RESUMO
The human foot is uniquely adapted to bipedal locomotion and has a deformable arch of variable stiffness. Intrinsic foot muscles regulate arch deformation, making them important for foot function. In this study we explore the hypothesis that normal daily activity in minimal footwear, which provides little or no support, increases foot muscle strength. Western adults wore minimal footwear for a six-month period (the "intervention" group). Foot strength, i.e., maximum isometric plantarflexion strength at the metatarsophalangeal joints, and foot biometrics were measured before and after the intervention. An additional group was investigated to add further insight on the long-term effects of footwear, consisting of Western adults with an average 2.5 years of experience in minimal footwear (the "experienced" group). This study shows that foot strength increases by, on average, 57.4% (p < 0.001) after six months of daily activity in minimal footwear. The experienced group had similar foot strength as the post intervention group, suggesting that six months of regular minimal footwear use is sufficient to gain full strength, which may aid healthy balance and gait.
Assuntos
Pé/fisiologia , Força Muscular/fisiologia , Sapatos/efeitos adversos , Adulto , Fenômenos Biomecânicos , Feminino , Marcha/fisiologia , Humanos , Locomoção , Masculino , Músculo Esquelético/fisiologia , Corrida/fisiologiaRESUMO
The endonuclease Artemis is responsible for opening DNA hairpins during V(D)J recombination and for processing a subset of pathological DNA double-strand breaks. Artemis is an attractive target for the development of therapeutics to manage various B cell and T cell tumors, because failure to open DNA hairpins and accumulation of chromosomal breaks may reduce the proliferation and viability of pre-T and pre-B cell derivatives. However, structure-based drug discovery of specific Artemis inhibitors has been hampered by a lack of crystal structures. Here, we report the structure of the catalytic domain of recombinant human Artemis. The catalytic domain displayed a polypeptide fold similar overall to those of other members in the DNA cross-link repair gene SNM1 family and in mRNA 3'-end-processing endonuclease CPSF-73, containing metallo-ß-lactamase and ß-CASP domains and a cluster of conserved histidine and aspartate residues capable of binding two metal atoms in the catalytic site. As in SNM1A, only one zinc ion was located in the Artemis active site. However, Artemis displayed several unique features. Unlike in other members of this enzyme class, a second zinc ion was present in the ß-CASP domain that leads to structural reorientation of the putative DNA-binding surface and extends the substrate-binding pocket to a new pocket, pocket III. Moreover, the substrate-binding surface exhibited a dominant and extensive positive charge distribution compared with that in the structures of SNM1A and SNM1B, presumably because of the structurally distinct DNA substrate of Artemis. The structural features identified here may provide opportunities for designing selective Artemis inhibitors.
Assuntos
Endonucleases/química , Dobramento de Proteína , Zinco/química , Animais , Domínio Catalítico , Proteínas de Ligação a DNA , Endonucleases/genética , Humanos , Células Sf9 , Spodoptera , Relação Estrutura-AtividadeRESUMO
Ghrelin influences a variety of metabolic functions through a direct action at its receptor, the GhrR (GhrR-1a). Ghrelin knockout (KO) and GhrR KO mice are resistant to the negative effects of high-fat diet (HFD) feeding. We have generated several classes of small-molecule GhrR antagonists and evaluated whether pharmacologic blockade of ghrelin signaling can recapitulate the phenotype of ghrelin/GhrR KO mice. Antagonist treatment blocked ghrelin-induced and spontaneous food intake; however, the effects on spontaneous feeding were absent in GhrR KO mice, suggesting target-specific effects of the antagonists. Oral administration of antagonists to HFD-fed mice improved insulin sensitivity in both glucose tolerance and glycemic clamp tests. The insulin sensitivity observed was characterized by improved glucose disposal with dramatically decreased insulin secretion. It is noteworthy that these results mimic those obtained in similar tests of HFD-fed GhrR KO mice. HFD-fed mice treated for 56 days with antagonist experienced a transient decrease in food intake but a sustained body weight decrease resulting from decreased white adipose, but not lean tissue. They also had improved glucose disposal and a striking reduction in the amount of insulin needed to achieve this. These mice had reduced hepatic steatosis, improved liver function, and no evidence of systemic toxicity relative to controls. Furthermore, GhrR KO mice placed on low- or high-fat diets had lifespans similar to the wild type, emphasizing the long-term safety of ghrelin receptor blockade. We have therefore demonstrated that chronic pharmacologic blockade of the GhrR is an effective and safe strategy for treating metabolic syndrome.
Assuntos
Resistência à Insulina/fisiologia , Insulina/metabolismo , Receptores de Grelina/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Fármacos Antiobesidade/farmacologia , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Células CHO , Cricetinae , Cricetulus , Gorduras na Dieta/farmacologia , Ingestão de Alimentos/efeitos dos fármacos , Grelina/antagonistas & inibidores , Grelina/farmacologia , Técnica Clamp de Glucose , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/tratamento farmacológico , Receptores de Grelina/fisiologia , Estresse Fisiológico/fisiologiaRESUMO
Identification of genes and pathways that alter lifespan has allowed for new insights into factors that control the aging process as well as disease. While strong molecular links exist between aging and metabolism, we hypothesize that targeting the mechanisms involved in aging will also give rise to therapeutics that treat other devastating age-related diseases, such as neurodegeneration, cancer, inflammation and cardiovascular disease. Insulin sensitivity, glycemic control and adiposity are not only hallmarks of the major metabolic diseases, type 2 diabetes and obesity, but they also represent significant risk factors for the development of Alzheimer's Disease and cognitive impairment. Insulin/IGF-1 signaling is an important pathway regulating aging and disease in a variety of species, including mammals. Here we describe an important role for the gut-derived peptide ghrelin in upstream signaling through the insulin/IGF-1 pathway and exemplify modulation of ghrelin signaling as an approach to mechanistic treatment of multiple age-related diseases by virtue of its ability to regulate key metabolic functions.
Assuntos
Adiposidade , Envelhecimento/fisiologia , Glicemia , Resistência à Insulina , Longevidade/fisiologia , Animais , HumanosRESUMO
Molecular genetics in lower organisms has allowed the elucidation of pathways that modulate the aging process. In certain instances, evolutionarily conserved genes and pathways have been shown to regulate lifespan in mammals as well. Many gene products known to affect lifespan are intimately involved in the control of energy metabolism, including the fuel sensor AMP-activated protein kinase (AMPK). We have shown previously that over-expression of an AMPK alpha subunit in Caenorhabditis elegans, designated aak-2, increases lifespan. Here we show the interaction of aak-2 with other pathways known to control aging in worms. Lifespan extension caused by daf-2/insulin-like signaling mutations was highly dependent on aak-2, as was the lifespan extension caused by over-expression of the deacetylase, sir-2.1. Similarly, there was partial requirement for aak-2 in lifespan extension by mitochondrial mutations (isp-1 and clk-1). Conversely, aak-2 was not required for lifespan extension in mutants lacking germline stem cells (glp-1) or mutants of the eating response (eat-2). These results show that aging is controlled by overlapping but distinct pathways and that AMPK/aak-2 represents a node in a network of evolutionarily conserved biochemical pathways that control aging.
Assuntos
Envelhecimento/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/fisiologia , Complexos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Nucleotídeos de Adenina/metabolismo , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Comportamento Alimentar , Fatores de Transcrição Forkhead , Regulação da Expressão Gênica no Desenvolvimento , Mutação em Linhagem Germinativa/genética , Larva/crescimento & desenvolvimento , Longevidade , Mitocôndrias/metabolismo , Modelos Biológicos , Ligação Proteica , Sirtuínas/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
Human SIRT1 is an enzyme that deacetylates the p53 tumor suppressor protein and has been suggested to modulate p53-dependent functions including DNA damage-induced cell death. In this report, we used EX-527, a novel, potent, and specific small-molecule inhibitor of SIRT1 catalytic activity to examine the role of SIRT1 in p53 acetylation and cell survival after DNA damage. Treatment with EX-527 dramatically increased acetylation at lysine 382 of p53 after different types of DNA damage in primary human mammary epithelial cells and several cell lines. Significantly, inhibition of SIRT1 catalytic activity by EX-527 had no effect on cell growth, viability, or p53-controlled gene expression in cells treated with etoposide. Acetyl-p53 was also increased by the histone deacetylase (HDAC) class I/II inhibitor trichostatin A (TSA). EX-527 and TSA acted synergistically to increase acetyl-p53 levels, confirming that p53 acetylation is regulated by both SIRT1 and HDACs. While TSA alone reduced cell survival after DNA damage, the combination of EX-527 and TSA had no further effect on cell viability and growth. These results show that, although SIRT1 deacetylates p53, this does not play a role in cell survival following DNA damage in certain cell lines and primary human mammary epithelial cells.
Assuntos
Dano ao DNA , Inibidores de Histona Desacetilases , Sirtuínas/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Catálise/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular , DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Humanos , Ácidos Hidroxâmicos/farmacologia , Sirtuína 1RESUMO
High-throughput screening against the human sirtuin SIRT1 led to the discovery of a series of indoles as potent inhibitors that are selective for SIRT1 over other deacetylases and NAD-processing enzymes. The most potent compounds described herein inhibit SIRT1 with IC50 values of 60-100 nM, representing a 500-fold improvement over previously reported SIRT inhibitors. Preparation of enantiomerically pure indole derivatives allowed for their characterization in vitro and in vivo. Kinetic analyses suggest that these inhibitors bind after the release of nicotinamide from the enzyme and prevent the release of deacetylated peptide and O-acetyl-ADP-ribose, the products of enzyme-catalyzed deacetylation. These SIRT1 inhibitors are low molecular weight, cell-permeable, orally bioavailable, and metabolically stable. These compounds provide chemical tools to study the biology of SIRT1 and to explore therapeutic uses for SIRT1 inhibitors.
Assuntos
Carbazóis/síntese química , Inibidores de Histona Desacetilases , Indóis/síntese química , Sirtuínas/antagonistas & inibidores , Animais , Disponibilidade Biológica , Células CHO , Carbazóis/química , Carbazóis/farmacologia , Permeabilidade da Membrana Celular , Cricetinae , Cricetulus , Estabilidade de Medicamentos , Fluorometria , Histona Desacetilases/química , Humanos , Técnicas In Vitro , Indóis/química , Indóis/farmacologia , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/metabolismo , NAD/química , NAD+ Nucleosidase/química , Niacinamida/química , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Sirtuína 1 , Sirtuínas/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
We describe a microplate-based assay for NAD-dependent Class III histone deacetylases (also known as SIRTs) that measures the enzyme-catalyzed release of nicotinamide from radiolabeled NAD, using a boronic acid resin to selectively capture the NAD. This method avoids the need for fluorogenic or radiolabeled peptides or separation of the reaction products using solvent extraction. The protocol reported here is rapid and uses commercially available materials. The use of a simple microplate filtration device allows for the simultaneous processing of 96 samples, facilitating enzyme kinetic analyses and inhibition studies. Furthermore, monitoring nicotinamide release rather than peptide deacetylation obviates the need for chemical modification of protein and peptide substrates. This assay is applicable to SIRTs and other enzymes that cleave nicotinamide from NAD.
Assuntos
Resinas Acrílicas/química , Ácidos Borônicos/química , NAD/metabolismo , Niacinamida/metabolismo , Sirtuínas/metabolismo , Animais , Radioisótopos de Carbono/química , Catálise , Técnicas de Laboratório Clínico , Filtração/métodos , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Humanos , Hidrólise , Cinética , Modelos Químicos , Estrutura Molecular , NAD/química , Niacinamida/química , Sirtuínas/químicaRESUMO
There has recently been significant progress in our understanding of the mechanisms that regulate ageing, and it has been shown that changes in single genes can dramatically extend lifespan and increase resistance to many diseases. Furthermore, many of these genes belong to evolutionarily conserved pathways that also control energy metabolism. In this review, we describe the shared molecular machinery that regulates ageing and energy metabolism. Although drugs to slow ageing face severe regulatory hurdles, it is likely that an understanding of ageing pathways will help to identify novel drug targets to treat metabolic disorders and other age-related diseases.
Assuntos
Envelhecimento/efeitos dos fármacos , Envelhecimento/fisiologia , Desenho de Fármacos , Metabolismo/efeitos dos fármacos , Metabolismo/fisiologia , Proteínas Quinases Ativadas por AMP , Adipócitos/efeitos dos fármacos , Envelhecimento/genética , Animais , Ingestão de Alimentos/fisiologia , Humanos , Insulina/fisiologia , Expectativa de Vida , Metabolismo/genética , Complexos Multienzimáticos/fisiologia , Proteínas Serina-Treonina Quinases/fisiologiaRESUMO
Resveratrol, a small molecule found in red wine, is reported to slow aging in simple eukaryotes and has been suggested as a potential calorie restriction mimetic. Resveratrol has also been reported to act as a sirtuin activator, and this property has been proposed to account for its anti-aging effects. We show here that resveratrol is a substrate-specific activator of yeast Sir2 and human SirT1. In particular, we observed that, in vitro, resveratrol enhances binding and deacetylation of peptide substrates that contain Fluor de Lys, a non-physiological fluorescent moiety, but has no effect on binding and deacetylation of acetylated peptides lacking the fluorophore. Consistent with these biochemical data we found that in three different yeast strain backgrounds, resveratrol has no detectable effect on Sir2 activity in vivo, as measured by rDNA recombination, transcriptional silencing near telomeres, and life span. In light of these findings, the mechanism accounting for putative longevity effects of resveratrol should be reexamined.
Assuntos
Antioxidantes/farmacologia , Histona Desacetilases/química , Sirtuínas/antagonistas & inibidores , Sirtuínas/química , Estilbenos/farmacologia , Ligação Competitiva , DNA Ribossômico/química , DNA Ribossômico/metabolismo , Relação Dose-Resposta a Droga , Proteínas Fúngicas/química , Inativação Gênica , Inibidores de Histona Desacetilases , Humanos , Técnicas In Vitro , Cinética , Modelos Químicos , Niacinamida/química , Peptídeos/química , Ligação Proteica , Recombinação Genética , Resveratrol , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 1 , Sirtuína 2 , Especificidade por Substrato , Telômero/metabolismo , Fatores de Tempo , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismoRESUMO
Although limiting energy availability extends lifespan in many organisms, it is not understood how lifespan is coupled to energy levels. We find that the AMP:ATP ratio, a measure of energy levels, increases with age in Caenorhabditis elegans and can be used to predict life expectancy. The C. elegans AMP-activated protein kinase alpha subunit AAK-2 is activated by AMP and functions to extend lifespan. In addition, either an environmental stressor that increases the AMP:ATP ratio or mutations that lower insulin-like signaling extend lifespan in an aak-2-dependent manner. Thus, AAK-2 is a sensor that couples lifespan to information about energy levels and insulin-like signals.
Assuntos
Monofosfato de Adenosina/metabolismo , Envelhecimento/fisiologia , Caenorhabditis elegans/fisiologia , Longevidade/efeitos dos fármacos , Complexos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Proteínas Quinases Ativadas por AMP , Trifosfato de Adenosina/metabolismo , Fatores Etários , Animais , Animais Geneticamente Modificados , Cruzamentos Genéticos , Primers do DNA , Metabolismo Energético/fisiologia , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde , Imunoprecipitação , Insulina/metabolismo , Complexos Multienzimáticos/genética , Mutagênese , Proteínas Serina-Treonina Quinases/genética , Análise de Sequência de DNA , TemperaturaRESUMO
BACKGROUND: Voltage-gated sodium channels composed of pore-forming alpha and auxiliary beta subunits are responsible for the rising phase of the action potential in cardiac muscle, but their localizations have not yet been clearly defined. METHODS AND RESULTS: Immunocytochemical studies show that the principal cardiac alpha subunit isoform Na(v)1.5 and the beta2 subunit are preferentially localized in intercalated disks, identified by immunostaining of connexin 43, the major protein of cardiac gap junctions. The brain alpha subunit isoforms Na(v)1.1, Na(v)1.3, and Na(v)1.6 are preferentially localized with beta1 and beta3 subunits in the transverse tubules, identified by immunostaining of alpha-actinin, a cardiac z-line protein. The beta1 subunit is also present in a small fraction of intercalated disks. The recently cloned beta4 subunit, which closely resembles beta2 in amino acid sequence, is also expressed in ventricular myocytes and is localized in intercalated disks as are beta2 and Na(v)1.5. CONCLUSIONS: Our results suggest that the primary sodium channels present in ventricular myocytes are composed of Na(v)1.5 plus beta2 and/or beta4 subunits in intercalated disks and Na(v)1.1, Na(v)1.3, and Na(v)1.6 plus beta1 and/or beta3 subunits in the transverse tubules.
Assuntos
Miócitos Cardíacos/química , Proteínas do Tecido Nervoso/análise , Canais de Sódio/análise , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Células Cultivadas/química , Conexina 43/análise , Ventrículos do Coração , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Dados de Sequência Molecular , Miócitos Cardíacos/ultraestrutura , Canal de Sódio Disparado por Voltagem NAV1.1 , Organelas/química , Isoformas de Proteínas/análise , Subunidades Proteicas/análise , Frações Subcelulares/química , TransfecçãoRESUMO
The principal alpha subunit of voltage-gated sodium channels is associated with auxiliary beta subunits that modify channel function and mediate protein-protein interactions. We have identified a new beta subunit termed beta4. Like the beta1-beta3 subunits, beta4 contains a cleaved signal sequence, an extracellular Ig-like fold, a transmembrane segment, and a short intracellular C-terminal tail. Using TaqMan reverse transcription-PCR analysis, in situ hybridization, and immunocytochemistry, we show that beta4 is widely distributed in neurons in the brain, spinal cord, and some sensory neurons.beta4 is most similar to the beta2 subunit (35% identity), and, like the beta2 subunit, the Ig-like fold of beta4 contains an unpaired cysteine that may interact with the alpha subunit. Under nonreducing conditions, beta4 has a molecular mass exceeding 250 kDa because of its covalent linkage to Nav1.2a, whereas on reduction, it migrates with a molecular mass of 38 kDa, similar to the mature glycosylated forms of the other beta subunits. Coexpression of beta4 with brain Nav1.2a and skeletal muscle Nav1.4 alpha subunits in tsA-201 cells resulted in a negative shift in the voltage dependence of channel activation, which overrode the opposite effects of beta1 and beta3 subunits when they were present. This novel, disulfide-linked beta subunit is likely to affect both protein-protein interactions and physiological function of multiple sodium channel alpha subunits.
Assuntos
Subunidades Proteicas/química , Subunidades Proteicas/fisiologia , Canais de Sódio/química , Canais de Sódio/fisiologia , Sequência de Aminoácidos , Animais , Adesão Celular , Linhagem Celular , Clonagem Molecular , Dissulfetos/química , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.2 , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Canais de Sódio/genética , Canais de Sódio/metabolismo , Distribuição Tecidual , Subunidade beta-2 do Canal de Sódio Disparado por Voltagem , Subunidade beta-4 do Canal de Sódio Disparado por VoltagemRESUMO
Transient receptor potential (TRP) proteins are cation-selective channels that function in processes as diverse as sensation and vasoregulation. Mammalian TRP channels that are gated by heat and capsaicin (>43 degrees C; TRPV1 (ref. 1)), noxious heat (>52 degrees C; TRPV2 (ref. 2)), and cooling (< 22 degrees C; TRPM8 (refs 3, 4)) have been cloned; however, little is known about the molecular determinants of temperature sensing in the range between approximately 22 degrees C and 40 degrees C. Here we have identified a member of the vanilloid channel family, human TRPV3 (hTRPV3) that is expressed in skin, tongue, dorsal root ganglion, trigeminal ganglion, spinal cord and brain. Increasing temperature from 22 degrees C to 40 degrees C in mammalian cells transfected with hTRPV3 elevated intracellular calcium by activating a nonselective cationic conductance. As in published recordings from sensory neurons, the current was steeply dependent on temperature, sensitized with repeated heating, and displayed a marked hysteresis on heating and cooling. On the basis of these properties, we propose that hTRPV3 is thermosensitive in the physiological range of temperatures between TRPM8 and TRPV1.
Assuntos
Cálcio/metabolismo , Proteínas de Transporte de Cátions , Permeabilidade da Membrana Celular , Ativação do Canal Iônico , Canais Iônicos/metabolismo , Temperatura , Sequência de Aminoácidos , Animais , Células CHO , Sinalização do Cálcio , Cátions Bivalentes/metabolismo , Clonagem Molecular , Cricetinae , Condutividade Elétrica , Eletrofisiologia , Evolução Molecular , Perfilação da Expressão Gênica , Humanos , Canais Iônicos/química , Canais Iônicos/genética , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Canais de Cátion TRPVRESUMO
The study of ion channel-mediated changes in membrane potential using the conventional bisoxonol fluorescent dye DiBAC(4)(3) has several limitations, including a slow onset of response and multistep preparation, that limit both the fidelity of the results and the throughput of membrane potential assays. Here, we report the characterization of the FLIPR Membrane Potential Assay Kit (FMP) in cells expressing voltage- and ligand-gated ion channels. The steady-state and kinetics fluorescence properties of FMP were compared with those of DiBAC(4)(3), using both FLIPR and whole-cell patch-clamp recording. Our experiments with the voltage-gated K(+) channel, hElk-1, revealed that FMP was 14-fold faster than DiBAC(4)(3) in response to depolarization. On addition of 60 mM KCl, the kinetics of fluorescence changes of FMP using FLIPR were identical to those observed in the electrophysiological studies using whole-cell current clamp. In addition, KCl concentration-dependent increases in FMP fluorescence correlated with the changes of membrane potential recorded in whole-cell patch clamp. In studies examining vanilloid receptor-1, a ligand-gated nonselective cation channel, FMP was superior to DiBAC(4)(3) with respect to both kinetics and amplitude of capsaicin-induced fluorescence changes. FMP has also been used to measure the activation of K(ATP) and hERG. Thus this novel membrane potential dye represents a powerful tool for developing high-throughput screening assays for ion channels.