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1.
Methods Enzymol ; 579: 51-86, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27572723

RESUMO

Imaging a material with electrons at near-atomic resolution requires a thin specimen that is stable in the vacuum of the transmission electron microscope. For biological samples, this comprises a thin layer of frozen aqueous solution containing the biomolecular complex of interest. The process of preparing a high-quality specimen is often the limiting step in the determination of structures by single-particle electron cryomicroscopy (cryo-EM). Here, we describe a systematic approach for going from a purified biomolecular complex in aqueous solution to high-resolution electron micrographs that are suitable for 3D structure determination. This includes a series of protocols for the preparation of vitrified specimens on various supports, including all-gold and graphene. We also describe techniques for troubleshooting when a preparation fails to yield suitable specimens, and common mistakes to avoid during each part of the process. Finally, we include recommendations for obtaining the highest quality micrographs from prepared specimens with current microscope, detector, and support technology.


Assuntos
Microscopia Crioeletrônica/métodos , Ouro/química , Grafite/química , Manejo de Espécimes/métodos , Coloração e Rotulagem/métodos , Microscopia Crioeletrônica/instrumentação , Desenho de Equipamento , Humanos , Processamento de Imagem Assistida por Computador , Proteínas/ultraestrutura , Manejo de Espécimes/instrumentação , Vitrificação
2.
Intern Med J ; 43(2): 174-82, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22471951

RESUMO

BACKGROUND: Lung cancer is the leading cause of cancer-related mortality in Australia. Screening using low-dose computed tomography (LDCT) can reduce lung cancer mortality. The feasibility of screening in Australia is unknown. This paper describes the rationale, design and methods of the Queensland Lung Cancer Screening Study. AIMS: The aim of the study is to describe the methodology for a feasibility study of lung cancer screening by LDCT in Australia. METHODS: The Queensland Lung Cancer Screening Study is an ongoing, prospective observational study of screening by LDCT at a single tertiary institution. Healthy volunteers at high risk of lung cancer (age 60-74 years; smoking history ≥30 pack years, current or quit within 15 years; forced expiratory volume in 1s ≥50% predicted) are recruited from the general public through newspaper advertisement and press release. Participants receive a LDCT scan of the chest at baseline, year 1 and year 2 using a multidetector helical computed tomography scanner and are followed up for a total of 5 years. Feasibility of screening will be assessed by cancer detection rates, lung nodule prevalence, optimal management strategies for lung nodules, economic costs, healthcare utilisation and participant quality of life. CONCLUSIONS: Studying LDCT screening in the Australian setting will help us understand how differences in populations, background diseases and healthcare structures modulate screening effectiveness. This information, together with results from overseas randomised studies, will inform and facilitate local policymaking.


Assuntos
Detecção Precoce de Câncer/métodos , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/epidemiologia , Tomografia Computadorizada por Raios X/métodos , Idoso , Detecção Precoce de Câncer/normas , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Queensland/epidemiologia , Fatores de Risco , Tomografia Computadorizada por Raios X/normas
3.
Int J Lab Hematol ; 30(4): 300-5, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18665827

RESUMO

Hemochromatosis has often been associated with progressive iron overload, but the natural history of iron accumulation in untreated C282Y homozygotes has been reported infrequently. The Hemochromatosis and Iron Overload Screening (HEIRS) Study screened 101 168 primary care participants for iron overload using transferrin saturation, unbound iron-binding capacity, Serum ferritin (SF), and HFE C282Y and H63D genotyping. SF was measured at initial screening (IS) and again when selected participants returned for a clinical examination (CE). The change in SF over the observation period (defined as ferritin rate of change) was analyzed according to age, gender, initial SF, initial SF/age, transferrin saturation, and iron removed by phlebotomy in C282Y homozygotes. Seventy-four male and 133 female untreated C282Y homozygotes were observed over a median of 112 days (34-924 days) between IS and CE. In men, SF increased in 54% and decreased in 46%. In women, SF increased in 50% and decreased in 50%. The significant variables affecting the SF rate were initial log SF (P = 0.0027) and transferrin saturation (P < 0.0001). Male C282Y homozygotes with higher SF rates (n = 27, upper 50th percentile) had significantly greater iron removed by phlebotomy (mean 4.93 g, range 1.0-17 g) than those with lower SF rates (n = 26, lower 50th percentile) (mean 2.6 g, 0.42-7.1, P < 0.05). SF was as likely to decrease as increase in untreated C282Y homozygotes over this relatively brief observation period. Incremental increases in SF are not inevitable in untreated C282Y homozygotes.


Assuntos
Ferritinas/sangue , Hemocromatose/sangue , Hemocromatose/genética , Antígenos de Histocompatibilidade Classe I/genética , Proteínas de Membrana/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Proteína da Hemocromatose , Homozigoto , Humanos , Ferro/metabolismo , Masculino , Pessoa de Meia-Idade
4.
Biochem Soc Trans ; 32(Pt 5): 724-7, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15493998

RESUMO

The APC (anaphase-promoting complex) is a multisubunit E3 ubiquitin ligase that targets cell-cycle-related proteins for degradation by the 26 S proteasome. The APC contains at least 13 subunits and is regulated by the binding of co-activator proteins and by phosphorylation. It is not known why the APC contains 13 subunits when many other ubiquitin ligases are small single-subunit enzymes. In the present study, the structures and functions of individual APC subunits are discussed. By dissecting the roles of its parts, we hope to gain insight into the mechanism of the intact APC.


Assuntos
Complexos Ubiquitina-Proteína Ligase/fisiologia , Motivos de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Domínio Catalítico , Ciclo Celular , Modelos Biológicos , Fosforilação , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Complexos Ubiquitina-Proteína Ligase/metabolismo
5.
Biochemistry ; 40(2): 395-402, 2001 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-11148033

RESUMO

PSE-4 is a class A beta-lactamase produced by strains of Pseudomonas aeruginosa and is highly active for the penicillin derivative carbenicillin. The crystal structure of the wild-type PSE-4 carbenicillinase has been determined to 1.95 A resolution by molecular replacement and represents the first structure of a carbenicillinase published to date. A superposition of the PSE-4 structure with that of TEM-1 shows a rms deviation of 1.3 A for 263 Calpha atoms. Most carbenicillinases are unique among class A beta-lactamases in that residue 234 is an arginine (ABL standard numbering scheme), while in all other class A enzymes this residue is a lysine. Kinetic characterization of a R234K PSE-4 mutant reveals a 50-fold reduction in k(cat)/K(m) and confirms the importance of Arg 234 for carbenicillinase activity. A comparison of the structure of the R234K mutant refined to 1.75 A resolution with the wild-type structure shows that Arg 234 stabilizes an alternate conformation of the Ser 130 side chain, not seen in other class A beta-lactamase structures. Our molecular modeling studies suggest that the position of a bound carbenicillin would be shifted relative to that of a bound benzylpenicillin in order to avoid a steric clash between the carbenicillin alpha-carboxylate group and the conserved side chain of Asn 170. The alternate conformation of the catalytic Ser 130 in wild-type PSE-4 may be involved in accommodating this shift in the bound substrate position.


Assuntos
Penicilinase/química , beta-Lactamases/química , Alanina/genética , Arginina/genética , Sítios de Ligação/genética , Cristalografia por Raios X , Ativação Enzimática , Hidrólise , Cinética , Lisina/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Penicilinase/metabolismo , Pseudomonas aeruginosa/enzimologia , beta-Lactamases/genética , beta-Lactamases/metabolismo
6.
Eur J Hum Genet ; 8(4): 286-92, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10854112

RESUMO

Recently, the VMD2 gene has been identified as the causative gene in juvenile-onset vitelliform macular dystrophy (Best disease), a central retinopathy primarily characterised by an impaired function of the retinal pigment epithelium. In this study we have further characterised the spectrum of VMD2 mutations in a series of 41 unrelated Best disease patients. Furthermore we expanded our analysis to include 32 unrelated patients with adult vitelliform macular dystrophy (AVMD) and 200 patients with age-related macular degeneration (AMD). Both AVMD and AMD share some phenotypic features with Best disease such as abnormal subretinal accumulation of lipofuscin material, progressive geographic atrophy and choroidal neovascularisation, and may be the consequence of a common pathogenic mechanism. In total, we have identified 23 distinct disease-associated mutations in Best disease and four different mutations in AVMD. Two of the mutations found in the AVMD patients were also seen in Best disease suggesting a considerable overlap in the aetiology of these two disorders. There were no mutations found in the AMD group. In addition, four frequent intragenic polymorphisms did not reveal allelic association of the VMD2 locus with AMD. These data exclude a direct role of VMD2 in the predisposition to AMD.


Assuntos
Oftalmopatias Hereditárias/genética , Proteínas do Olho/genética , Degeneração Macular/genética , Adolescente , Adulto , Idade de Início , Idoso , Substituição de Aminoácidos , Bestrofinas , Canais de Cloreto , DNA/química , DNA/genética , Análise Mutacional de DNA , Proteínas do Olho/química , Saúde da Família , Humanos , Pessoa de Meia-Idade , Modelos Moleculares , Mutação , Mutação Puntual
7.
Hum Genet ; 105(3): 200-10, 1999 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10987646

RESUMO

Albinism is a heterogeneous group of genetic disorders resulting from deficiencies in pigmentation. Clinically, it is divided into ocular (OA) and oculocutaneous albinism (OCA). OCA involves lack of pigment in the skin, hair, and eyes and results from mutations in the tyrosinase gene or in the P gene. OA mainly affects pigmentation in the visual system and may be a mild form of OCA or may be caused by other genetic defects. Clinical diagnosis of albinism type is difficult, because of the observed range of phenotypic variation. Thus, genetic analysis may be helpful with respect to a more accurate diagnosis. Here, we report the mutational profile, determined by genetic analysis of the tyrosinase and P genes, of a large German albino population. We have revealed a total of 42 distinct mutations, 19 of which are novel. Of the 74 unrelated patients screened, 32 (43%) had mutations in the tyrosinase gene, 16 (22%) had P gene mutations, and 26 (35%) patients had no detectable genetic abnormalities. This defines a population of albino patients who are tyrosinase-gene- and P-gene-negative and who thus may represent a good study group for searching for additional genes associated with albinism.


Assuntos
Albinismo Oculocutâneo/genética , Proteínas de Transporte/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Monofenol Mono-Oxigenase/genética , Albinismo Oculocutâneo/patologia , Processamento Alternativo/genética , Substituição de Aminoácidos , DNA/química , DNA/genética , Análise Mutacional de DNA , Feminino , Mutação da Fase de Leitura , Alemanha , Humanos , Masculino , Mutação , Linhagem , Polimorfismo Genético , Polimorfismo Conformacional de Fita Simples , Deleção de Sequência
8.
Mol Cell Biol ; 18(12): 6995-7008, 1998 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9819387

RESUMO

As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H- and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester- and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol- and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerol-enriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.


Assuntos
Proteínas de Ligação a DNA/genética , Fatores de Troca do Nucleotídeo Guanina , Acetato de Tetradecanoilforbol/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Análise Mutacional de DNA , DNA Complementar/genética , Ativação Enzimática/fisiologia , Expressão Gênica/genética , Humanos , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação Puntual/genética , RNA Mensageiro/genética , Retroviridae/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Transformação Genética/genética
9.
Hum Mol Genet ; 7(9): 1517-25, 1998 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9700209

RESUMO

Vitelliform macular dystrophy (Best's disease) is an autosomal dominant, early-onset form of macular degeneration in which the primary defect is thought to occur at the level of the retinal pigment epithelium. Genetic linkage has mapped the disease locus to chromosome 11q12-q13.1 within a 980 kb interval flanked by markers at loci D11S4076 and uteroglobin. To identify the disease gene, we systematically characterized genes from within the critical region and analysed the coding regions for mutations in 12 patients from large multigeneration Best's disease families. Following this approach, we identified a novel gene of unknown function carrying heterozygous mutations in all 12 probands. Of these, 10 result in distinct missense mutations of amino acids that are highly conserved throughout evolution, spanning a phylogenetic distance from Caenorhabditis elegans to human, and include V9M, A10T, W24C, R25Q, R218Q, Y227N, Y227C, V235M, P297A and F305S. One deletion mutation, DeltaI295, was found in two families and segregates with the disease in both cases. Northern blot analysis reveals tissue-specific expression for this gene, exclusively in the retinal pigment epithelium. In conclusion, our data provide strong evidence that mutations in the gene that we have identified cause Best's disease.


Assuntos
Proteínas de Ligação a DNA , Oftalmopatias Hereditárias/genética , Proteínas do Olho/genética , Degeneração Macular/genética , Mutação , Sequência de Aminoácidos , Sequência de Bases , Bestrofinas , Canais de Cloreto , Mapeamento Cromossômico , Cromossomos Humanos Par 11/genética , Primers do DNA/genética , DNA Complementar/genética , Éxons , Feminino , Expressão Gênica , Heterozigoto , Humanos , Íntrons , Masculino , Dados de Sequência Molecular , Proteínas Nucleares/genética , Linhagem , Epitélio Pigmentado Ocular/metabolismo , Homologia de Sequência de Aminoácidos
10.
Biochemistry ; 35(33): 10974-84, 1996 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-8718891

RESUMO

The steady state tryptophan fluorescence of apo-human cyclooxygenase-2 (hCox-2) is quenched approximately 40%-50% by the slow binding inhibitors diclofenac, indomethacin, ketoprofen, NS-398, and DuP-697. The effects of these inhibitors on tryptophan fluorescence are both time and concentration dependent. Addition of each inhibitor results in a rapid fluorescence decrease, followed by a slower time dependent quenching. The slow, time dependent loss of fluorescence follows first-order kinetics, the rate constants for the process increasing with inhibitor concentration in a saturation-type manner. The rapid fluorescence loss also increases with increasing inhibitor concentration in the same manner. These results are consistent with the initial formation of a rapid equilibrium complex of enzyme and inhibitor (EI), followed by the slower formation of a tightly bound enzyme-inhibitor complex (EI*). The fluorescence of the EI complex is not significantly different from that of the EI* complex. The kinetic parameters of each inhibitor derived for this process (Ki and kon) are close to those obtained by determination of the rate constants for the onset of enzyme inhibition, thereby linking the fluorescence changes with inhibitor binding. The reversible inhibitors ibuprofen and docosahexaenoic acid do not quench the protein fluorescence but do decrease both the rate of the slow fluorescence loss and the magnitude of the initial rapid fluorescence decrease caused by the slow binding inhibitors, consistent with their competitive behavior. ASA-acetylated apo-hCox-2 shows the same fluorescence-quenching behavior in the presence of most of the above inhibitors. However, acetylation apparently blocks the binding of diclofenac, whereas the affinity of ibuprofen is increased. The effects of the collisional quenching agents iodide and acrylamide on both the native and inhibited enzyme are small (< 20% quenching at 0.3 M), showing that inhibitor binding does not result in an increased solvent accessibility of protein tryptophans. The cause of the inhibitor-induced quenching of the intrinsic apo-hCox-2 fluorescence is likely energy transfer to the bound inhibitor. Calculations based on the inhibitor-tryptophan distances in ovine Cox-1 indicate that the distances are within the required range for significant quenching to occur.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Isoenzimas/química , Prostaglandina-Endoperóxido Sintases/química , Acetilação , Animais , Baculoviridae/genética , Linhagem Celular , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Humanos , Cinética , Proteínas de Membrana , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometria de Fluorescência , Spodoptera , Triptofano/química
11.
Health Psychol ; 10(4): 252-8, 1991.
Artigo em Inglês | MEDLINE | ID: mdl-1915211

RESUMO

Investigated the social support available to families of children born with craniofacial anomalies and the perceived degree of satisfaction derived from these relationships. Thirty-six children (1 month to 5 years old) born with craniofacial deformities (FD) were matched by age and sex to 36 children with no significant physical or behavioral problems. The Social Support Questionnaire-Revised, the Revised Denver Developmental Screening Test, and a semistructured interview were administered. Results indicated that parents of FD children reported less available social support and were significantly less satisfied with their support. Parents of children who had more severe physical impairments and were rated as less attractive reported having less available and less satisfying social support. In particular, the social competence of the child was the most important predictor of parental social support. This result is interesting as the parents of FD children appeared to underreport the presence of behavioral-psychological problems in their children.


Assuntos
Anormalidades Múltiplas/psicologia , Adaptação Psicológica , Fenda Labial/psicologia , Fissura Palatina/psicologia , Disostose Craniofacial/psicologia , Ossos Faciais/anormalidades , Crânio/anormalidades , Apoio Social , Desenvolvimento Infantil , Pré-Escolar , Avaliação da Deficiência , Feminino , Assistência Domiciliar/psicologia , Humanos , Lactente , Masculino , Relações Mãe-Filho , Papel do Doente
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