RESUMO
The three-dimensional crystal structure of pi class glutathione S-transferase YfYf from mouse liver complexed with the inhibitor S-(p-nitrobenzyl)glutathione has been determined at 1.8 A resolution by X-ray diffraction. In addition two complexes with glutathione sulphonic acid and S-hexylglutathione have been determined at resolutions of 1.9 and 2.2 A, respectively. The high resolution of the S-(p-nitrobenzyl)glutathione complex allows a detailed analysis of the active site including the hydrophobic (H-) subsite. The nitrobenzyl moiety occupies a hydrophobic pocket with its aromatic ring sandwiched between Phe8 and the hydroxyl group of Tyr108. An insertion of two residues Gly41 and Leu42, with respect to the pig enzyme, splits helix alpha B into an alpha-helix and a 3(10) helix. Water bridges between carbonyl oxygen atoms of the alpha-helix at its C terminus and the amide NH groups of the 3(10) helix at its N terminus provide structural continuity between these two secondary elements. Tyr7 appears to be the only residue close to the sulphur atom of glutathione, while three conserved water molecules lie in the surrounding area in all complexes. The enzyme mechanism is discussed on the basis of the structural analysis.
Assuntos
Glutationa Transferase/química , Glutationa/análogos & derivados , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Glutationa/química , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/metabolismo , Ligação de Hidrogênio , Fígado/enzimologia , Masculino , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de ProteínaRESUMO
Cardiac disease is a well-known complication of myotonic dystrophy, understanding of which has been increased by recent advances in both molecular techniques and cardiological investigations. Conduction disturbances and tachyarrhythmias occur commonly in myotonic dystrophy. These have been shown to have a broad correlation in severity with both neuromuscular disease and the extent of the molecular defect in some, but not all, studies. Clinical evidence of generalised cardiomyopathy is unusual. The rate of progression differs widely between individuals; sudden death may be caused by ventricular arrhythmias or complete heart block, and this can be at an early stage of disease. A familial tendency towards cardiac complications has been shown in some studies. The histopathology is of fibrosis, primarily in the conducting system and sino-atrial node, myocyte hypertrophy and fatty infiltration. Electron microscopy shows prominent I-bands and myofibrillar degeneration. Myotonin protein kinase, the primary product of the myotonic dystrophy gene, may be located at the intercalated discs and have a different isoform in cardiac tissue. The role of other genes or the normal myotonic dystrophy allele in myotonic heart disease has yet to be determined. Suggestions for clinical management include a careful cardiac history and a 12-lead ECG at least every year, with a low threshold for use of 24 h Holter monitoring. Extra care should be taken before, during and after general anaesthetics, which carry a high frequency of cardiorespiratory complications. Finally, myotonic dystrophy should be considered in previously undiagnosed patients presenting to a cardiologist or general physician with suspected arrhythmia or conduction block.
Assuntos
Arritmias Cardíacas/etiologia , Distrofia Miotônica/complicações , Arritmias Cardíacas/patologia , Arritmias Cardíacas/fisiopatologia , Sistema de Condução Cardíaco/fisiopatologia , Humanos , Miocárdio/patologia , Distrofia Miotônica/patologia , Distrofia Miotônica/fisiopatologiaRESUMO
We describe a family with a proximal myopathy, subclinical EMG myotonia, cataracts and deafness. Transmission through two generations and down the male line confirms autosomal dominant inheritance. There was no abnormal expansion of the CTG triplet repeat in the last exon of the dystrophia myotonica protein kinase (DMPK) gene associated with myotonic dystrophy. Heteroduplex analysis of all but the promoter region of the DMPK gene has excluded point mutations in this gene as an underlying cause for this myotonic disorder. The family was not sufficiently informative to exclude linkage to the sodium channel gene SCN4A or the chloride channel gene CLC1. This family clearly fulfils the recently established diagnostic criteria for PROMM (proximal myotonic myopathy) and in addition shows consistent severe deafness as a hitherto undescribed feature of PROMM. We discuss the diagnostic criteria of PROMM in relation to this family and other recent papers, all of which would now fulfil the aforementioned diagnostic criteria for PROMM.
Assuntos
Miotonia/genética , Adulto , Idoso , Audiometria , Catarata/genética , Catarata/patologia , Surdez/genética , Surdez/patologia , Eletromiografia , Saúde da Família , Feminino , Genes Dominantes/genética , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Musculares/genética , Doenças Musculares/patologia , Miotonia/patologia , Linhagem , FenótipoRESUMO
Somnolence in myotonic dystrophy (DM) has not been measured using a reliable daytime somnolence scale. The aim of this study was to compare somnolence in DM patients with healthy controls and Charcot-Marie-Tooth disease (CMT) patients using such a scale and to compare this with potential contributory factors. We investigated 35 subjects with adult-onset DM, 16 healthy controls and 13 CMT controls. The Epworth Sleepiness Scale (ESS) was the principal measurement of daytime somnolence. Nocturnal sleep was assessed using a sleep diary. Other assessments measured daytime respiratory function, cognitive function, motor impairment, disability, swallowing capacity and depression. DM and CMT patients had greater daytime sleepiness than unaffected controls. In the DM group significant correlations were found between somnolence and measures of disability, sleep quality and some measures of depression. It was concluded that there is an abnormal level of daytime somnolence in DM, which is partially associated with disability.
Assuntos
Estado de Consciência , Distrofia Miotônica/complicações , Qualidade de Vida , Transtornos do Sono-Vigília/etiologia , Adulto , Deglutição , Transtorno Depressivo/etiologia , Pessoas com Deficiência , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Distrofia Miotônica/psicologiaRESUMO
Mouse liver glutathione S-transferase YfYf (Pi class) reacts with [14C]ethacrynic acid to form a covalent adduct with a stoichiometry of 1 mol per mol of subunit. Proteolytic digestion of the enzyme-[14C]ethacrynic acid adduct with V8 protease produced an 11 kDa fragment containing radioactivity. Sequencing revealed this to be an N-terminal peptide (minus the first 15 residues, terminating at Glu-112) which contains only one cysteine residue (Cys-47). This is tentatively identified as the site of ethacrynic attachment. Kinetic studies reveal that glutathione S-conjugates protect against inactivation by ethacrynic acid, but the level of protection is not consistent with their potency as product inhibitors. A model is proposed in which glutathione S-conjugates and ethacrynic acid compete for the free enzyme, and a second molecule of ethacrynic acid reacts covalently with the enzyme-ethacrynic acid complex. The native protein contains one thiol reactive with 5,5'-dithiobis-(2-nitrobenzoic acid) at neutral pH. The resultant mixed disulphide, like the ethacrynic acid adduct, is inactive, but treatment with cyanide (which incorporates on a mol for mol basis) restores activity to 35% of that of the native enzyme.
Assuntos
Ácido Ditionitrobenzoico/farmacologia , Ácido Etacrínico/farmacologia , Glutationa Transferase/antagonistas & inibidores , Isoenzimas/antagonistas & inibidores , Fígado/enzimologia , Sequência de Aminoácidos , Animais , Ácido Etacrínico/antagonistas & inibidores , Glutationa/análogos & derivados , Glutationa/farmacologia , Glutationa Transferase/química , Concentração de Íons de Hidrogênio , Isoenzimas/química , Cinética , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Mapeamento de Peptídeos , Compostos de Sulfidrila/químicaRESUMO
Previously, it has been reported that energy consumed as alcohol prior to lunch does not result in subsequent reductions in voluntary food intake, and in some situations alcohol can increase subsequent appetite. The present study extends these findings by examining the effects of beliefs about alcohol content. Eighteen unrestrained men ate lunch 20 min after a preload of either water, an alcoholic beer or a non-alcoholic beer matched for energy content. Food intake was significantly less following the non-alcoholic beer than after alcohol or water, but when preload energy was included subjects had a higher overall energy intake on the day they consumed alcohol compared with both water and no-alcohol conditions. There were no significant differences in hunger or fullness ratings following the three drinks before or after the test meal, but the specific relationship between rated hunger and intake within the test meal was altered by the drink manipulation. The rate at which hunger decreased, and fullness increased, was slower after alcohol than after water or no-alcohol. The drinks did not alter the pleasantness of the test meal or increase hunger at the start of eating. When contrasted with previous work, these data confirm that alcohol consumed before lunch fails to reduce subsequent food intake, but also suggests that changes in rated appetite are influenced by beliefs about alcohol content.
Assuntos
Apetite/efeitos dos fármacos , Etanol/administração & dosagem , Percepção , Adulto , Afeto , Cerveja/análise , Ingestão de Alimentos , Etanol/análise , Alimentos , Humanos , Fome , Masculino , Psicofarmacologia , Saciação , PaladarRESUMO
Mouse glutathione S-transferase GST YfYf (an orthologue of GST P or 7-7 in the rat and of GST pi in the human) was found to have a subunit Mr of 24,500 and cross-reacted with anti-(rat GST YfYf). N-Terminal analysis showed a close similarity to the rat, human and bovine orthologues. On isoelectric focusing the native enzyme had a pI of 8.3 and a pI of 7.3 in the presence of urea. Initial-rate studies with 1-chloro-2,4-dinitrobenzene (CDNB) and GSH as substrates and inhibition studies with the product of the enzyme-catalysed conjugation of CDNB and GSH, S-(2,4-dinitrophenyl)glutathione, indicated a rapid-equilibrium random mechanism for the enzyme. The diuretic drug ethacrynic acid was found to be simultaneously a competitive inhibitor and an uncompetitive activator of the enzyme (with CDNB as the substrate whose concentration was varied). By using a computer simulation program (EKPLOT) a model was developed that would explain the experimental data. It is proposed that ethacrynic acid can compete with CDNB at the active site but simultaneously bind to an allosteric site on the enzyme, causing an elevation in the Vmax. for the conjugation of CDNB and GSH. The implications of such an activation mechanism for an enzyme potentially conjugating a range of xenobiotic compounds are discussed.
Assuntos
Ácido Etacrínico/metabolismo , Glutationa Transferase/metabolismo , Sítio Alostérico , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Bovinos , Simulação por Computador , Dinitroclorobenzeno/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ácido Etacrínico/farmacologia , Glutationa/metabolismo , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/química , Humanos , Ponto Isoelétrico , Cinética , Masculino , Camundongos , Dados de Sequência Molecular , Ratos , Homologia de Sequência do Ácido NucleicoRESUMO
Duchenne muscular dystrophy (DMD) causes a progressive impairment of muscle function leading to hypercapnic respiratory failure. Most studies of respiratory function in DMD have been cross-sectional rather than longitudinal, and these data have not been related to survival. We retrospectively studied 58 patients with DMD with at least 2 yr of follow-up spirometry and known vital status. Spirometry was abnormal at entry: median FEV(1) 1.60 L (range 0.4 to 2.6 L), FVC 1.65 L (range 0.45 to 2.75 L), FVC 64% predicted (range 29 to 97%). Individual rates of change of vital capacity varied, with a median annual change of -0.18 L (range 0.04 to -0.74 L), -8.0% predicted FVC (range 2 to -39%). During the study 37 patients died; the median age of death, calculated by Kaplan-Meier analysis, was 21.5 yr (range 15 to 28.5 yr). The age when vital capacity fell below 1 L was a strong marker of subsequent mortality (5-yr survival 8%). The maximal vital capacity recorded and its rate of decline (however expressed) predicted survival time. Repeated spirometric measurement provides a simple and relatively powerful means of assessing disease progression in these patients and should be considered when planning treatment trials.
Assuntos
Distrofia Muscular de Duchenne/fisiopatologia , Espirometria , Adolescente , Criança , Progressão da Doença , Eletromiografia , Volume Expiratório Forçado , Humanos , Masculino , Distrofia Muscular de Duchenne/complicações , Distrofia Muscular de Duchenne/mortalidade , Prognóstico , Insuficiência Respiratória/diagnóstico , Insuficiência Respiratória/etiologia , Músculos Respiratórios/fisiopatologia , Estudos Retrospectivos , Taxa de Sobrevida , Capacidade VitalRESUMO
REM-related oxygen desaturation occurs in advanced Duchenne muscular dystrophy (DMD) and might be an independent predictor of disease progression. We have followed 18 patients for 10 yr after an initial respiratory sleep study or until death or onset of nasal ventilation. We measured baseline spirometry, blood gas tensions, maximal respiratory pressures, and body mass index. In 11 cases, VC was recorded serially. Median survival was 50 (range, 13 to 89) mo from initial study and unrelated to age at time of study, BMI, or mouth pressures but correlated with PaCO2 (r = -0.72, p < 0.005, n = 17), minimal nocturnal SaO2 (r = 0.62, p < 0.007, n = 18) and VC (r = 0. 65, p < 0.005, n = 17). Cox regression analysis showed a VC of less than 1 L at the time of study to be the best single predictor of subsequent survival. The only measure associated with age of death was the age at which the VC fell below 1 L (r = 0.79, p < 0.004). These data suggest measurement of PaCO2 or serial assessment of VC should be studied further as valid methods of assessing prognosis in DMD.