RESUMO
BACKGROUND: Statins reduce LDL cholesterol and prevent vascular events, but their net effects in people at low risk of vascular events remain uncertain. METHODS: This meta-analysis included individual participant data from 22 trials of statin versus control (n=134,537; mean LDL cholesterol difference 1·08 mmol/L; median follow-up 4·8 years) and five trials of more versus less statin (n=39,612; difference 0·51 mmol/L; 5·1 years). Major vascular events were major coronary events (ie, non-fatal myocardial infarction or coronary death), strokes, or coronary revascularisations. Participants were separated into five categories of baseline 5-year major vascular event risk on control therapy (no statin or low-intensity statin) (<5%, ≥5% to <10%, ≥10% to <20%, ≥20% to <30%, ≥30%); in each, the rate ratio (RR) per 1·0 mmol/L LDL cholesterol reduction was estimated. FINDINGS: Reduction of LDL cholesterol with a statin reduced the risk of major vascular events (RR 0·79, 95% CI 0·77-0·81, per 1·0 mmol/L reduction), largely irrespective of age, sex, baseline LDL cholesterol or previous vascular disease, and of vascular and all-cause mortality. The proportional reduction in major vascular events was at least as big in the two lowest risk categories as in the higher risk categories (RR per 1·0 mmol/L reduction from lowest to highest risk: 0·62 [99% CI 0·47-0·81], 0·69 [99% CI 0·60-0·79], 0·79 [99% CI 0·74-0·85], 0·81 [99% CI 0·77-0·86], and 0·79 [99% CI 0·74-0·84]; trend p=0·04), which reflected significant reductions in these two lowest risk categories in major coronary events (RR 0·57, 99% CI 0·36-0·89, p=0·0012, and 0·61, 99% CI 0·50-0·74, p<0·0001) and in coronary revascularisations (RR 0·52, 99% CI 0·35-0·75, and 0·63, 99% CI 0·51-0·79; both p<0·0001). For stroke, the reduction in risk in participants with 5-year risk of major vascular events lower than 10% (RR per 1·0 mmol/L LDL cholesterol reduction 0·76, 99% CI 0·61-0·95, p=0·0012) was also similar to that seen in higher risk categories (trend p=0·3). In participants without a history of vascular disease, statins reduced the risks of vascular (RR per 1·0 mmol/L LDL cholesterol reduction 0·85, 95% CI 0·77-0·95) and all-cause mortality (RR 0·91, 95% CI 0·85-0·97), and the proportional reductions were similar by baseline risk. There was no evidence that reduction of LDL cholesterol with a statin increased cancer incidence (RR per 1·0 mmol/L LDL cholesterol reduction 1·00, 95% CI 0·96-1·04), cancer mortality (RR 0·99, 95% CI 0·93-1·06), or other non-vascular mortality. INTERPRETATION: In individuals with 5-year risk of major vascular events lower than 10%, each 1 mmol/L reduction in LDL cholesterol produced an absolute reduction in major vascular events of about 11 per 1000 over 5 years. This benefit greatly exceeds any known hazards of statin therapy. Under present guidelines, such individuals would not typically be regarded as suitable for LDL-lowering statin therapy. The present report suggests, therefore, that these guidelines might need to be reconsidered. FUNDING: British Heart Foundation; UK Medical Research Council; Cancer Research UK; European Community Biomed Programme; Australian National Health and Medical Research Council; National Heart Foundation, Australia.
Assuntos
LDL-Colesterol/sangue , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Doenças Vasculares/prevenção & controle , Humanos , Guias de Prática Clínica como Assunto , Ensaios Clínicos Controlados Aleatórios como Assunto , Medição de Risco/métodos , Resultado do Tratamento , Doenças Vasculares/sangue , Doenças Vasculares/epidemiologiaRESUMO
BACKGROUND: Lowering of LDL cholesterol with standard statin regimens reduces the risk of occlusive vascular events in a wide range of individuals. We aimed to assess the safety and efficacy of more intensive lowering of LDL cholesterol with statin therapy. METHODS: We undertook meta-analyses of individual participant data from randomised trials involving at least 1000 participants and at least 2 years' treatment duration of more versus less intensive statin regimens (five trials; 39â612 individuals; median follow-up 5·1 years) and of statin versus control (21 trials; 129â526 individuals; median follow-up 4·8 years). For each type of trial, we calculated not only the average risk reduction, but also the average risk reduction per 1·0 mmol/L LDL cholesterol reduction at 1 year after randomisation. FINDINGS: In the trials of more versus less intensive statin therapy, the weighted mean further reduction in LDL cholesterol at 1 year was 0·51 mmol/L. Compared with less intensive regimens, more intensive regimens produced a highly significant 15% (95% CI 11-18; p<0·0001) further reduction in major vascular events, consisting of separately significant reductions in coronary death or non-fatal myocardial infarction of 13% (95% CI 7-19; p<0·0001), in coronary revascularisation of 19% (95% CI 15-24; p<0·0001), and in ischaemic stroke of 16% (95% CI 5-26; p=0·005). Per 1·0 mmol/L reduction in LDL cholesterol, these further reductions in risk were similar to the proportional reductions in the trials of statin versus control. When both types of trial were combined, similar proportional reductions in major vascular events per 1·0 mmol/L LDL cholesterol reduction were found in all types of patient studied (rate ratio [RR] 0·78, 95% CI 0·76-0·80; p<0·0001), including those with LDL cholesterol lower than 2 mmol/L on the less intensive or control regimen. Across all 26 trials, all-cause mortality was reduced by 10% per 1·0 mmol/L LDL reduction (RR 0·90, 95% CI 0·87-0·93; p<0·0001), largely reflecting significant reductions in deaths due to coronary heart disease (RR 0·80, 99% CI 0·74-0·87; p<0·0001) and other cardiac causes (RR 0·89, 99% CI 0·81-0·98; p=0·002), with no significant effect on deaths due to stroke (RR 0·96, 95% CI 0·84-1·09; p=0·5) or other vascular causes (RR 0·98, 99% CI 0·81-1·18; p=0·8). No significant effects were observed on deaths due to cancer or other non-vascular causes (RR 0·97, 95% CI 0·92-1·03; p=0·3) or on cancer incidence (RR 1·00, 95% CI 0·96-1·04; p=0·9), even at low LDL cholesterol concentrations. INTERPRETATION: Further reductions in LDL cholesterol safely produce definite further reductions in the incidence of heart attack, of revascularisation, and of ischaemic stroke, with each 1·0 mmol/L reduction reducing the annual rate of these major vascular events by just over a fifth. There was no evidence of any threshold within the cholesterol range studied, suggesting that reduction of LDL cholesterol by 2-3 mmol/L would reduce risk by about 40-50%. FUNDING: UK Medical Research Council, British Heart Foundation, European Community Biomed Programme, Australian National Health and Medical Research Council, and National Heart Foundation.
Assuntos
LDL-Colesterol/sangue , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Doença das Coronárias/mortalidade , Doença das Coronárias/prevenção & controle , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Infarto do Miocárdio/prevenção & controle , Ensaios Clínicos Controlados Aleatórios como Assunto , Acidente Vascular Cerebral/prevenção & controleRESUMO
BACKGROUND: Although statin therapy reduces the risk of occlusive vascular events in people with diabetes mellitus, there is uncertainty about the effects on particular outcomes and whether such effects depend on the type of diabetes, lipid profile, or other factors. We undertook a prospective meta-analysis to help resolve these uncertainties. METHODS: We analysed data from 18 686 individuals with diabetes (1466 with type 1 and 17,220 with type 2) in the context of a further 71,370 without diabetes in 14 randomised trials of statin therapy. Weighted estimates were obtained of effects on clinical outcomes per 1.0 mmol/L reduction in LDL cholesterol. FINDINGS: During a mean follow-up of 4.3 years, there were 3247 major vascular events in people with diabetes. There was a 9% proportional reduction in all-cause mortality per mmol/L reduction in LDL cholesterol in participants with diabetes (rate ratio [RR] 0.91, 99% CI 0.82-1.01; p=0.02), which was similar to the 13% reduction in those without diabetes (0.87, 0.82-0.92; p<0.0001). This finding reflected a significant reduction in vascular mortality (0.87, 0.76-1.00; p=0.008) and no effect on non-vascular mortality (0.97, 0.82-1.16; p=0.7) in participants with diabetes. There was a significant 21% proportional reduction in major vascular events per mmol/L reduction in LDL cholesterol in people with diabetes (0.79, 0.72-0.86; p<0.0001), which was similar to the effect observed in those without diabetes (0.79, 0.76-0.82; p<0.0001). In diabetic participants there were reductions in myocardial infarction or coronary death (0.78, 0.69-0.87; p<0.0001), coronary revascularisation (0.75, 0.64-0.88; p<0.0001), and stroke (0.79, 0.67-0.93; p=0.0002). Among people with diabetes the proportional effects of statin therapy were similar irrespective of whether there was a prior history of vascular disease and irrespective of other baseline characteristics. After 5 years, 42 (95% CI 30-55) fewer people with diabetes had major vascular events per 1000 allocated statin therapy. INTERPRETATION: Statin therapy should be considered for all diabetic individuals who are at sufficiently high risk of vascular events.
Assuntos
Doenças Cardiovasculares , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/mortalidade , Doenças Cardiovasculares/prevenção & controle , LDL-Colesterol/sangue , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Multicêntricos como Assunto , Ensaios Clínicos Controlados Aleatórios como Assunto , Triglicerídeos/sangueRESUMO
BACKGROUND: Results of previous randomised trials have shown that interventions that lower LDL cholesterol concentrations can significantly reduce the incidence of coronary heart disease (CHD) and other major vascular events in a wide range of individuals. But each separate trial has limited power to assess particular outcomes or particular categories of participant. METHODS: A prospective meta-analysis of data from 90,056 individuals in 14 randomised trials of statins was done. Weighted estimates were obtained of effects on different clinical outcomes per 1.0 mmol/L reduction in LDL cholesterol. FINDINGS: During a mean of 5 years, there were 8186 deaths, 14,348 individuals had major vascular events, and 5103 developed cancer. Mean LDL cholesterol differences at 1 year ranged from 0.35 mmol/L to 1.77 mmol/L (mean 1.09) in these trials. There was a 12% proportional reduction in all-cause mortality per mmol/L reduction in LDL cholesterol (rate ratio [RR] 0.88, 95% CI 0.84-0.91; p<0.0001). This reflected a 19% reduction in coronary mortality (0.81, 0.76-0.85; p<0.0001), and non-significant reductions in non-coronary vascular mortality (0.93, 0.83-1.03; p=0.2) and non-vascular mortality (0.95, 0.90-1.01; p=0.1). There were corresponding reductions in myocardial infarction or coronary death (0.77, 0.74-0.80; p<0.0001), in the need for coronary revascularisation (0.76, 0.73-0.80; p<0.0001), in fatal or non-fatal stroke (0.83, 0.78-0.88; p<0.0001), and, combining these, of 21% in any such major vascular event (0.79, 0.77-0.81; p<0.0001). The proportional reduction in major vascular events differed significantly (p<0.0001) according to the absolute reduction in LDL cholesterol achieved, but not otherwise. These benefits were significant within the first year, but were greater in subsequent years. Taking all years together, the overall reduction of about one fifth per mmol/L LDL cholesterol reduction translated into 48 (95% CI 39-57) fewer participants having major vascular events per 1000 among those with pre-existing CHD at baseline, compared with 25 (19-31) per 1000 among participants with no such history. There was no evidence that statins increased the incidence of cancer overall (1.00, 0.95-1.06; p=0.9) or at any particular site. INTERPRETATION: Statin therapy can safely reduce the 5-year incidence of major coronary events, coronary revascularisation, and stroke by about one fifth per mmol/L reduction in LDL cholesterol, largely irrespective of the initial lipid profile or other presenting characteristics. The absolute benefit relates chiefly to an individual's absolute risk of such events and to the absolute reduction in LDL cholesterol achieved. These findings reinforce the need to consider prolonged statin treatment with substantial LDL cholesterol reductions in all patients at high risk of any type of major vascular event.
Assuntos
LDL-Colesterol/sangue , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hipercolesterolemia/tratamento farmacológico , Causas de Morte , Doença das Coronárias/mortalidade , Doença das Coronárias/prevenção & controle , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Infarto do Miocárdio/mortalidade , Ensaios Clínicos Controlados Aleatórios como Assunto , Fatores de Risco , Acidente Vascular Cerebral/mortalidade , Acidente Vascular Cerebral/prevenção & controle , Resultado do TratamentoRESUMO
OBJECTIVE: To determine whether the mode of anesthesia used during the tension-free vaginal tape procedure affects postoperative voiding function. METHODS: A retrospective cohort study was performed using cases in which tension-free vaginal tape placement was the sole procedure performed. Of the 173 cases reviewed, we were able to use the data from 163. Hierarchal linear regression was used to identify independent predictors of our dependent variable: days to complete voiding. In the first block, established predictors of postoperative voiding dysfunction were entered into the model. In the second block, potential confounders of the relationship between anesthesia type and days to complete voiding identified during univariate analysis (P <.15) were entered into the model. In the third block, anesthesia type was entered into the model to determine whether it added any unique variance after controlling for previously established predictors of postoperative voiding dysfunction. RESULTS: The mean days to complete voiding was similar in our local or regional anesthesia (n = 90) and general anesthesia groups (n = 73) (2.3 [0-21] versus 2.3 [0-14], P =.95). Our final regression model (F = 2.74, P =.011) included age, prior pelvic organ prolapse surgery, and preoperative urge symptoms and explained 22.2% of the variance in days to complete voiding. Anesthesia type did not add any predictive improvement after controlling for these variables. CONCLUSION: General anesthesia, and therefore lack of a cough-stress test, does not increase the chance of postoperative voiding dysfunction associated with tension-free vaginal tape.
Assuntos
Anestesia Geral/efeitos adversos , Próteses e Implantes , Transtornos Urinários/etiologia , Procedimentos Cirúrgicos Urológicos , Prolapso Uterino/cirurgia , Estudos de Coortes , Feminino , Humanos , Modelos Logísticos , Pessoa de Meia-Idade , Complicações Pós-Operatórias , Estudos Retrospectivos , UrodinâmicaRESUMO
The role of MutS ATPase in mismatch repair is controversial. To clarify further the function of this activity, we have examined adenine nucleotide effects on interactions of Escherichia coli MutS with homoduplex and heteroduplex DNAs. In contrast to previous results with human MutS alpha, we find that a physical block at one end of a linear heteroduplex is sufficient to support stable MutS complex formation in the presence of ATP.Mg(2+). Surface plasmon resonance analysis at low ionic strength indicates that the lifetime of MutS complexes with heteroduplex DNA depends on the nature of the nucleotide present when MutS binds. Whereas complexes prepared in the absence of nucleotide or in the presence of ADP undergo rapid dissociation upon challenge with ATP x Mg(2+), complexes produced in the presence of ATP x Mg(2+), adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP) x Mg(2+), or ATP (no Mg(2+)) are resistant to dissociation upon ATP challenge. AMPPNP x Mg(2+) and ATP (no Mg(2+)) reduce MutS affinity for heteroduplex but have little effect on homoduplex affinity, resulting in abolition of specificity for mispaired DNA at physiological salt concentrations. Conversely, the highest mismatch specificity is observed in the absence of nucleotide or in the presence of ADP. ADP has only a limited effect on heteroduplex affinity but reduces MutS affinity for homoduplex DNA.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli , Adenina/química , Adenosina Trifosfatases/metabolismo , Adenilil Imidodifosfato/farmacologia , Pareamento Incorreto de Bases , Cátions/metabolismo , DNA/metabolismo , Reparo do DNA , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Hidrólise , Indicadores e Reagentes/farmacologia , Cinética , Magnésio/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento , Ácidos Nucleicos Heteroduplexes/metabolismo , Cloreto de Potássio/farmacologia , Ligação Proteica , Estreptavidina/farmacologia , Ressonância de Plasmônio de Superfície , Fatores de TempoRESUMO
Formation of a ternary complex between human MutSalpha, MutLalpha, and heteroduplex DNA has been demonstrated by surface plasmon resonance spectroscopy and electrophoretic gel shift methods. Formation of the hMutLalpha.hMutSalpha.heteroduplex complex requires a mismatch and ATP hydrolysis, and depends on DNA chain length. Ternary complex formation was supported by a 200-base pair G-T heteroduplex, a 100-base pair substrate was somewhat less effective, and a 41-base pair heteroduplex was inactive. As judged by surface plasmon resonance spectroscopy, ternary complexes produced with the 200-base pair G-T DNA contained approximately 0.8 mol of hMutLalpha/mol of heteroduplex-bound hMutSalpha. Although the steady-state levels of the hMutLalpha.hMutSalpha. heteroduplex were substantial, this complex was found to turn over, as judged by surface plasmon resonance spectroscopy and electrophoretic gel shift analysis. With the former method, the majority of the complexes dissociated rapidly upon termination of protein flow, and dissociation occurred in the latter case upon challenge with competitor DNA. However, ternary complex dissociation as monitored by gel shift assay was prevented if both ends of the heteroduplex were physically blocked with streptavidin.biotin complexes. This observation suggests that, like hMutSalpha, the hMutLalpha.hMutSalpha complex can migrate along the helix contour to dissociate at DNA ends.
Assuntos
Adenosina Trifosfatases , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/química , DNA/química , Proteínas de Neoplasias/química , Proteínas Proto-Oncogênicas/química , Proteínas Adaptadoras de Transdução de Sinal , Trifosfato de Adenosina/metabolismo , Pareamento de Bases , Proteínas de Transporte , DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Dimerização , Guanina , Humanos , Cinética , Endonuclease PMS2 de Reparo de Erro de Pareamento , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ressonância de Plasmônio de Superfície , TiminaRESUMO
This article summarizes the results of the Ohio University Sexual Assault Risk Reduction Project, which is a program designed to reduce college women's risk for sexual assault. The program was evaluated at 2 separate universities with 762 women. Participants were randomly assigned either to the program or to the no-treatment comparison group, and they completed measures that assessed sexual victimization, dating behaviors, sexual communication, and rape empathy at the pretest and at the 2-month and 6-month follow-ups. At the 2-month follow-up, there were no differences between the groups on any of the outcome measures. However, those women who were moderately victimized during the 2-month follow-up were significantly less likely to be revictimized during the 6-month follow-up period if they participated in the program.
Assuntos
Estupro/prevenção & controle , Adolescente , Adulto , Conscientização , Comunicação , Empatia , Feminino , Humanos , Avaliação de Programas e Projetos de Saúde , Distribuição Aleatória , Fatores de RiscoRESUMO
This article describes the potential of the Longitudinal Study to analyse different ethnic groups by broad period of entry. This is illustrated with the use of one variable (childlessness), but could be the basis for further analytical work on other variables. Each group has a unique demographic profile, which is largely explained by its migration history. Migration influences important life course transitions. Differences between more established residents (present in 1981 and 1991) and less established residents (present in 1991 only) imply that generalisations for the entire ethnic group or future generations should not be made.
Assuntos
Censos , Emigração e Imigração/estatística & dados numéricos , Emigração e Imigração/tendências , Etnicidade/estatística & dados numéricos , Família/etnologia , Crescimento Demográfico , Adulto , Distribuição por Idade , Idoso , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Pais , Grupos Raciais , Distribuição por Sexo , Mudança Social , Reino UnidoRESUMO
Estrone glucuronide conjugates of hen egg white lysozyme were prepared by the mixed anhydride and active ester coupling procedures. Both methods gave good yields of conjugates, but the active ester procedure gave a more diverse range of products, making it less suitable for preparing conjugates for homogeneous enzyme immunoassay. Conjugation of lysozyme with estrone glucuronide by the mixed anhydride procedure gave one major derivative exclusively acylated at lysine residue 33 whereas conjugation by the active ester method gave six derivatives which were acylated at one or more of lysine residues 33, 97, and 116. None of the lysine residues 1, 13, and 96, or the N-terminal alpha-amino group, were acylated in any of the conjugates isolated. The correlation of the conjugate structures with the protein environments of the amino groups in the crystal structure of lysozyme suggested that the sites of acylation were determined not only by the chemical nature of the acylating reagent but also by the surface accessibility and nucleophilicity of the individual lysine residues.
Assuntos
Estrona/química , Muramidase/química , Acilação , Alquilação , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Clara de Ovo , Glucuronatos , Hidrólise , Indicadores e Reagentes , Oxirredução , Peptídeos/química , TripsinaRESUMO
Acid-polyacrylamide gel electrophoresis (acid-PAGE) was used for analysis of lysozyme-estrone glucuronide conjugates. The resolution of the system allowed the identification of individual conjugate families which differed only in the position of acylation or in the number of estrone glucuronide units. Acid-PAGE was a good alternative to denaturing cation-exchange chromatography for the analysis, separation, and small-scale purification of lysozyme-estrone glucuronide conjugates. It revealed the true order of the relative degree of positive charge on the lysozyme-estrone glucuronide conjugates.
Assuntos
Estrogênios Conjugados (USP)/química , Estrona/química , Muramidase/química , Grupo dos Citocromos c/metabolismo , Eletroforese em Gel de Poliacrilamida , Glucuronatos/química , Desnaturação ProteicaRESUMO
ATP hydrolysis by MutS homologs is required for function of these proteins in mismatch repair. However, the function of ATP hydrolysis in the repair reaction is controversial. In this paper we describe a steady-state kinetic analysis of the DNA-activated ATPase of human MutSalpha. Comparison of salt concentration effects on mismatch repair and mismatch-provoked excision in HeLa nuclear extracts with salt effects on the DNA-activated ATPase suggests that ATP hydrolysis by MutSalpha is involved in the rate determining step in the repair pathway. While the ATPase is activated by homoduplex and heteroduplex DNA, the half-maximal concentration for activation by heteroduplex DNA is significantly lower under physiological salt concentrations. Furthermore, at optimal salt concentration, heteroduplex DNA increases the kcat for ATP hydrolysis to a greater extent than does homoduplex DNA. We also demonstrate that the degree of ATPase activation is dependent on DNA chain length, with the kcat for hydrolysis increasing significantly with chain length of the DNA cofactor. These results are discussed in terms of the translocation (Allen, D. J., Makhov, A., Grilley, M., Taylor, J., Thresher, R., Modrich, P., and Griffith, J. D. (1997) EMBO J. 16, 4467-4476) and the molecular switch (Gradia, S., Acharya, S., and Fishel, R. (1997) Cell 91, 995-1005) models that invoke distinct roles for ATP hydrolysis in MutS homolog function.
Assuntos
Adenosina Trifosfatases/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Complexos Multienzimáticos/metabolismo , Oligodesoxirribonucleotídeos/metabolismo , Pareamento Incorreto de Bases , Sequência de Bases , Núcleo Celular/metabolismo , Reparo do DNA/efeitos dos fármacos , Ativação Enzimática , Células HeLa , Humanos , Cinética , Dados de Sequência Molecular , Proteína 3 Homóloga a MutS , Ácidos Nucleicos Heteroduplexes/metabolismo , Oligodesoxirribonucleotídeos/química , Cloreto de Potássio/farmacologia , Especificidade por SubstratoRESUMO
ATP hydrolysis by bacterial and eukaryotic MutS activities is required for their function in mismatch correction, and two different models for the role of ATP in MutS function have been proposed. In the translocation model, based on study of bacterial MutS, ATP binding reduces affinity of the protein for a mismatch and activates secondary DNA binding sites that are subsequently used for movement of the protein along the helix contour in a reaction dependent on nucleotide hydrolysis (Allen, D. J., Makhov, A., Grilley, M., Taylor, J., Thresher, R., Modrich, P., and Griffith, J. D. (1997) EMBO J. 16, 4467-4476). The molecular switch model, based on study of human MutSalpha, invokes mismatch recognition by the MutSalpha.ADP complex. After recruitment of downstream repair activities to the MutSalpha.mismatch complex, ATP binding results in release of MutSalpha from the heteroduplex (Gradia, S., Acharya, S., and Fishel, R.(1997) Cell 91, 995-1005). To further clarify the function of ATP binding and hydrolysis in human MutSalpha action, we evaluated the effects of ATP, ADP, and nonhydrolyzable ATP analogs on the lifetime of protein.DNA complexes. All of these nucleotides were found to increase the rate of dissociation of MutSalpha from oligonucleotide heteroduplexes. These experiments also showed that ADP is not required for mismatch recognition by MutSalpha, but that the nucleotide alters the dynamics of formation and dissociation of specific complexes. Analysis of the mechanism of ATP-promoted dissociation of MutSalpha from a 200-base pair heteroduplex demonstrated that dissociation occurs at DNA ends in a reaction dependent on ATP hydrolysis, implying that release from this molecule involves movement of the protein along the helix contour as predicted for a translocation mechanism. In order to reconcile the relatively large rate of movement of MutS homologs along the helix with their modest rate of ATP hydrolysis, we propose a novel mechanism for protein translocation along DNA that supports directional movement over long distances with minimal energy input.
Assuntos
Trifosfato de Adenosina/metabolismo , Pareamento Incorreto de Bases , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Difosfato de Adenosina/metabolismo , Sítios de Ligação , DNA/química , Proteínas de Ligação a DNA/química , Guanina , Humanos , Cinética , Modelos Moleculares , Proteína 3 Homóloga a MutS , Ligação Proteica , Especificidade por Substrato , TiminaRESUMO
Application of time series analysis to a database containing serial pregnanediol data from 113 complete ovulatory menstrual cycles contributed by 83 women of proven fertility and 68 cycles for which pregnanediol values were available over the ovulatory period, detected the first statistically significant risk in pregnanediol excretion for all cycles for which a baseline was available (n = 170). However, even at the 99% confidence level, for 22% of cycles a rise was observed before the presumed day of ovulation. Therefore, a threshold value for pregnanediol was sought from the database as a better marker for the end of fertility. A value of 1.4 mg per 24 h was not reached before day 2 after the pre-ovulatory estrogen peak day for 96% of the cycles. In the remaining 4% of cycles it was reached one day after the total estrogen peak day. The validity of this threshold was confirmed in extensive studies using the Ovarian Monitor where the equivalent is 6.3 mumol per 24 h of pregnanediol glucuronide and measurements are performed on timed urine specimens with a minimum collection time of three hours. These studies were as follows: 1) a World Health Organization study on the use of the Ovarian Monitor as a fertility self test in the home (108 cycles), 2) a multicenter study on returning fertility during breast feeding conducted by Family Health International (73 women), and 3) the general application of the Ovarian Monitor for pregnancy achievement and avoidance during the past ten years (over 250,000 PdG assays performed in ten countries). With rare exceptions, the use of these threshold values is applicable for all women provided correction is made for urine volume.
Assuntos
Glucuronatos/urina , Ciclo Menstrual/urina , Menstruação/urina , Pregnanodiol/urina , Esteroides/urina , Biomarcadores/urina , Bases de Dados Factuais , Feminino , Previsões , Humanos , Monitorização Fisiológica/métodos , Estudos Multicêntricos como Assunto , Detecção da Ovulação/métodos , AutocuidadoRESUMO
We found that with oral supplementation by a liquid soy-based protein hydrolysate in malnourished COPD patients (BMI <= 20), it possible to increase weight over a 6-week period, and body water and an index of muscle mass (MAMC), but not total body nitrogen (TBN judged by Nitrogen Index) which identifies a particular challenge for nutrition support in COPD patients. There was no associated improvement in pulmonary function but we found that better nourished COPD patients (BMI > 20) had some pulmonary function advantage; it is suggested that TBN may need to improve with nutrition support for pulmonary function to improve.
RESUMO
Despite the existence of several protocols, problems appear to persist in the small scale chemical synthesis of radiolabeled 11-ketotestosterone from cortisol. We investigated the possibilities of using the mild oxidant pyridinium dichromate for the oxidative cleavage of the dihydroxyacetone side chain of cortisol and 17 beta-hydroxysteroid dehydrogenase for the subsequent reduction of the resulting 17-keto group. Our protocol has resulted in consistently high yields of both the intermediate, adrenosterone (70-80%), and the product, 11-ketotestosterone (up to 60%). This, taken together with the convenience and relatively low cost of our method, recommends the protocol for its use for the synthesis of [3H]-11-ketotestosterone for endocrine studies.
Assuntos
Hidrocortisona/química , Testosterona/análogos & derivados , Androstenos/química , Androstenos/metabolismo , Hidroxiesteroide Desidrogenases/metabolismo , Oxirredução , Compostos de Piridínio , Testosterona/síntese química , Testosterona/metabolismo , TrítioRESUMO
Subspecific typing of clinical meningococcal strains is important in the investigation of outbreaks and for disease surveillance. Serogrouping, typing, and subtyping of strains currently require isolation of a meningococcus from one or more clinical specimens. However, the increasing widespread practice of preadmission administration of parenteral antibiotics has resulted in a decrease in the frequency of positive cultures obtained from blood and cerebrospinal fluid. Confirmation of meningococcal disease can be obtained by meningococcus-specific PCR from both cerebrospinal fluid (H. Ni et al., Lancet 340:1432-1434, 1992) and peripheral blood (J. Newcombe et al., J. Clin. Microbiol. 34:1637-1640, 1996) specimens. However, current PCR protocols do not yield epidemiologically useful typing information. We report here the use of PCR-single-stranded confirmational polymorphism (PCR-SSCP) analysis to amplify and type meningococcal DNA present in clinical specimens. PCR-SSCP analysis with the VR1 region of the Neisseria meningitidis porA gene as the target produced unique banding patterns for each serosubtype. Direct PCR-SSCP of clinical specimens can therefore provide typing data that can be used to investigate the epidemiology of clusters of cases and outbreaks and for disease surveillance in situations in which culture of patient specimens proves negative.
Assuntos
Técnicas de Tipagem Bacteriana , Infecções Meningocócicas/microbiologia , Neisseria meningitidis/classificação , Reação em Cadeia da Polimerase/métodos , Humanos , Neisseria meningitidis/isolamento & purificaçãoRESUMO
Human replication protein A (hRPA) is an essential single-stranded-DNA-binding protein that stimulates the activities of multiple DNA replication and repair proteins through physical interaction. To understand DNA binding and its role in hRPA heterologous interaction, we examined the physical structure of hRPA complexes with single-stranded DNA (ssDNA) by scanning transmission electron microscopy. Recent biochemical studies have shown that hRPA combines with ssDNA in at least two binding modes: by interacting with 8 to 10 nucleotides (hRPA8nt) and with 30 nucleotides (hRPA30nt). We find the relatively unstable hRPA8nt complex to be notably compact with many contacts between hRPA molecules. In contrast, on similar lengths of ssDNA, hRPA30nt complexes align along the DNA and make few intermolecular contacts. Surprisingly, the elongated hRPA30nt complex exists in either a contracted or an extended form that depends on ssDNA length. Therefore, homologous-protein interaction and available ssDNA length both contribute to the physical changes that occur in hRPA when it binds ssDNA. We used activated DNA-dependent protein kinase as a biochemical probe to detect alterations in conformation and demonstrated that formation of the extended hRPA30nt complex correlates with increased phosphorylation of the hRPA 29-kDa subunit. Our results indicate that hRPA binds ssDNA in a multistep pathway, inducing new hRPA alignments and conformations that can modulate the functional interaction of other factors with hRPA.