RESUMO
The design of structurally diverse enzymes is constrained by long-range interactions that are necessary for accurate folding. We introduce an atomistic and machine learning strategy for the combinatorial assembly and design of enzymes (CADENZ) to design fragments that combine with one another to generate diverse, low-energy structures with stable catalytic constellations. We applied CADENZ to endoxylanases and used activity-based protein profiling to recover thousands of structurally diverse enzymes. Functional designs exhibit high active-site preorganization and more stable and compact packing outside the active site. Implementing these lessons into CADENZ led to a 10-fold improved hit rate and more than 10,000 recovered enzymes. This design-test-learn loop can be applied, in principle, to any modular protein family, yielding huge diversity and general lessons on protein design principles.
Assuntos
Técnicas de Química Combinatória , Endo-1,4-beta-Xilanases , Engenharia de Proteínas , Catálise , Domínio Catalítico , Engenharia de Proteínas/métodos , Endo-1,4-beta-Xilanases/químicaRESUMO
Bacteroides vulgatus is a member of the human microbiota whose abundance is increased in patients with Crohn's disease. We show that a B. vulgatus glycoside hydrolase from the carbohydrate active enzyme family GH123, BvGH123, is an N-acetyl-ß-galactosaminidase that acts with retention of stereochemistry, and, through a 3-D structure in complex with Gal-thiazoline, provide evidence in support of a neighbouring group participation mechanism.
Assuntos
Bacteroides/enzimologia , beta-N-Acetil-Galactosaminidase/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Sítios de Ligação , Domínio Catalítico , Doença de Crohn/etiologia , Doença de Crohn/microbiologia , Humanos , Simulação de Dinâmica Molecular , Estereoisomerismo , Especificidade por Substrato , Tiazóis/química , Tiazóis/metabolismo , beta-N-Acetil-Galactosaminidase/químicaRESUMO
The modification of nucleocytoplasmic proteins with O-linked N-acetylglucosamine (O-GlcNAc) plays diverse roles in multicellular organisms. Inhibitors of O-GlcNAc hydrolase (OGA), the enzyme that removes O-GlcNAc from proteins, lead to increased O-GlcNAc levels in cells and are seeing widespread adoption in the field as a research tool used in cells and in vivo. Here we synthesize and study a series of tight binding carbohydrate-based inhibitors of human OGA (hOGA). The most potent of these 2'-aminothiazolines binds with a sub-nanomolar Ki value to hOGA (510 ± 50 pM) and the most selective has greater than 1 800 000-fold selectivity for hOGA over mechanistically related human lysosomal ß-hexosaminidase. Structural data of inhibitors in complex with an hOGA homologue reveals the basis for variation in binding among these compounds. Using linear free energy analyses, we show binding of these 2'-aminothiazoline inhibitors depends on the pKa of the aminothiazoline ring system, revealing the protonation state of the inhibitor is a key driver of binding. Using series of inhibitors and synthetic substrates, we show that 2'-aminothiazoline inhibitors are transition state analogues of hOGA that bind to the enzyme up to 1-million fold more tightly than the substrate. These collective data support an oxazoline, rather than a protonated oxazolinium ion, intermediate being formed along the reaction pathway. Inhibitors from this series will prove generally useful tools for the study of O-GlcNAc. The new insights gained here, into the catalytic mechanism of hOGA and the fundamental drivers of potency and selectivity of OGA inhibitors, should enable tuning of hOGA inhibitors with desirable properties.
RESUMO
Complex regulatory decisions about risk rely on the brokering of evidence between providers and recipients, and involve personality and power relationships that influence the confidence that recipients may place in the sufficiency of evidence and, therefore, the decision outcome. We explore these relationships in an agent-based model; drawing on concepts from environmental risk science, decision psychology and computer simulation. A two-agent model that accounts for the sufficiency of evidence is applied to decisions about salt intake, animal carcass disposal and radioactive waste. A dynamic version of the model assigned personality traits to agents, to explore their receptivity to evidence. Agents with 'aggressor' personality sets were most able to imbue fellow agents with enhanced receptivity (with 'avoider' personality sets less so) and clear confidence in the sufficiency of evidence. In a dynamic version of the model, when both recipient and provider were assigned the 'aggressor' personality set, this resulted in 10 successful evidence submissions in 71 days, compared with 96 days when both agents were assigned the 'avoider' personality set. These insights suggest implications for improving the efficiency and quality of regulatory decision making by understanding the role of personality and power.
Assuntos
Tomada de Decisões , Personalidade , Poder Psicológico , Animais , Cadáver , Ingestão de Alimentos , Humanos , Modelos Teóricos , Resíduos Radioativos/análise , Medição de Risco , Cloreto de Sódio/metabolismo , Gerenciamento de Resíduos/métodosRESUMO
Silks are fibres produced by spiders, some insects and even a crustacean, and are formed from protein solution by a pulltrusion process that is not well understood. Here we describe three aspects of the functional anatomy of the spinning apparatus in a spider: (i) changes in the diameter of the duct of the silk gland along its length for individuals at different stages of development, (ii) the correlation between the morphology of the duct and size and (iii) changes in the thickness of the wall of the duct. We conclude that in the distal part of the duct both the lumen's geometry and change in diameter with distance remains remarkably constant as the duct increases in length from moult to moult as the spider grows. This suggests constancy in the region where the nascent silk filament is drawn down within the lumen of the duct, which is likely to be fundamental for forming strong and tough fibres.
Assuntos
Seda/biossíntese , Aranhas/anatomia & histologia , Aranhas/crescimento & desenvolvimento , Animais , Feminino , Aranhas/metabolismoRESUMO
AIM: Lunges are commonly included in rehabilitation and strength training programs; however limited information regarding differences between lateral and forward lunges with varying step lengths in young adults exists. The current study compared sagittal plane joint kinematics and kinetics between forward and lateral lunges using self-selected and standardized (60% height) step lengths. METHODS: Thirty-two young adults (16 men, 16 women) completed six lunges of each direction/distance combination while stepping (dominant) limb ankle, knee, and hip peak flexion and net joint extensor moment impulse were quantified. RESULTS: While lateral direction (P=0.063) step lengths were statistically equal between self-selected and standardized lunges, forward self-selected distances were 10% less than the standardized (P<0.001). Compared to forward lunges, lateral lunge ankle flexion was 83.5% greater (P<0.001) for standard and 55.3% greater (P<0.001) for self-selected distances. Knee flexion was 12.8% greater (P<0.001) during forward lunges compared to lateral lunges, with no significant hip direction differences. Ankle impulse during the lateral lunges was 71.3% greater (P<0.001) compared to forward lunges. Lateral lunge knee impulse was 47.6% greater (P<0.001) for standardized and 16.9% greater (P=0.001) for self-selected distances compared to forward lunges. Forward lunge hip impulse was 64.5% greater for the standardized (P<0.001) and 44.6% greater for self-selected (P<0.001) distances compared to lateral lunges. CONCLUSION: Forward lunges, particularly using 60% body height step length, appear to place the greatest demands on the hip extensors. Lateral lunges prompted greater ankle flexion and greater ankle and knee extensor kinetic contributions. These data provide rationale for lunge variation selection for young adults.
Assuntos
Fenômenos Biomecânicos/fisiologia , Atividade Motora/fisiologia , Treinamento Resistido/métodos , Adulto , Análise de Variância , Articulação do Tornozelo/fisiologia , Feminino , Articulação do Quadril/fisiologia , Humanos , Articulação do Joelho/fisiologia , Masculino , Militares , Músculo Esquelético/fisiologiaRESUMO
BACKGROUND: The possible influence of diet and body weight on bowel habit in children is unknown. The present study aimed to investigate the inter-relationships between bowel function, excess body weight and dietary intake in a group of preadolescent children. METHODS: Eighty-four preadolescent children aged 7-10 years were recruited [mean (SD) age 9.7 (1.0) years]. All children completed a bowel habit diary, examining specific parameters of bowel function and a weighed food inventory concurrently for seven consecutive days. Height and weight measurements were also taken. Children were grouped according to whether they met dietary recommendations and by overweight status; differences in bowel function between the groups were then analysed. RESULTS: Children who exceeded reference values for fat were more likely to report an incidence of straining to start (P = 0.005) and pain during defaecation (P = 0.021). Subjects who met protein recommendations were less likely to report incomplete evacuation (P = 0.000) and those who met zinc recommendations were less likely to report pain during defaecation (P = 0.044). Excess body weight (according to International Obesity Task Force cut-offs) was also associated with poor bowel habit, with overweight and obese children reporting lower defaecation frequency and a higher incidence of straining and feelings of incomplete evacuation, although these findings were not statistically significant. Defaecation frequency in healthy children was 1.4 defaecations per day compared to 1.2 defaecations for overweight and obese children. CONCLUSION: A poor diet that fails to meet dietary recommendations as well as being overweight and obese appears to be associated with increased defaecation problems in preadolescent children.
Assuntos
Defecação , Dieta , Sobrepeso/complicações , Índice de Massa Corporal , Peso Corporal , Criança , Colo/fisiopatologia , Constipação Intestinal/complicações , Constipação Intestinal/epidemiologia , Estudos Transversais , Registros de Dieta , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/efeitos adversos , Proteínas Alimentares/administração & dosagem , Feminino , Grupos Focais , Humanos , Masculino , Sobrepeso/fisiopatologia , Prevalência , Autorrelato , Reino Unido/epidemiologia , Zinco/administração & dosagem , Zinco/efeitos adversosRESUMO
Micronutrient status is of fundamental importance both upon conception and throughout pregnancy. There is an abundance of literature investigating nutrient intakes during individual trimesters of pregnancy but few studies have investigated baseline intakes of nutrients throughout gestation as a continuum. The current investigation set out to measure habitual micronutrient intakes at weeks 13, 25, 35 of pregnancy and 6 weeks postpartum using a prospective background information questionnaire, 4-7-day weighed food diary and postnatal questionnaire. Seventy-two primiparous, Caucasian Londoners were recruited at the study start with 42 completing the first, second, third trimester and postpartum study stages respectively. Study findings indicated that sodium intakes were significantly higher than UK guidelines throughout and after pregnancy (P < 0.001). Intakes of folate, iron, vitamin D, potassium, iodine and selenium were lower than UK recommendations during and after pregnancy, but to varying levels of statistical significance (P < 0.05). Only 23-38% of women met UK recommendations for folate (300 microg day(-1)) through dietary sources. Similarly, only a small percentage of women met dietary guidelines for iron (19-28%). The findings from the current study indicate that public health interventions may be required to help expectant mothers achieve an optimal diet, particularly after birth when dietary recommendations increase for some micronutrients.
Assuntos
Inquéritos sobre Dietas , Comportamento Alimentar , Fenômenos Fisiológicos da Nutrição Materna/fisiologia , Micronutrientes/administração & dosagem , Necessidades Nutricionais , Estado Nutricional , Adulto , Inglaterra , Feminino , Humanos , Política Nutricional , Período Pós-Parto , Gravidez , Resultado da Gravidez , Estudos Prospectivos , Inquéritos e Questionários , Aumento de Peso , Adulto JovemRESUMO
Recent trends in risk-based decision making are reviewed in relation to novel developments in comparative risk analysis, strategic risk analysis, weight of evidence frameworks, and participative decision making. Delivery of these innovations must take account of organisational capabilities in risk management and the institutional culture that implements decision on risk. We stress the importance of managing risk knowledge within organisations, and emphasise the use of core criteria for effective risk-based decisions by reference to decision process, implementation and the security of strategic added value.
Assuntos
Tomada de Decisões , Ecologia/tendências , Meio Ambiente , Conservação dos Recursos Naturais/legislação & jurisprudência , Ecologia/legislação & jurisprudência , Modelos Teóricos , Formulação de Políticas , Medição de Risco/métodos , Medição de Risco/tendênciasRESUMO
Glycosyltransferases catalyze glycosidic bond formation using sugar donors containing a nucleoside phosphate or a lipid phosphate leaving group. Only two structural folds, GT-A and GT-B, have been identified for the nucleotide sugar-dependent enzymes, but other folds are now appearing for the soluble domains of lipid phosphosugar-dependent glycosyl transferases. Structural and kinetic studies have provided new insights. Inverting glycosyltransferases utilize a direct displacement S(N)2-like mechanism involving an enzymatic base catalyst. Leaving group departure in GT-A fold enzymes is typically facilitated via a coordinated divalent cation, whereas GT-B fold enzymes instead use positively charged side chains and/or hydroxyls and helix dipoles. The mechanism of retaining glycosyltransferases is less clear. The expected two-step double-displacement mechanism is rendered less likely by the lack of conserved architecture in the region where a catalytic nucleophile would be expected. A mechanism involving a short-lived oxocarbenium ion intermediate now seems the most likely, with the leaving phosphate serving as the base.
Assuntos
Glicosiltransferases/química , Glicosiltransferases/fisiologia , Animais , Bacillus subtilis/metabolismo , Carboidratos/química , Enzimas/química , Evolução Molecular , Genômica , Glicômica , Glicosilação , Humanos , Íons , Conformação Molecular , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de ProteínaAssuntos
Testes de Função Plaquetária/métodos , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Sequências de Repetição em Tandem/genética , Aspirina/farmacologia , Ensaio de Imunoadsorção Enzimática , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores da Agregação Plaquetária/farmacologiaRESUMO
BACKGROUND: The diagnosis of functional constipation according to Rome II criteria includes assessment of straining. However the prevalence in older adults is unknown. AIMS: To assess the prevalence of straining and its association with stool frequency in free-living (FL) and institutionalised (INS) older adults. METHODS: 50 FL subjects (mean age 74 years, range (65-97), 42% male) and 42 INS subjects (mean age 84 years, range (69-101) 36% male) were recruited. Stool frequency and straining to start and to finish were prospectively recorded by subjects for 7 consecutive days in a bowel habit diary. Statistical analyses were performed using the Chi-square or the Pearson correlation coefficient as appropriate. RESULTS: The mean stool frequency (n/week) was significantly higher (p <0.001) in the FL group compared with the INS group (11.7 and 4.9 respectively). The percentage of subjects experiencing straining to start on more than 25% of occasions was significantly lower in the FL compared with the INS group (34% and 64% respectively, chi2 = 8.4, p = 0.004, df = 1). The percentage of subjects experiencing straining to finish on more than 25% of occasions was significantly lower in the FL compared with the INS group (16% and 41% respectively, chi2 = 6.9, p = 0.009, df = 1). CONCLUSIONS: FL subjects had significantly higher stool frequency and had to strain passing a stool (to start and to finish) less often than their INS counterparts. Moreover, straining to start was experienced more often than straining to finish in both groups.
Assuntos
Envelhecimento/fisiologia , Constipação Intestinal/epidemiologia , Defecação/fisiologia , Instituição de Longa Permanência para Idosos , Institucionalização , Idoso , Idoso de 80 Anos ou mais , Distribuição de Qui-Quadrado , Constipação Intestinal/diagnóstico , Feminino , Avaliação Geriátrica , Humanos , Masculino , Prevalência , Estudos Prospectivos , Inquéritos e QuestionáriosRESUMO
BACKGROUND: The diagnosis of functional constipation according to Rome II criteria includes assessment of straining. However the prevalence in older adults is unknown. Moreover, laxative use increases with age, especially in the elderly. AIMS: to assess the prevalence of straining and its association with laxative use in free-living (FL) and institutionalised (INS) older adults. METHODS: 50 FL (mean age 74 years, 42% male) and 42 INS subjects (mean age 84 years, 36% male) were recruited. Straining to start and to finish defecation were prospectively recorded by subjects for 7 consecutive days in a bowel habit diary. Concurrently, the subjects recorded any laxative use during the 7 days study. Statistical analyses were performed using the Chi-square statistic. RESULTS: 20% of FL and 65% of INS subjects recorded taking laxatives during the study week. Of the 40 FL subjects not taking laxatives, 30 had to strain to start on 25% or less of occasions and 36 had to strain to finish on 25% or less of occasions (chi(2) = 7.2; p = 0.012 and chi(2) = 5.4; p = 0.041, respectively). In the INS group, although 64% of subjects taking laxatives had to strain on more than 25% of occasions, the Chi-square test was not statistically significant. CONCLUSIONS: From our results, it seems that laxatives were used appropriately in the FL, with the majority of those taking laxatives having to strain to start on more than 25% of occasions.
Assuntos
Atividades Cotidianas , Catárticos/uso terapêutico , Constipação Intestinal/tratamento farmacológico , Constipação Intestinal/fisiopatologia , Defecação/fisiologia , Instituição de Longa Permanência para Idosos , Idoso , Feminino , Humanos , Londres , Masculino , Casas de SaúdeRESUMO
The conformational agenda harnessed by different glycosidases along the reaction pathway has been mapped by X-ray crystallography. The transition state(s) formed during the enzymic hydrolysis of glycosides features strong oxocarbenium-ion-like character involving delocalization across the C-1-O-5 bond. This demands planarity of C-5, O-5, C-1 and C-2 at or near the transition state. It is widely, but incorrectly, assumed that the transition state must be (4)H(3) (half-chair). The transition-state geometry is equally well supported, for pyranosides, by both the (4)H(3) and (3)H(4) half-chair and (2,5)B and B(2,5) boat conformations. A number of retaining beta-glycosidases acting on gluco -configured substrates have been trapped in Michaelis and covalent intermediate complexes in (1)S(3) (skew-boat) and (4)C(1) (chair) conformations, respectively, pointing to a (4)H(3)-conformed transition state. Such a (4)H(3) conformation is consistent with the tight binding of (4)E- (envelope) and (4)H(3)-conformed transition-state mimics to these enzymes and with the solution structures of compounds bearing an sp (2) hybridized anomeric centre. Recent work reveals a (1)S(5) Michaelis complex for beta-mannanases which, together with the (0)S(2) covalent intermediate, strongly implicates a B(2,5) transition state for beta-mannanases, again consistent with the solution structures of manno -configured compounds bearing an sp (2) anomeric centre. Other enzymes may use different strategies. Xylanases in family GH-11 reveal a covalent intermediate structure in a (2,5)B conformation which would also suggest a similarly shaped transition state, while (2)S(0)-conformed substrate mimics spanning the active centre of inverting cellulases from family GH-6 may also be indicative of a (2,5)B transition-state conformation. Work in other laboratories on both retaining and inverting alpha-mannosidases also suggests non-(4)H(3) transition states for these medically important enzymes. Three-dimensional structures of enzyme complexes should now be able to drive the design of transition-state mimics that are specific for given enzymes, as opposed to being generic or merely fortuitous.
Assuntos
Glicosídeo Hidrolases/química , Celulases/química , Celulases/metabolismo , Cristalografia por Raios X , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Glicosídeo Hidrolases/metabolismo , Hidrólise , Conformação Proteica , beta-Manosidase/química , beta-Manosidase/metabolismoRESUMO
The drinking habits of children aged 11 to 16 years attending a comprehensive school are reported. Pupils were able to access alcoholic drinks readily despite being underage. A general awareness of the health and social risks of drinking did not act as a deterrent and, alarmingly, only less than 10% of the group reported that they did not drink. Health education and the role that schools can play are highlighted.
Assuntos
Consumo de Bebidas Alcoólicas/epidemiologia , Adolescente , Consumo de Bebidas Alcoólicas/efeitos adversos , Consumo de Bebidas Alcoólicas/prevenção & controle , Área Programática de Saúde , Criança , Feminino , Educação em Saúde , Pesquisa sobre Serviços de Saúde , Humanos , Masculino , Inquéritos e Questionários , Reino Unido/epidemiologiaRESUMO
OBJECTIVE: To examine the effects of L-arginine on basal coronary tone and flow mediated dilatation induced by atrial pacing in patients with coronary artery disease and stable angina. DESIGN: Atrial pacing was performed during intracoronary infusions of normal saline and L-arginine (150 micromol/min) in 8 patients with coronary artery disease and stable angina. The luminal diameter of epicardial coronary arteries was assessed by quantitative angiography. RESULTS: L-arginine administration significantly increased the diameter of all the coronary segments and stenoses. During atrial pacing with saline infusion, luminal diameter of the proximal, distal, and stenosis reference segments increased significantly (p < 0.01 versus saline) but stenosis diameter did not change. L-arginine administration did not change the magnitude (NS) of atrial pacing induced dilatation in proximal and distal segments and in coronary stenoses and their reference segments. CONCLUSIONS: Non-stenotic segments of diseased coronary arteries dilate in response to atrial pacing but stenoses do not. L-arginine dilates coronary segments and stenoses but does not increase the magnitude of the response to atrial pacing in proximal and distal segments and in coronary stenoses and their reference segments. These findings provide evidence that the shear stress responsive mechanism is absent at stenoses but present in non-stenotic segments of diseased coronary arteries. They also indicate a relative deficiency of L-arginine, except in the shear response mechanism.
Assuntos
Angina Pectoris/terapia , Arginina/uso terapêutico , Estimulação Cardíaca Artificial , Circulação Coronária/efeitos dos fármacos , Doença das Coronárias/terapia , Vasodilatadores/uso terapêutico , Angina Pectoris/fisiopatologia , Velocidade do Fluxo Sanguíneo , Angiografia Coronária , Doença das Coronárias/fisiopatologia , Estenose Coronária/tratamento farmacológico , Estenose Coronária/fisiopatologia , Vasos Coronários/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Evidence from epidemiological studies overwhelmingly indicates that fruit, vegetables and cereals prevent cancer at a wide range of sites in the body, not just the large bowel. However, recent important papers have shown that an intervention diet rich in fruit, vegetables and fibre failed to decrease the risk of new colorectal adenoma formation. The adenoma-cancer sequence and the role of diet are discussed in the light of these new findings.
Assuntos
Adenoma/prevenção & controle , Neoplasias Colorretais/prevenção & controle , Fibras na Dieta , Dieta , Feminino , Frutas , Humanos , Masculino , VerdurasRESUMO
To investigate a possible association between the human prothrombin gene G20210A polymorphism and coronary artery thrombosis, we screened 172 consecutive patients with ischaemic heart disease admitted for coronary arteriography. The patients were divided into two groups on the basis of their clinical history and examination of their hospital records: 66 patients with a definite previous myocardial infarction, and 106 with angina-like chest pain but no evidence of myocardial infarction. The overall frequency of the G20210A polymorphism was 0.011, four out of the 172 patients being heterozygous for the mutation. The allelic frequency was 0.015 in the group with myocardial infarction and 0.009 in the group without myocardial infarction (P = 0.622). The results of this study suggest that the single nucleotide polymorphism at position 20210 of the prothrombin gene is unlikely to be a risk factor for coronary thrombosis.
Assuntos
Infarto do Miocárdio/genética , Polimorfismo de Nucleotídeo Único , Protrombina/genética , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Frequência do Gene , Genótipo , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/etiologia , Isquemia Miocárdica , Trombose/etiologia , Trombose/genéticaRESUMO
OBJECTIVE: To compare iodine intakes estimated from weighed dietary records with iodine intakes obtained by direct analysis of duplicate diets in a group of vegans. DESIGN: Cross-sectional study. SETTING: London and the south-east of England. SUBJECTS: Thirty-three vegans consuming their habitual diet were recruited through the UK Vegan Society; 26 subjects (11 males, 15 females), age 21-84 y, completed the study. INTERVENTIONS: Iodine intakes were estimated from 4 day weighed dietary records and compared with iodine intakes obtained by direct analysis of concurrent 4 day duplicate diets. RESULTS: There was wide variation in iodine intakes. Mean daily iodine intake in males was significantly lower (P<0.05) when estimated from dietary records (42 microg) compared with that analysed from duplicate diets (137 microg). Conversely, in females the mean daily iodine intake from dietary records (1448 microg) was higher (P=0.43) than from duplicate diets (216 microg). Variation in iodine intakes determined by the two different methods may be attributed to the absence of iodine content of some foods, in particular foods suitable for vegan consumption, in food composition tables and the variability in iodine content of seaweed. CONCLUSIONS: The use of current food tables to estimate iodine intake in vegans is limited. It is not always practical to determine iodine intake using the duplicate portion technique, therefore more reliable information on iodine content of foods, incorporating the variation within foods, is needed.