RESUMO
Formation of many site-specific protein-nucleic acid complexes involves sequential conformational changes subsequent to initial binding which create functionally active assemblies. Characterization of population distributions and structural characteristics of intermediate and product conformations is necessary to understand both the mechanisms and the thermodynamics of these processes. For these purposes, here we develop the quantitative method of multiple hit footprinting (MHF), where chemical or enzymatic probing is performed as a function of either concentrations of the footprinting agent and/or time of exposure to it, in the multiple hit regime where many of the population or subpopulation of reactive DNA molecules are modified at more than one site. Properly controlled MHF experiments yield both the population distribution of different conformers and reactivity rate constants of the footprinting agent at all reactive positions in each conformer, which may be interpreted in terms of the accessibility of the site or the local concentration of the reagent. MHF experiments are particularly well-suited for dissecting effects at sites where unbound DNA is non-reactive and bound DNA is reactive with base-specific probes (e.g. KMnO4, DMS). We suggest that this method will also be applicable to analysis of enhancements in reactivity of other footprinting agents (e.g. DNase I, HO.). To demonstrate the utility of the MHF analysis, we quantify fragment distributions and individual site reactivities from multiple-hit KMnO4 footprinting of the non-template strand of Esigma70 RNA polymerase-lambdaPR promoter DNA complexes populated at binding equilibrium at 37 degreesC and transiently populated at a fixed time after a temperature downshift from 37 degreesC to 0 degreesC. For this system, a MHF analysis directly addresses the following questions: (i) what fraction of the population of promoter DNA molecules is open in the vicinity of the transcription start site (RPo) both at 37 degreesC and (transiently) after a downshift to 0 degreesC; (ii) does opening of the start site region in RPo occur entirely in one mechanistic step at the lambdaPR promoter and (iii) does the structure of RPo vary with temperature? In addition, we use the MHF-determined population distribution of KMnO4-reactive (RPo) and non-reactive promoter DNA to normalize the biphasic kinetics of decay of RPo to free promoter DNA after a 37 degrees to 0 degreesC temperature downshift, and thereby characterize the kinetics of the conformational changes involved in forming RPo.
Assuntos
Pegada de DNA/métodos , RNA Polimerases Dirigidas por DNA/química , DNA/química , Escherichia coli/enzimologia , Conformação de Ácido Nucleico , Cinética , Oligodesoxirribonucleotídeos/análise , Permanganato de Potássio/metabolismo , TemperaturaRESUMO
Kinetic studies of formation and dissociation of open-promoter complexes (RPo) involving Esigma70 RNA polymerase (R) and the lambdaPR promoter (P) demonstrate the existence of two kinetically significant intermediates, designated I1 and I2, and facilitate the choice of conditions under which each accumulates. For such conditions, we report the results of equilibrium and transient DNase I and KMnO4 footprinting studies which characterize I1 and I2. At 0 degreesC, where extrapolation of equilibrium data indicates I1 is the dominant complex, DNA bases in the vicinity of the transcription start site (+1) do not react with KMnO4, indicating that this region is closed in I1. However, the DNA backbone in I1 is extensively protected from DNase I cleavage; the DNase I footprint extends approximately 30 bases downstream and at least approximately 40 bases upstream from the start site. I1 has a short lifetime (=15 seconds), based on its sensitivity to competition with heparin. Shortly after a temperature downshift from 37 degreesC to 0 degreesC, in the time-range where we conclude that the dominant, transiently accumulated complex is I2, DNase I and KMnO4 footprinting reveal a complex with a closed-start site and an extended DNase I footprint like that of I1. However, unlike I1, I2 is insensitive to heparin competition and has a much longer dissociation lifetime at 0 degreesC. Based on footprinting, kinetic and thermodynamic studies, we conclude that in the short-lived intermediate I1 the promoter start site and downstream region are bound in a cleft defined by the open clamp-like jaws of Esigma70. We propose that binding of the start site and downstream DNA in this cleft triggers massive, relatively slow conformational changes which likely include RNA polymerase jaw closing with coupled folding. These proposed conformational changes occur prior to opening of the promoter start site region, and are responsible for the much longer lifetime of I2. Closing of the jaws of polymerase around the downstream region of promoter DNA appears to trigger opening of the start site region. From a quantitative analysis of the biphasic decay of KMnO4 reactivity of RPo at 0 degreesC, we obtain the equilibrium constant K3 for the conversion of I2 to RPo and the rate constant k-2 for the conversion of I2 to I1 (i.e. jaw opening). These quantitative results were previously unavailable at any temperature, and are necessary for the dissection of dissociation kinetic data at higher temperatures.
Assuntos
Pegada de DNA , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/enzimologia , Regiões Promotoras Genéticas/genética , Bacteriófago lambda/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/química , Desoxirribonuclease I/metabolismo , Cinética , Conformação de Ácido Nucleico , Permanganato de Potássio/metabolismo , Temperatura , Transcrição Gênica/genéticaRESUMO
High avidity anti-dsDNA IgG antibodies are believed to play an important role in the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE) and therefore attempts have been made to reduce the concentration of these antibodies in the bloodstream of SLE patients. Previously we reported the development of an antigen based heteropolymer (AHP), a bispecific complex prepared by using the avidin-biotin system to crosslink dsDNA to a mAb specific for the human erythrocyte (E) complement receptor. Our studies indicated that this AHP could bind anti-dsDNA antibodies to E and facilitate clearance of these autoantibodies from the circulation of a monkey without E destruction. Here we report an improved covalent crosslinking procedure and purification scheme in which the AHP construct is isolated by precipitation in 50% saturated ammonium sulfate. We used a dsDNA binding dye, PicoGreen, to demonstrate specificity of binding of dsDNA to E via the AHP. The efficacy of the AHP in binding IgG anti-dsDNA antibodies to E was demonstrated in a sensitive and quantitative assay, based on the time resolved fluorescence properties of europium-labeled anti-human IgG mAbs used to probe the E. We also used this assay to screen SLE patient and normal plasmas for levels of anti-dsDNA IgG. The results of this assay correlate very well with the Farr assay, and therefore this approach may be useful in the development of informative and specific assays for a variety of autoantibodies. Treatment of SLE plasmas with E-AHP under conditions close to physiological led to substantial reductions (> or = 90%) in anti-dsDNA titers. It should be possible to test these new AHP for their ability to target and safely remove IgG anti-dsDNA antibodies from the circulation in animal models.
Assuntos
Anticorpos Antinucleares/análise , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , DNA/imunologia , Imunoglobulina G/análise , Lúpus Eritematoso Sistêmico/terapia , Receptores de Complemento 3b/imunologia , Anticorpos Antinucleares/imunologia , Anticorpos Antinucleares/metabolismo , Anticorpos Biespecíficos/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Relação Dose-Resposta Imunológica , Eritrócitos/metabolismo , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Lúpus Eritematoso Sistêmico/imunologiaRESUMO
Chemical and enzymatic probing (footprinting) of the reactivity of the promoter DNA backbone is applied to characterize two binary open complexes RPo1 (-Mg2+) and RPo2 (+Mg2+), formed by Escherichia coli RNA polymerase (E sigma 70) at the lambda PR promoter. We report that HO. detects major differences in backbone reactivity between RPo1 and RPo2 in the open region from -4 to +1 relative to the transcription start site. Deoxyribose sugars at positions -4 to +1 of the t (template) strand react with HO. in RPo2 but are relatively protected in RPo1. Binding of Mg2+ to convert RPo1 to RPo2 therefore increases the reactivity of two negatively charged footprinting agents [MnO4-; Suh, W.-C., Ross, W., & Record, M. T., Jr., (1993) Science 259, 358-361; and Fe(EDTA)2-/HO.] at the start site and is required for binding of the negatively-charged initiating nucleotides to the polymerase and the t strand at the start site. We propose that these effects result from binding of two Mg2+ ions to the catalytic carboxyls in the nucleotide binding sites. Except for the key region on the t strand at the start site, the promoter DNA of both RPo1 and RPo2 is continuously protected from DNase I and hydroxyl radical (HO.) cleavage between the -12 and +25 promoter positions. Protection in the upstream region, extending from -13 to about -70, is periodic, with an 11 base pair periodicity indicative of binding of polymerase to a single face of the DNA helix.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Bacteriófago lambda/genética , DNA Viral/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Magnésio/metabolismo , Regiões Promotoras Genéticas , Fator sigma/metabolismo , Transcrição Gênica , Sequência de Bases , Pegada de DNA , DNA Viral/genética , Desoxirribonuclease I , Radical Hidroxila , Magnésio/química , Dados de Sequência Molecular , Conformação de Ácido NucleicoRESUMO
OBJECTIVE: To develop an in vitro model for investigating the mechanism by which autoantibodies in immune complexes (ICs) that are bound to primate erythrocytes via antigen-based heteropolymers (AHPs) are cleared from the circulation and localized to the liver. METHODS: IgG anti-double-stranded DNA (anti-dsDNA) antibodies in ICs with dsDNA were bound to human erythrocytes via complement receptor 1 (CR1) either by opsonization with normal human serum as a complement source or through the use of an AHP, which consists of an anti-CR1 monoclonal antibody (mAb) that is chemically crosslinked with dsDNA. We performed parallel investigations of the mechanism of transfer of both types of erythrocyte-bound ICs to a monocytic cell line (U937). Erythrocytes with CR1-bound ICs were incubated with U937 cells under a variety of conditions, and subsequently, the levels of IgG anti-dsDNA, CR1, AHP, or C3b on both erythrocytes and U937 cells were measured by flow cytometry with appropriate fluorescently labeled probes. RESULTS: In the presence of U937 cells, both the AHP-anti-dsDNA and C3b-opsonized ICs were rapidly removed from the erythrocytes; at 37 degrees C, more than half of the complexes were removed in 2 minutes. Monomeric mouse IgG2a mAb blocked the transfer of both types of complexes by 75%, suggesting that Fcgamma receptor type I (FcgammaRI) is the main phagocyte receptor responsible for the removal of ICs from erythrocytes. Levels of CR1 on the erythrocyte surface were reduced during transfer of the AHP-anti-dsDNA ICs, suggesting that transfer involves a concomitant removal of CR1, presumably by proteolysis. CONCLUSION: Transfer of AHP-anti-dsDNA ICs from erythrocyte CR1 to model phagocytes occurs by a mechanism that is similar to the natural mechanism of IC clearance, involving recognition by FcgammaRI and removal of erythrocyte CR1 as key steps.
Assuntos
Anticorpos Antinucleares/metabolismo , DNA/imunologia , Animais , Complexo Antígeno-Anticorpo/efeitos dos fármacos , Complexo Antígeno-Anticorpo/metabolismo , Doenças Autoimunes/metabolismo , Eritrócitos/química , Eritrócitos/imunologia , Humanos , Imunoglobulina G/farmacologia , Lúpus Eritematoso Sistêmico/imunologia , Metaloendopeptidases/antagonistas & inibidores , Camundongos , Fagócitos/imunologia , Receptores de Complemento 3b/imunologia , Células U937RESUMO
Heteropolymers (HP), bispecific mAbs which bind target pathogens to primate erythrocytes via complement receptor 1, facilitate clearance of pathogens from the bloodstream by targeting them for destruction in the liver without causing lysis or clearance of the erythrocytes. We show that when HP prepared with mouse IgG are intravenously infused into monkeys one or more times prior to exposure to a prototype pathogen, they bind to erythrocytes and remain in the circulation long enough to act as "sentinels," preventing pathogen invasion of the bloodstream. The effectiveness of HP as sentinels is limited both by the monkey's immune response to the HP and, prior to the immune response, by a gradual loss of the HP from monkey erythrocytes over a period of 1 week, and we have investigated possible causes of this HP loss. In overview, our results suggest that HP prepared with mouse IgG are able to effectively function as sentinels for a minimum of 4 days and, after repeat infusion, possibly for up to 2 weeks.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Complexo Antígeno-Anticorpo/imunologia , Bacteriófago phi X 174/imunologia , Receptores de Complemento 3b/imunologia , Animais , Haplorrinos , Fragmentos Fc das Imunoglobulinas/imunologia , Infusões Intravenosas , Camundongos , Polímeros , Fatores de TempoRESUMO
OBJECTIVE: To identify requirements for vocational training and continuing education programs in rural general practice. DESIGN: A questionnaire was sent to all 487 rural doctors and 140 metropolitan and 140 provincial city general practitioners (GPs) in Queensland. A sample of medical educators, health professional and consumer representatives and rural doctors was also interviewed. Responses were compared by geographical area, practice characteristics and level of postgraduate training. RESULTS: There are significant differences between rural and urban practice profiles. Rural doctors have to practise a range of clinical skills in an environment with restricted access to health professional support, although the need for advanced training in procedural or other skills depends on the type of rural practice. Rural and urban doctors want more influence in determining continuing medical education (CME) programs. Interactive learning methods were rated as the most effective education methods by both rural and urban GPs. Rural doctors were less likely to consider that they spent enough time on CME. CONCLUSION: Vocational training programs should accommodate various rural career objectives, including those requiring advanced levels of procedural work. There is a significant unmet demand for CME tailored to the needs of individual doctors, both rural and urban, but distance and isolation may make this more critical in rural practice. These issues need to be addressed as training opportunities can contribute to improved retention of the rural medical workforce.
Assuntos
Educação Médica Continuada , Medicina de Família e Comunidade/educação , Saúde da População Rural , Austrália , HumanosRESUMO
A pilot survey by telephone interview, followed by a questionnaire of all rural doctors identified in Queensland, was used develop both a definition of rural practice that distinguishes it from urban general practice and a classification of rural and remote practice which assists in sampling of rural doctors. Questionnaire responses in specific geographic areas were compared using chi-square and Mantel-Haenszel chi-square tests. Several factors were found to differentiate rural from urban general practice consistently, thereby enabling a functional definition of rural practice to be developed. Within the broad group of rural doctors, gradients in practice characteristics were found to differentiate doctors in larger rural centres from those in smaller, more remote communities. These gradients were related to the distance and time of travel from support services. They formed the basis of a complex classification of rural and remote general practice. This functional definition of rural and remote medical practice should be considered by researchers of rural medicine issues when sampling rural and remote doctors. The strategies used in this study could be adapted for use in considering practice characteristics of other rural health professions.