochemical examination revealed significantly elevated numbers of activated astrocytes and an increase in the astrocyte to neuron ratio in SCN of transgenic compared to wild-type mice. This increase was more substantial in female than in male transgenic mice. In addition, low GABA production in SCN of transgenic mice was detected. Our results offer a brief appraisal of SCN dysfunction in AD and demonstrate that inflammatory responses may be an underlying perpetrator for the changes in circadian rhythmicity and sleep disturbance in AD and could also be at the root of marked sex disparities observed in AD subjects.

3.
J Biol Chem ; 293(17): 6482-6496, 2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29514982

RESUMO

DNA interstrand cross-links (ICLs) block the progress of the replication and transcription machineries and can weaken chromosomal stability, resulting in various diseases. FANCD2-FANCI-associated nuclease (FAN1) is a conserved structure-specific nuclease that unhooks DNA ICLs independently of the Fanconi anemia pathway. Recent structural studies have proposed two different mechanistic features for ICL unhooking by human FAN1: a specific basic pocket that recognizes the terminal phosphate of a 1-nucleotide (nt) 5' flap or FAN1 dimerization. Herein, we show that despite lacking these features, Pseudomonas aeruginosa FAN1 (PaFAN1) cleaves substrates at ∼3-nt intervals and resolves ICLs. Crystal structures of PaFAN1 bound to various DNA substrates revealed that its conserved basic Arg/Lys patch comprising Arg-228 and Lys-260 recognizes phosphate groups near the 5' terminus of a DNA substrate with a 1-nt flap or a nick. Substitution of Lys-260 did not affect PaFAN1's initial endonuclease activity but significantly decreased its subsequent exonuclease activity and ICL unhooking. The Arg/Lys patch also interacted with phosphates at a 3-nt gap, and this interaction could drive movement of the scissile phosphates into the PaFAN1-active site. In human FAN1, the ICL-resolving activity was not affected by individual disruption of the Arg/Lys patch or basic pocket. However, simultaneous substitution of both FAN1 regions significantly reduced its ICL-resolving activity, suggesting that these two basic regions play a complementary role in ICL repair. On the basis of these findings, we propose a conserved role for two basic regions in FAN1 to guide ICL unhooking and to maintain genomic stability.


Assuntos
Proteínas de Bactérias/química , Desoxirribonuclease I/química , Simulação de Dinâmica Molecular , Pseudomonas aeruginosa/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Domínios Proteicos , Pseudomonas aeruginosa/genética , Relação Estrutura-Atividade
4.
Neurobiol Aging ; 62: 168-179, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29154037

RESUMO

Age and sex are risk factors of Alzheimer's disease (AD). Among the neurotransmitter systems, gamma-aminobutyric acid (GABA) has been implicated in AD pathogenesis but the relevance of sex-specific GABAergic dysfunction during AD progression remains unknown. In the present study, we utilized state-of-the-art high-resolution magic angle spinning nuclear magnetic resonance to systematically monitor the brain region-, age-, and sex-specific modulation of GABA levels in wild-type and Tg2576 mice with amyloid pathology. In addition, we followed the possible role of reactive astrocytes in sex-specific GABA modulation. In female Tg2576 mice, hippocampal GABA levels were significantly elevated, along with higher number of reactive astrocytes and amyloid deposition. The elevated GABA was found to be produced via the monoamine oxidase-B route from putrescine in reactive astrocytes, more substantially in female than male mice, thus suggesting a role of astrocytes in memory impairment and sex-related differences in AD. Our results paint a coherent model of memory impairment in AD and signify that dynamic changes in regional GABA may be at the root of marked sex disparities observed in AD.


Assuntos
Envelhecimento/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Caracteres Sexuais , Ácido gama-Aminobutírico/metabolismo , Doença de Alzheimer/patologia , Proteínas Amiloidogênicas/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Astrócitos/fisiologia , Modelos Animais de Doenças , Feminino , Estudos Longitudinais , Espectroscopia de Ressonância Magnética/métodos , Masculino , Transtornos da Memória/genética , Transtornos da Memória/metabolismo , Transtornos da Memória/patologia , Camundongos Transgênicos , Monoaminoxidase/metabolismo , Putrescina/metabolismo , Fatores de Risco
5.
Sci Rep ; 7(1): 17305, 2017 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-29230019

RESUMO

ß-methylamino-L-alanine (BMAA) has been linked to several interrelated neurodegenerative diseases. Despite considerable research, specific contributions of BMAA toxicity to neurodegenerative diseases remain to be fully resolved. In the present study, we utilized state-of-the-art high-resolution magic-angle spinning nuclear magnetic resonance (HRMAS NMR), applied to intact zebrafish (Danio rerio) embryos, as a model of vertebrate development, to elucidate changes in metabolic profiles associated with BMAA exposure. Complemented by several alternative analytical approaches (i.e., in vivo visualization and in vitro assay), HRMAS NMR identified robust and dose-dependent effect of BMAA on several relevant metabolic pathways suggesting a multifaceted toxicity of BMAA including: (1) localized production of reactive oxygen species (ROS), in the developing brain, consistent with excitotoxicity; (2) decreased protective capacity against excitotoxicity and oxidative stress including reduced taurine and glutathione; (3) inhibition of several developmentally stereotypical energetic and metabolic transitions, i.e., metabolic reprogramming; and (4) inhibition of lipid biosynthetic pathways. Matrix-assisted laser desorption time-of-flight (MALDI-ToF) mass spectrometry further identified specific effects on phospholipids linked to both neural development and neurodegeneration. Taken together, a unified model of the neurodevelopmental toxicity of BMAA in the zebrafish embryo is presented in relation to the potential contribution of BMAA to neurodegenerative disease.


Assuntos
Diamino Aminoácidos/toxicidade , Embrião não Mamífero/patologia , Agonistas de Aminoácidos Excitatórios/toxicidade , Espectroscopia de Ressonância Magnética/métodos , Metaboloma/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Espectrometria de Massas , Metabolômica , Peixe-Zebra/metabolismo
6.
Cell Rep ; 21(5): 1375-1385, 2017 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-29091773

RESUMO

DNA interstrand crosslinks (ICLs) that are repaired in non-dividing cells must be recognized independently of replication-associated DNA unwinding. Using cell-free extracts from Xenopus eggs that support neither replication nor transcription, we establish that ICLs are recognized and processed by the mismatch repair (MMR) machinery. We find that ICL repair requires MutSα (MSH2-MSH6) and the mismatch recognition FXE motif in MSH6, strongly suggesting that MutSα functions as an ICL sensor. MutSα recruits MutLα and EXO1 to ICL lesions, and the catalytic activity of both these nucleases is essential for ICL repair. As anticipated for a DNA unwinding-independent recognition process, we demonstrate that least distorting ICLs fail to be recognized and repaired by the MMR machinery. This establishes that ICL structure is a critical determinant of repair efficiency outside of DNA replication.


Assuntos
Reparo de Erro de Pareamento de DNA/fisiologia , DNA/metabolismo , Animais , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Proteínas MutL/metabolismo , Oócitos/metabolismo , Xenopus/crescimento & desenvolvimento , Proteínas de Xenopus/metabolismo
7.
Methods Enzymol ; 591: 415-431, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28645378

RESUMO

Nitrogen mustards (NMs) react with two bases on opposite strands of a DNA duplex to form a covalent linkage, yielding adducts called DNA interstrand cross-links (ICLs). This prevents helix unwinding, blocking essential processes such as replication and transcription. Accumulation of ICLs causes cell death in rapidly dividing cells, especially cancer cells, making ICL-forming agents like NMs valuable in chemotherapy. However, the repair of ICLs can contribute to chemoresistance through a number of pathways that remain poorly understood. One of the impediments in studying NM ICL repair mechanisms has been the difficulty of generating site-specific and stable NM ICLs. Here, we describe two methods to synthesize stable NM ICL analogs that make it possible to study DNA ICL repair. As a proof of principle of the suitability of these NM ICLs for biochemical and cell biological studies, we use them in primer extension assays with Klenow polymerase. We show that the NM ICL analogs block the polymerase activity and remain intact under our experimental conditions.


Assuntos
Reagentes para Ligações Cruzadas/química , DNA/química , Mecloretamina/química , Eletroforese em Gel de Poliacrilamida
8.
PLoS Genet ; 13(5): e1006813, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28542210

RESUMO

Double strand breaks (DSBs) and interstrand crosslinks (ICLs) are toxic DNA lesions that can be repaired through multiple pathways, some of which involve shared proteins. One of these proteins, DNA Polymerase θ (Pol θ), coordinates a mutagenic DSB repair pathway named microhomology-mediated end joining (MMEJ) and is also a critical component for bypass or repair of ICLs in several organisms. Pol θ contains both polymerase and helicase-like domains that are tethered by an unstructured central region. While the role of the polymerase domain in promoting MMEJ has been studied extensively both in vitro and in vivo, a function for the helicase-like domain, which possesses DNA-dependent ATPase activity, remains unclear. Here, we utilize genetic and biochemical analyses to examine the roles of the helicase-like and polymerase domains of Drosophila Pol θ. We demonstrate an absolute requirement for both polymerase and ATPase activities during ICL repair in vivo. However, similar to mammalian systems, polymerase activity, but not ATPase activity, is required for ionizing radiation-induced DSB repair. Using a site-specific break repair assay, we show that overall end-joining efficiency is not affected in ATPase-dead mutants, but there is a significant decrease in templated insertion events. In vitro, Pol θ can efficiently bypass a model unhooked nitrogen mustard crosslink and promote DNA synthesis following microhomology annealing, although ATPase activity is not required for these functions. Together, our data illustrate the functional importance of the helicase-like domain of Pol θ and suggest that its tethering to the polymerase domain is important for its multiple functions in DNA repair and damage tolerance.


Assuntos
Domínio Catalítico , Reparo do DNA por Junção de Extremidades , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Animais , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/genética , DNA Polimerase Dirigida por DNA , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética
9.
Zebrafish ; 13(5): 456-65, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27348393

RESUMO

Techniques based on nuclear magnetic resonance (NMR) for imaging and chemical analyses of in vivo, or otherwise intact, biological systems are rapidly emerging and finding diverse applications within a wide range of fields. Very recently, several NMR-based techniques have been developed for the zebrafish as a model animal system. In the current study, the novel application of high-resolution magic angle spinning (HR-MAS) NMR is presented as a means of metabolic profiling of intact zebrafish embryos. Toward investigating the utility of HR-MAS NMR as a toxicological tool, these studies specifically examined metabolic changes of embryos exposed to polymethoxy-1-alkenes (PMAs)-a recently identified family of teratogenic compounds from freshwater algae-as emerging environmental contaminants. One-dimensional and two-dimensional HR-MAS NMR analyses were able to effectively identify and quantify diverse metabolites in early-stage (≤36 h postfertilization) embryos. Subsequent comparison of the metabolic profiles between PMA-exposed and control embryos identified several statistically significant metabolic changes associated with subacute exposure to the teratogen, including (1) elevated inositol as a recognized component of signaling pathways involved in embryo development; (2) increases in several metabolites, including inositol, phosphoryl choline, fatty acids, and cholesterol, which are associated with lipid composition of cell membranes; (3) concomitant increase in glucose and decrease in lactate; and (4) decreases in several biochemically related metabolites associated with central nervous system development and function, including γ-aminobutyric acid, glycine, glutamate, and glutamine. A potentially unifying model/hypothesis of PMA teratogenicity based on the data is presented. These findings, taken together, demonstrate that HR-MAS NMR is a promising tool for metabolic profiling in the zebrafish embryo, including toxicological applications.


Assuntos
Alcenos/toxicidade , Teratogênios/toxicidade , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Animais , Embrião não Mamífero/efeitos dos fármacos , Espectroscopia de Ressonância Magnética
10.
DNA Repair (Amst) ; 44: 33-41, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27311543

RESUMO

DNA interstrand crosslinks (ICLs) covalently join the two strands of a DNA duplex and block essential processes such as DNA replication and transcription. Several important anti-tumor drugs such as cisplatin and nitrogen mustards exert their cytotoxicity by forming ICLs. However, multiple complex pathways repair ICLs and these are thought to contribute to the development of resistance towards ICL-inducing agents. While the understanding of many aspects of ICL repair is still rudimentary, studies in recent years have provided significant insights into the pathways of ICL repair. In this perspective we review the recent advances made in elucidating the mechanisms of ICL repair with a focus on the role of TLS polymerases. We describe the emerging models for how these enzymes contribute to and are regulated in ICL repair, discuss the key open questions and examine the implications for this pathway in anti-cancer therapy.


Assuntos
Reparo do DNA , DNA de Neoplasias/genética , DNA Polimerase Dirigida por DNA/genética , DNA/genética , Neoplasias/genética , Animais , Antineoplásicos/uso terapêutico , Cisplatino/uso terapêutico , Reagentes para Ligações Cruzadas/uso terapêutico , DNA/antagonistas & inibidores , DNA/metabolismo , Replicação do DNA/efeitos dos fármacos , DNA de Neoplasias/antagonistas & inibidores , DNA de Neoplasias/metabolismo , DNA Polimerase Dirigida por DNA/classificação , DNA Polimerase Dirigida por DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Mecloretamina/uso terapêutico , Neoplasias/enzimologia , Neoplasias/patologia , Neoplasias/terapia , Transcrição Genética/efeitos dos fármacos , Xenopus
11.
Nucleic Acids Res ; 44(15): 7281-91, 2016 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-27257072

RESUMO

Several important anti-tumor agents form DNA interstrand crosslinks (ICLs), but their clinical efficiency is counteracted by multiple complex DNA repair pathways. All of these pathways require unhooking of the ICL from one strand of a DNA duplex by nucleases, followed by bypass of the unhooked ICL by translesion synthesis (TLS) polymerases. The structures of the unhooked ICLs remain unknown, yet the position of incisions and processing of the unhooked ICLs significantly influence the efficiency and fidelity of bypass by TLS polymerases. We have synthesized a panel of model unhooked nitrogen mustard ICLs to systematically investigate how the state of an unhooked ICL affects pol η activity. We find that duplex distortion induced by a crosslink plays a crucial role in translesion synthesis, and length of the duplex surrounding an unhooked ICL critically affects polymerase efficiency. We report the synthesis of a putative ICL repair intermediate that mimics the complete processing of an unhooked ICL to a single crosslinked nucleotide, and find that it provides only a minimal obstacle for DNA polymerases. Our results raise the possibility that, depending on the structure and extent of processing of an ICL, its bypass may not absolutely require TLS polymerases.


Assuntos
Reagentes para Ligações Cruzadas/química , Dano ao DNA , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , DNA/química , Conformação de Ácido Nucleico , DNA/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/genética , Relação Estrutura-Atividade
12.
Endocrinology ; 156(5): 1757-69, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25756310

RESUMO

In humans and zebrafish, 2 glucocorticoid (GC) receptor (GR) splice variants exist: the canonical GR α-isoform (GRα), and the GRß. In the present study, we have used the zebrafish model system in order to reveal genes affected by each of these 2 receptor isoforms. By injecting zebrafish embryos with different splice-blocking morpholinos, we could knock down both GR isoforms or could target the alternative splicing of the GR pre-mRNA in favor of the GRß. In addition, specific GRß overexpression was achieved by injecting mRNA. Embryos were treated with the synthetic GC dexamethasone, and transcriptome analysis was performed. Two distinct gene clusters were found that were regulated by GRα: one that was regulated by GRα under basal conditions (presence of endogenous cortisol only), and one that was regulated upon increased activation of GRα (using a pharmacological dose of dexamathasone). GRß may act as a dominant-negative inhibitor of GRα when GRß is overexpressed and the GRα expression level is knocked down simultaneously. However, without GRα knockdown, no evidence for this activity was found. In addition, the data indicate regulation of gene transcription through other mechanisms of action by GRß. We also investigated the concentrations of several metabolites using nuclear magnetic resonance spectroscopy. We found that dexamethasone treatment and knockdown of GRα together with overexpression of GRß had opposite effects on glucose, amino acid, and fatty acid levels. Thus, we have shed new light on the molecular mechanisms of GC-induced effects on metabolism, which are known to increase the risk of obesity, hyperglycemia, and diabetes.


Assuntos
Metaboloma/genética , Receptores de Glucocorticoides/genética , Transcriptoma/genética , Animais , Dexametasona/farmacologia , Embrião não Mamífero , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Glucocorticoides/farmacologia , Metaboloma/efeitos dos fármacos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/metabolismo , Transcriptoma/efeitos dos fármacos , Peixe-Zebra
13.
Hum Mol Genet ; 23(19): 5069-86, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-24824219

RESUMO

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a leukodystrophy characterized by myelin vacuolization and caused by mutations in MLC1 or GLIALCAM. Patients with recessive mutations in either MLC1 or GLIALCAM show the same clinical phenotype. It has been shown that GLIALCAM is necessary for the correct targeting of MLC1 to the membrane at cell junctions, but its own localization was independent of MLC1 in vitro. However, recent studies in Mlc1(-/-) mice have shown that GlialCAM is mislocalized in glial cells. In order to investigate whether the relationship between Mlc1 and GlialCAM is species-specific, we first identified MLC-related genes in zebrafish and generated an mlc1(-/-) zebrafish. We have characterized mlc1(-/-) zebrafish both functionally and histologically and compared the phenotype with that of the Mlc1(-/-) mice. In mlc1(-/-) zebrafish, as in Mlc1(-/-) mice, Glialcam is mislocalized. Re-examination of a brain biopsy from an MLC patient indicates that GLIALCAM is also mislocalized in Bergmann glia in the cerebellum. In vitro, impaired localization of GlialCAM was observed in astrocyte cultures from Mlc1(-/-) mouse only in the presence of elevated potassium levels, which mimics neuronal activity. In summary, here we demonstrate an evolutionary conserved role for MLC1 in regulating glial surface levels of GLIALCAM, and this interrelationship explains why patients with mutations in either gene (MLC1 or GLIALCAM) share the same clinical phenotype.


Assuntos
Cistos/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Proteínas de Membrana/metabolismo , Neuroglia/metabolismo , Proteínas/metabolismo , Animais , Animais Geneticamente Modificados , Astrócitos/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Ciclo Celular , Linhagem Celular , Membrana Celular/metabolismo , Cistos/genética , Modelos Animais de Doenças , Epêndima/citologia , Epêndima/metabolismo , Epêndima/ultraestrutura , Expressão Gênica , Genótipo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Humanos , Junções Intercelulares/metabolismo , Junções Intercelulares/ultraestrutura , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mutação , Fenótipo , Transporte Proteico , Proteínas/genética , Retina/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
14.
Zebrafish ; 11(3): 240-7, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24707799

RESUMO

Lipids play an important role in many neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, and Huntington's disease. Zebrafish models for these diseases have been recently developed. The detailed brain lipid composition of the adult zebrafish is not known, and therefore, the representativeness of these models cannot be properly evaluated. In this study, we characterized the total lipid composition of healthy adult zebrafish using (1)H nuclear magnetic resonance spectroscopy. A close resemblance of the zebrafish brain composition is shown in comparison to the human brain. Moreover, several lipids involved in the pathogenesis of neurodegenerative diseases (i.e., cholesterol, phosphatidylcholine, docosahexaenoic acid, and further, polyunsaturated fatty acids) are detected and quantified. These lipids might represent useful biomarkers in future research toward human therapies. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry coupled with high-performance thin-layer chromatography was used for further characterization of zebrafish brain lipids. Our results show that the lipid composition of the zebrafish brain is rather similar to the human brain and thus confirms that zebrafish represents a good model for studying various brain diseases.


Assuntos
Química Encefálica , Lipídeos/análise , Lipídeos/química , Espectroscopia de Ressonância Magnética , Doenças Neurodegenerativas/fisiopatologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Peixe-Zebra/metabolismo , Animais , Biomarcadores/metabolismo , Encéfalo/metabolismo , Cromatografia em Camada Delgada , Modelos Animais de Doenças , Humanos , Metabolismo dos Lipídeos
15.
FEBS J ; 278(7): 1167-74, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21288303

RESUMO

MicroRNA mir-9 is speculated to be involved in insulin secretion because of its ability to regulate exocytosis. Sirt1 is an NAD-dependent protein deacetylase and a critical factor in the modulation of cellular responses to altered metabolic flux. It has also been shown recently to control insulin secretion from pancreatic ß-islets. However, little is known about the regulation of Sirt1 and mir-9 levels in pancreatic ß-cells, particularly during glucose-dependent insulin secretion. In this article, we report that mir-9 and Sirt1 protein levels are actively regulated in vivo in ß-islets during glucose-dependent insulin secretion. Our data also demonstrates that mir-9 targets and regulates Sirt1 expression in insulin-secreting cells. This targeting is relevant in pancreatic ß-islets, where we show a reduction in Sirt1 protein levels when mir-9 expression is high during glucose-dependent insulin secretion. This functional interplay between insulin secretion, mir-9 and Sirt1 expression could be relevant in diabetes. It also highlights the crosstalk between an NAD-dependent protein deacetylase and microRNA in pancreatic ß-cells.


Assuntos
Glucose/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , MicroRNAs/metabolismo , Sirtuína 1/metabolismo , Animais , Sequência de Bases , Regulação da Expressão Gênica , Células HEK293 , Humanos , Secreção de Insulina , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/fisiologia , Masculino , Camundongos , MicroRNAs/genética , Dados de Sequência Molecular , Células NIH 3T3 , Alinhamento de Sequência , Sirtuína 1/genética
16.
Biochim Biophys Acta ; 1799(10-12): 681-93, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20713194

RESUMO

Cellular processes such as proliferation, differentiation and death are intrinsically dependent upon the redox status of a cell. Among other indicators of redox flux, cellular NAD(H) levels play a predominant role in transcriptional reprogramming. In addition to this, normal physiological functions of a cell are regulated in response to perturbations in NAD(H) levels (for example, due to alterations in diet/metabolism) to maintain homeostatic conditions. Cells achieve this homeostasis by reprogramming various components that include changes in chromatin structure and function (transcription). The interdependence of changes in gene expression and NAD(H) is evolutionarily conserved and is considered crucial for the survival of a species (by affecting reproductive capacity and longevity). Proteins that bind and/or use NAD(H) as a co-substrate (such as, CtBP and PARPs/Sirtuins respectively) are known to induce changes in chromatin structure and transcriptional profiles. In fact, their ability to sense perturbations in NAD(H) levels has been implicated in their roles in development, stress responses, metabolic homeostasis, reproduction and aging or age-related diseases. It is also becoming increasingly clear that both the levels/activities of these proteins and the availability of NAD(H) are equally important. Here we discuss the pivotal role of NAD(H) in controlling the functions of some of these proteins, the functional interplay between them and physiological implications during calorie restriction, energy homeostasis, circadian rhythm and aging.


Assuntos
Regulação da Expressão Gênica/fisiologia , Homeostase/fisiologia , NAD/metabolismo , Transcrição Genética/fisiologia , Envelhecimento/fisiologia , Animais , Metabolismo Energético/fisiologia , Humanos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA