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The potential for Ni toxicity in seawater is of concern because of mining and processing activities in coastal regions. Determining Ni speciation is vital to understanding and predicting Ni toxicity and for bioavailability-based nickel risk assessment. The goal of this study was to characterize the complexation of Ni in relation to toxicity using embryological development of purple sea urchin (S. purpuratus). It was predicted that free ion [Ni2+] would be a better predictor of toxicity than total dissolved Ni concentrations (NiD). Synthetic ligands with known logKf values (Ethylenediaminetetraacetic acid (EDTA), Nitrilotriacetic acid (NTA), tryptophan (TRP), glutamic acid (GA), histidine (HD), and citric acid (CA)) were used to test the assumptions of the biotic ligand model (BLM) for Ni in seawater. [NiD] was measured by graphite furnace atomic absorption spectroscopy (GFAAS) and Ni2+ was first quantified using the ion-exchange technique (IET) and then concentrations were measured by GFAAS; [Ni2+] was also estimated using aquatic geochemistry modelling software (Visual Minteq). The mean EC50 values for [NiD] in unmodified artificial seawater control was 3.6 µM (95% CI 3.0-4.5) [211 µg/L 95% CI 176-264] and the addition of ligands provided protection, up to 6.5-fold higher [NiD] EC50 for EDTA. Compared to the control, measured EC50 values based on total dissolved nickel were higher in the presence of ligands. As predicted by BLM theory, [Ni2+] was a better predictor of Ni toxicity with 17% variability in EDTA and CA media while there was 72% variability in the prediction of Ni toxicity with total dissolved Ni. The results of this research provide support for the application of BLM- based prediction models for estimating Ni impacts in seawater.
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BACKGROUND: Integration of mental health services into primary healthcare is proliferating in low-resource countries. We aimed to evaluate the impact of different compositions of primary care mental health services for depression and alcohol use disorder (AUD), when compared to usual primary care services. METHODS: We conducted a non-randomized controlled study in rural Nepal. We compared treatment outcomes among patients screening positive and receiving: (a) primary care mental health services without a psychological treatment component (TG); (b) the same services including a psychological treatment (TG + P); and (c) primary care treatment as usual (TAU). Primary outcomes included change in depression and AUD symptoms, as well as disability. Disability was measured using the 12-item WHO Disability Assessment Schedule. Symptom severity was assessed using the 9-item Patient Health Questionnaire for depression, the 10-item Alcohol Use Disorders Identification Test for AUD. We used negative binomial regression models for the analysis. RESULTS: For depression, when combining both treatment groups (TG, n = 77 and TG + P, n = 60) compared to TAU (n = 72), there were no significant improvements. When only comparing the psychological treatment group (TG + P) with TAU, there were significant improvements for symptoms and disability (aß = - 2.64; 95%CI - 4.55 to - 0.74, p = 0.007; aß = - 12.20; 95%CI - 19.79 to - 4.62; p = 0.002, respectively). For AUD, when combining both treatment groups (TG, n = 92 and TG + P, n = 80) compared to TAU (n = 57), there were significant improvements in AUD symptoms and disability (aß = - 15.13; 95%CI - 18.63 to - 11.63, p < 0.001; aß = - 9.26; 95%CI - 16.41 to - 2.12, p = 0.011; respectively). For AUD, there were no differences between TG and TG + P. Patients' perceptions of health workers' skills in common psychological factors were associated with improvement in depression patient outcomes (ß = - 0.36; 95%CI - 0.55 to - 0.18; p < 0.001) but not for AUD patients. CONCLUSION: Primary care mental health services for depression may only be effective when psychological treatments are included. Health workers' competencies as perceived by patients may be an important indicator for treatment effect. AUD treatment in primary care appears to be beneficial even without additional psychological services.
Assuntos
Alcoolismo , Serviços de Saúde Mental , Alcoolismo/terapia , Depressão/terapia , Humanos , Saúde Mental , NepalRESUMO
BACKGROUND: We use the Global Multidimensional Poverty Index (MPI) to explore how different dimensions of poverty more directly linked to young people are associated with depressive symptoms among South African youth. METHODS: Data came from the 2017 wave of the nationally-representative National Income Dynamics Study (NIDS) in South Africa. We focused on a sample of 15-24-year-olds whose depressive symptoms were assessed using an adapted version of the 10-item Centre for Epidemiological Studies Depression Scale. We examine how individual dimensions and indicators of the MPI relate to depression, in comparison to more conventional measures, including household income, subjective social standing, overcrowding and personal assets. Cross-sectional analyses were adjusted for clustering to account for sampling design. RESULTS: The MPI index was not associated with probable depression (OR = 1.02, 95 % CI 0.81-1.29). Only lack of access to the labour market emerged as a key individual dimension associated with probable depression (OR = 5.29, 95 % CI 1.70-16.47), a relationship driven by an increased odds for those not in employment, education or training. Lack of household assets, living in an informal dwelling and lower perceived social standing were also associated with increased odds for depression. No gender differences were noted. LIMITATIONS: The study is cross-sectional and not suitable to examine the causal nature of the association between multidimensional poverty and depression. CONCLUSIONS: Poverty dimensions that measure youth's access to employment or training have a strong association with depression. Further research is needed to assess whether improved access to employment or training contributes to improving mental health among young South Africans.
Assuntos
Depressão , Pobreza , Adolescente , Humanos , Estudos Transversais , Depressão/epidemiologia , Depressão/psicologia , África do Sul/epidemiologia , Pobreza/psicologia , RendaRESUMO
This study examines the longitudinal impact of the South African Child Support Grant (CSG) on risk for depression and life satisfaction among young people (15-19 years). We analysed data from the last three waves of the National Income Dynamics Study (NIDS), a nationally representative panel survey that took place every two years from 2008 to 2017. We used an instrumental variable (IV) approach that exploits multiple changes in age eligibility from 1998 to 2012. Depressive symptoms were assessed using an 8-item version of the Centre for Epidemiological Studies Depression Scale; participants who scored above 8 were considered at risk for depression. Life satisfaction was rated on a scale of 1 ('very dissatisfied') to 10 ('very satisfied'); participants who scored 8 or above were classified as satisfied. We also examined impacts on educational deficit (≥2 years behind) and not being in education, employment or training (NEET) as secondary outcomes, as these are also important for mental health. Age eligibility strongly predicted CSG receipt at Wave 3. In instrumental variable models, CSG receipt did not influence the risk for depression (ß = 0.10, SE = 0.10, p = 0.316), nor life satisfaction (ß = -0.07, SE = 0.09, p = 0.420) at Wave 3, nor at Waves 4 or 5. Some improvements in educational deficit were observed at Wave 3 among CSG beneficiaries compared to non-beneficiaries. These results were robust to multiple specifications. CSG receipt did not improve the psychological wellbeing of adolescents and young adults, nor did it improve their education or employment outcomes. Our findings highlight the need to identify alternative social policies that address the root causes of youth social disadvantage, in conjunction with targeted approaches to improve the mental health of young South Africans living in poverty.
Assuntos
Custódia da Criança , Saúde Mental , Adolescente , Criança , Organização do Financiamento , Humanos , Pobreza , África do Sul/epidemiologia , Adulto JovemRESUMO
AIM: There is limited evidence of the safety and impact of task-shared care for people with severe mental illnesses (SMI; psychotic disorders and bipolar disorder) in low-income countries. The aim of this study was to evaluate the safety and impact of a district-level plan for task-shared mental health care on 6 and 12-month clinical and social outcomes of people with SMI in rural southern Ethiopia. METHODS: In the Programme for Improving Mental health carE, we conducted an intervention cohort study. Trained primary healthcare (PHC) workers assessed community referrals, diagnosed SMI and initiated treatment, with independent research diagnostic assessments by psychiatric nurses. Primary outcomes were symptom severity and disability. Secondary outcomes included discrimination and restraint. RESULTS: Almost all (94.5%) PHC worker diagnoses of SMI were verified by psychiatric nurses. All prescribing was within recommended dose limits. A total of 245 (81.7%) people with SMI were re-assessed at 12 months. Minimally adequate treatment was received by 29.8%. All clinical and social outcomes improved significantly. The impact on disability (standardised mean difference 0.50; 95% confidence interval (CI) 0.35-0.65) was greater than impact on symptom severity (standardised mean difference 0.28; 95% CI 0.13-0.44). Being restrained in the previous 12 months reduced from 25.3 to 10.6%, and discrimination scores reduced significantly. CONCLUSIONS: An integrated district level mental health care plan employing task-sharing safely addressed the large treatment gap for people with SMI in a rural, low-income country setting. Randomised controlled trials of differing models of task-shared care for people with SMI are warranted.
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Transtornos Psicóticos Afetivos/terapia , Transtorno Bipolar/terapia , Serviços Comunitários de Saúde Mental/métodos , Atenção Primária à Saúde/métodos , Enfermagem Psiquiátrica , Transtornos Psicóticos/terapia , Esquizofrenia/terapia , Adulto , Transtornos Psicóticos Afetivos/diagnóstico , Transtornos Psicóticos Afetivos/fisiopatologia , Transtorno Bipolar/diagnóstico , Transtorno Bipolar/fisiopatologia , Estudos de Coortes , Serviços Comunitários de Saúde Mental/organização & administração , Atenção à Saúde , Etiópia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Preconceito , Transtornos Psicóticos/diagnóstico , Transtornos Psicóticos/fisiopatologia , Regionalização da Saúde , Restrição Física , População Rural , Esquizofrenia/diagnóstico , Esquizofrenia/fisiopatologia , Índice de Gravidade de Doença , Estigma Social , Adulto JovemRESUMO
BACKGROUND: The burden of mental disorders in low- and middle-income countries is large. Yet there is a major treatment gap for these disorders which can be reduced by integrating the care of mental disorders in primary care. AIM: We aimed to evaluate the impact of a district mental health care plan (MHCP) on contact coverage for and detection of mental disorders, as well as impact on mental health symptom severity and individual functioning in rural Uganda. RESULTS: For adults who attended primary care facilities, there was an immediate positive effect of the MHCP on clinical detection at 3 months although this was not sustained at 12 months. Those who were treated in primary care experienced significant reductions in symptom severity and functional impairment over 12 months. There was negligible change in population-level contact coverage for depression and alcohol use disorder. CONCLUSION: The study found that it is possible to integrate mental health care into primary care in rural Uganda. Treatment by trained primary care workers improves clinical and functioning outcomes for depression, psychosis and epilepsy. Challenges remain in accessing the men for care, sustaining the improvement in detection over time, and creating demand for services among those with presumed need.
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BACKGROUND: Vibrio cholerae neuraminidase is part of a mucinase complex which may function in pathogenesis by degrading the mucin layer of the gastrointestinal tract. The neuraminidase, which has been the target of extensive inhibitor studies, plays a subtle role in the pathology of the bacterium, by processing higher order gangliosides to GM1, the receptor for cholera toxin. RESULTS: We report here the X-ray crystal structure of V. cholerae neuraminidase at 2.3 A resolution. The 83 kDa enzyme folds into three distinct domains. The central catalytic domain has the canonical neuraminidase beta-propeller fold, and is flanked by two domains which possess identical legume lectin-like topologies but without the usual metal-binding loops. The active site has many features in common with other viral and bacterial neuraminidases but, uniquely, has an essential Ca2+ ion which plays a crucial structural role. CONCLUSIONS: The environment of the small intestine requires V. cholerae to secrete several adhesins, and it is known that its neuraminidase can bind to cell surfaces, and remain active. The unexpected lectin-like domains possibly mediate this attachment. These bacterial lectin folds represent additional members of a growing lectin superfamily.
Assuntos
Neuraminidase/química , Vibrio cholerae/enzimologia , Aderência Bacteriana , Sítios de Ligação , Lectinas/química , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Relação Estrutura-Atividade , Difração de Raios XRESUMO
BACKGROUND: Control of intracellular events by protein phosphorylation is promoted by specific protein kinases. All the known protein kinase possess a common structure that defines a catalytically competent entity termed the 'kinase catalytic core'. Within this common structural framework each kinase displays its own unique substrate specificity, and a regulatory mechanism that may be modulated by association with other proteins. Structural studies of phosphorylase kinase (Phk), the major substrate of which is glycogen phosphorylase, may be expected to shed light on its regulation. RESULTS: We report two crystal structures of the catalytic core (residues 1-298; Phk gamma trnc) of the gamma-subunit of rabbit muscle phosphorylase kinase: the binary complex with Mn2+/beta-gamma-imidoadenosine 5'-triphosphate (AMPPNP) to a resolution of 2.6 A and the binary complex with Mg2+/ADP to a resolution of 3.0 A. The structures were solved by molecular replacement using the cAMP-dependent protein kinase (cAPK) as a model. CONCLUSIONS: The overall structure of Phk gamma trnc is similar to that of the catalytic core of other protein kinases. It consists of two domians joined on one edge by a 'hinge', with the catalytic site located in the cleft between the domains. Phk gamma trnc is constitutively active, and lacks the need for an activatory phosphorylation event that is essential for many kinases. The structure exhibits an essentially 'closed' conformation of the domains which is similar to that of cAPK complexed with substrates. The phosphorylated residue that is located at the domain interface in many protein kinases and that is believed to stabilize an active conformation is substituted by a glutamate in Phk gamma trnc. The glutamate, in a similar manner to the phosphorylated residue in other protein kinases, interacts with an arginine adjacent to the catalytic aspartate but does not participate in interdomain contacts. The interactions between the enzyme and the nucleotide product of its activity, Mg2+/ADP, explain the inhibitory properties of the nucleotides that are observed in kinetic studies.
Assuntos
Adenilil Imidodifosfato/metabolismo , Fosforilase Quinase/química , Estrutura Terciária de Proteína , Difosfato de Adenosina/metabolismo , Adenilil Imidodifosfato/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Catálise , Cristalografia por Raios X , Proteínas Quinases Dependentes de AMP Cíclico/química , Magnésio/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Fosforilase Quinase/metabolismo , Conformação Proteica , Coelhos , Especificidade por SubstratoRESUMO
BACKGROUND: Eukaryotic cell cycle progression is regulated by cyclin dependent protein kinases (CDKs) whose activity is regulated by association with cyclins and by reversible phosphorylation. Cyclins also determine the subcellular location and substrate specificity of CDKs. Cyclins exhibit diverse sequences but all share homology over a region of approximately 100 amino acids, termed the cyclin box. From the determination of the structure of cyclin A, together with results from biochemical and genetic analyses, we can identify which parts of the cyclin molecular may contribute to cyclin A structure and function. RESULTS: We have solved the crystal structure, at 2.0 A resolution, of an active recombinant fragment of bovine cyclin A, cyclin A-3, corresponding to residues 171-432 of human cyclin A. The cyclin box has an alpha-helical fold comprising five alpha helices. This fold is repeated in the C-terminal region, although this region shares negligible sequence similarity with the cyclin box. CONCLUSIONS: Analysis of residues that are conserved throughout the A, B, and E cyclins identifies two exposed clusters of residues, one of which has recently been shown to be involved in the association with human CDK2. The second cluster may identify another site of cyclin A-protein interaction. Comparison of the structure of the unbound cyclin with the structure of cyclin A complexed with CDK2 reveals that cyclin A does not undergo any significant conformational changes on complex formation. Threading analysis shows that the cyclin-box fold is consistent with the sequences of the transcription factor TFIIB and other functionally related proteins. The structural results indicate a role for the cyclin-box fold both as a template for the cyclin family and as a generalised adaptor molecule in the regulation of transcription.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Ciclina A , Ciclinas/química , Modelos Moleculares , Fragmentos de Peptídeos/química , Conformação Proteica , Algoritmos , Sequência de Aminoácidos , Animais , Bovinos , Ciclo Celular , Simulação por Computador , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Humanos , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Fator de Transcrição TFIIB , Fatores de Transcrição/química , Transcrição GênicaRESUMO
The nanH genes of Vibrio cholerae and Salmonella typhimurium LT2 coding neuraminidase were cloned separately in Escherichia coli, and the expression products purified. Single crystals of the V. cholerae neuraminidase were obtained using the hanging drop vapour diffusion method with polyethylene glycol as precipitant at pH 7.2. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 71.9 A, b = 79.0 A, c = 165.7 A, and with one molecule in the asymmetric unit. Diffraction extends to at least 2.5 A. Single crystals of the S. typhimurium neuraminidase were obtained by hanging drop with potassium phosphate as precipitant at pH 7.2. The crystals also belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 47.4 A, b = 82.8 A, c = 92.4 A, and with one molecule in the asymmetric unit. Diffraction extends to at least 1.8 A.
Assuntos
Neuraminidase/química , Proteínas Recombinantes/biossíntese , Salmonella typhimurium/enzimologia , Vibrio cholerae/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , Cristalização , Escherichia coli/genética , Dados de Sequência Molecular , Neuraminidase/genética , Neuraminidase/isolamento & purificação , Difração de Raios XRESUMO
Lipase from Pseudomonas glumae has been purified and crystallized in two forms, using the hanging drop method of vapour diffusion at 4 degrees C and 15 degrees C. Both forms grew at pH 9.0 from 0.1 M-Tris buffer in the presence of 10% (v/v) acetone. Form 1 was crystallized from 27 to 29% polyethylene glycol in the presence of less than 0.5% (v/v) n-dodecyl-beta-D-glucopyranoside. Form 2 was grown from 17 to 19% ammonium sulphate in the presence of 1% n-octyl-beta-D-glucopyranoside. Form 1 is orthorhombic with space group P2(1)2(1)2(1), and cell dimensions of a = 158.1 A, b = 158.6 A, c = 63.4 A, Form 2 is tetragonal with space group P4(1)2(1)2 (or P4(3)2(1)2) and cell dimensions of a = 89.3 A, c = 180.4 A. Form 1 probably has four molecules per asymmetric unit and diffracts to at least 2.5 A. Form 2 has two molecules per asymmetric unit and diffracts to at least 3.0 A.
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Lipase/química , Pseudomonas/enzimologia , Cristalização , Lipase/isolamento & purificação , Lipase/metabolismo , Difração de Raios XRESUMO
The AT-selective drug berenil has been co-crystallized with the dodecanucleotide sequence d(CGCAAATTTGCG)2. The crystal structure has been solved to a resolution of 2.0 A and an R factor of 18.3%, with the location of 65 water molecules. The drug is symmetrically bound in the 5'-AATT region of the minor groove, with its amidinium groups hydrogen-bonding to O-2 atoms of the thymine base at each end of the binding site. This arrangement is distinct from that previously found for berenil with the sequence d(CGCGAATTCGCG)2, which has the drug bound to the sequencing 5'-ATT via hydrogen bonds to adenine N-3 atoms with the involvement of a bridging water molecule at one end of the binding site. The reasons for these differences are discussed in terms of changes in helical parameters; in particular propeller twist and base-pair roll are considered to be important. The conformational and base-pair geometry of the dodecanucleotide in the structure reported here, is closely similar to that for the native structure, suggesting that the 5'-AAATTT sequence does not significantly alter during drug binding, either because of its inflexibility or because its geometry is nearly ideal for berenil binding.
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DNA/metabolismo , Diminazena/análogos & derivados , Oligodesoxirribonucleotídeos , Sequência de Bases , Sítios de Ligação , Gráficos por Computador , Cristalografia , Diminazena/química , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Relação Estrutura-Atividade , Água/químicaRESUMO
3-phosphoglycerate kinase (PGK) is a typical kinase with two structural domains. The domains each bind one of the two substrates, 3-phosphoglycerate (3-PG) and MgATP. For the phospho-transfer reaction to take place the substrates must be brought closer by a hinge-bending domain closure. Open and closed structures of the enzyme with different relative domain positions have been determined from different species, but a comprehensive description of this conformational transition is yet to be attained. Crystals of pig muscle PGK in complex with MgADP and 3-phosphoglycerate were grown under the conditions which have previously resulted in crystals of the closed, catalytically competent conformation of Trypanosoma brucei PGK. The X-ray structure of the pig muscle ternary complex was determined at 1.8 A and the model was refined to R=20.8% and Rfree=24.1%. Contrary to expectation, however, it represents an essentially open conformation compared to that of T. brucei PGK. In addition, the beta-phosphate group of ADP is mobile in the new structure, in contrast to its well-defined position in T. brucei PGK. An extensive comparison of the ternary complexes from these remote species has been carried out in order to establish general differences between the two conformations and is reported here. A second pair of the open and closed structures was also compared. These analyses have made it possible to define several characteristic changes which accompany the structural transition, in addition to those identified previously: (1) the operation of a hinge at beta-strand L in the inter-domain region which greatly affects the relative domain positions; (2) the rearrangement and movement of helix 8, regulated through the interactions with the nucleotide phosphate; and (3) the existence of another hinge between helix 14 and the rest of the C-terminal part of the chain, which allows fine adjustment of the N-domain position. The main hinge at beta-strand L acts in concert with the C-terminal hinge at helix 7 described previously. Simultaneous interactions of the nucleotide phosphate groups with the loop that precedes helix 8, beta-strand J and the N terminus of helix 13 are required for propagation of the nucleotide effect towards the beta-strand L molecular hinge. A detailed description of the role of nucleotide binding in the hinge operation is presented.
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Difosfato de Adenosina/metabolismo , Ácidos Glicéricos/metabolismo , Músculos/enzimologia , Fosfoglicerato Quinase/química , Fosfoglicerato Quinase/metabolismo , Suínos , Difosfato de Adenosina/análise , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Movimento (Física) , Fosfatos/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Neuraminidase heads from the following subtypes of influenza A virus have been crystallized: N5, N6, N8 and whale virus N9. The last three yielded crystals of X-ray diffraction quality; the N5 crystals obtained so far are not suitable for high resolution studies. The cell dimensions and space groups of crystals grown from the four subtypes have been determined.
Assuntos
Vírus da Influenza A/enzimologia , Neuraminidase/química , Cristalografia , Neuraminidase/isolamento & purificação , Proteínas Virais/química , Proteínas Virais/isolamento & purificação , Difração de Raios XRESUMO
The catalytic subunit of phosphorylase kinase is composed of a kinase catalytic core domain (residues 1 to 298), which has a 33% identity with the kinase core of the cyclic AMP-dependent protein kinase, and a C-terminal calmodulin binding domain. The kinase domain of the catalytic subunit has been expressed in Escherichia coli, purified and crystallised in the presence of ATP and magnesium from 5% (w/v) polyethylene glycol 8000, 10% (v/v) glycerol, 50 mM Hepes/NaOH (pH 6.9). A three-fold excess of magnesium to ATP was used for crystal growth. The inclusion of glycerol in the crystallization medium produced a marked reduction in mosaic spread of the diffraction spots from greater than 1 degree to 0.3 degree. The crystals are orthorhombic, space group P2(1)2(1)2(1) with unit cell dimensions a = 47.1 A, b = 69.1 A, c = 112.9 A and one molecule per asymmetric unit. Data to 3 A resolution have been collected and structure determination is in progress.
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Fosforilase Quinase/metabolismo , Animais , Catálise , Cromatografia de Afinidade , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Músculo Esquelético/enzimologia , Mutagênese Sítio-Dirigida , Fosforilase Quinase/química , Fosforilase Quinase/genética , Fosforilase Quinase/isolamento & purificação , CoelhosRESUMO
An NAD(P)H quinone reductase isolated from Walker rat 256 carcinoma cells has been crystallized in a form suitable for high-resolution structural analysis. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with cell parameters a = 168.15 A, b = 105.09 A and c = 67.38 A and contain four monomeric or two dimeric enzyme molecules per asymmetric unit. Diffraction extends beyond 2.3 A resolution.
Assuntos
Quinona Redutases , Animais , Linhagem Celular , NAD(P)H Desidrogenase (Quinona) , Ratos , Células Tumorais Cultivadas/enzimologia , Difração de Raios XRESUMO
The structure of Salmonella typhimurium LT2 neuraminidase (STNA) is reported here to a resolution of 1.6 angstroms together with the structures of three complexes of STNA with different inhibitors. The first is 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid (Neu5Ac2en or DANA), the second and third are phosphonate derivatives of N-acetyl-neuraminic acid (NANA) which have phosphonate groups at the C2 position equatorial (ePANA) and axial (aPANA) to the plane of the sugar ring. The complex structures are at resolutions of 1.6 angstroms, 1.6 angstroms and 1.9 angstroms, respectively. These analyses show the STNA active site to be topologically inflexible and the interactions to be dominated by the arginine triad, with the pyranose rings of the inhibitors undergoing distortion to occupy the space available. Solvent structure differs only around the third phosphonate oxygen, which attracts a potassium ion. The STNA structure is topologically identical to the previously reported influenza virus neuraminidase structures, although very different in detail; the root-mean-square (r.m.s) deviation for 210 C alpha positions considered equivalent is 2.28 angstroms (out of a total of 390 residues in influenza and 381 in STNA). The active site residues are more highly conserved, in that both the viral and bacterial structures contain an arginine triad, a hydrophobic pocket, a tyrosine and glutamic acid residue at the base of the site and a potential proton-donating aspartic acid. However, differences in binding to O4 and to the glycerol side-chain may reflect the different kinetics employed by the two enzymes.
Assuntos
Inibidores Enzimáticos/química , Ácido N-Acetilneuramínico/análogos & derivados , Neuraminidase/química , Salmonella typhimurium/enzimologia , Ácidos Siálicos/química , Sítios de Ligação , Catálise , Modelos Moleculares , Neuraminidase/antagonistas & inibidores , Conformação Proteica , Ácidos Siálicos/farmacologiaRESUMO
Reverse transcriptase (RT) from the human immunodeficiency virus type 1 has been crystallized in four closely related forms, the best of which diffract X-rays to 2.2 A resolution. The RT was crystallized as a complex with a non-nucleoside inhibitor, either nevirapine or a nevirapine analogue. Crystals grew from 6% PEG 3400 buffered at pH 5. These were of space group P2(1)2(1)2(1) with unit cell parameters a = 147 A, b = 112 A, c = 79 A (form A), with one RT heterodimer in the asymmetric unit. Changes in unit cell parameters and degree of crystalline order were observed on soaking pregrown crystals in various solutions, giving three further sets of unit cells. These were a = 143 A, b = 112, A, c = 79 A (form B), a = 141 A, b = 111 A, c = 73 A (form C), a = 143 A, b = 117 A, c = 66.5 A (form D). The last two forms diffract X-rays to 2.2 A resolution. Structure determinations of these latter crystal forms of RT should give a detailed atomic model for this therapeutically important drug target.
Assuntos
HIV-1/enzimologia , DNA Polimerase Dirigida por RNA/química , Cristalografia por Raios X , Transcriptase Reversa do HIV , Nevirapina , Piridinas/química , Piridinas/farmacologia , Inibidores da Transcriptase ReversaRESUMO
A glucopyranose spirohydantoin (a pyranose analogue of the potent herbicide, hydantocidin) has been identified as the highest affinity glucose analogue inhibitor of glycogen phosphorylase b (GPb). In order to elucidate the structural features that contribute to the binding, the structures of GPb in the native T state conformation and in complex with glucopyranose spirohydantoin have been determined at 100 K to 2.0 A and 1.8 A resolution, respectively, and refined to crystallographic R values of 0.197 (R[free] 0.248) and 0.182 (R[free] 0.229), respectively. The low temperature structure of GPb is almost identical to that of the previously determined room temperature structure, apart from a decrease in overall atomic temperature factors ((B) room temperature GPb = 34.9 A2; (B) 100 K GPb = 23.4 A2). The glucopyranose spirohydantoin inhibitor (Ki = 3.0 microM) binds at the catalytic site and induces small changes in two key regions of the protein: the 280s loop (residues 281-286) that results in a decrease in mobility of this region, and the 380s loop (residues 377-385) that undergoes more significant shifts in order to optimize contact to the ligand. The hydantoin group, that is responsible for increasing the affinity of the glucose compound by a factor of 10(3), makes only one hydrogen bond to the protein, from one of its NH groups to the main chain oxygen of His377. The other polar groups of the hydantoin group form hydrogen bonds to five water molecules. These waters are involved in extensive networks of hydrogen bonds and appear to be an integral part of the protein structure. Analysis of the water structure at the catalytic site of the native enzyme, shows that five waters are displaced by ligand binding and that there is a significant decrease in mobility of the remaining waters on formation of the GPb-hydantoin complex. The ability of the inhibitor to exploit existing waters, to displace waters and to recruit new waters appears to be important for the high affinity of the inhibitor.
Assuntos
Glucose/química , Hidantoínas/química , Fosforilases/química , Água/química , Animais , Sítios de Ligação/fisiologia , Cristalografia por Raios X , Inibidores Enzimáticos/química , Glucose/análogos & derivados , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Músculos/enzimologia , Ligação Proteica/fisiologia , Conformação Proteica , Coelhos , TemperaturaRESUMO
The three-dimensional structure of the papain-leupeptin complex has been determined by X-ray crystallography to a resolution of 2.1 A (overall R-factor = 19.8%). The structure indicates that: (i) leupeptin contacts the S subsites of the papain active site and not the S' subsites; (ii) the 'carbonyl' carbon atom of the inhibitor is covalently bound by the Cys-25 sulphur atom of papain and is tetrahedrally coordinated; (iii) the 'carbonyl' oxygen atom of the inhibitor faces the oxyanion hole and makes hydrogen bond contacts with Gln-19 and Cys-25.