Dose-response relations of azimilide in the management of symptomatic, recurrent, atrial fibrillation.

We evaluated the efficacy and safety of azimilide, a new class III antiarrhythmic agent that blocks both the slow and fast components of the cardiac-delayed rectifier potassium currents in 4 randomized, double-blind, placebo-controlled trials with similar protocols. The purpose of this study was to assess the relation between dose and effect. A total of 1,380 patients with a documented history of symptomatic atrial fibrillation (AF), atrial flutter, or both, were enrolled. After a 3-day loading period during which the assigned dose was given twice a day, subjects received placebo or azimilide (35, 50, 75, 100, or 125 mg once a day) for the duration of the study period. The primary end point of the studies was the time to symptomatic arrhythmia recurrence with a transtelephonic electrocardiogram typical of AF, atrial flutter, or paroxysmal supraventricular tachycardia. For each study, Kaplan-Meier estimates of the median time to recurrence were computed for placebo and for each azimilide dose. Cox proportional-hazards modeling was used to estimate hazard ratios for each active dose. Each of the 2 highest azimilide doses (100 and 125 mg/day) significantly prolonged the time to recurrence of arrhythmia. For the 100 mg/day dose, the hazard ratio was 1.34, 95% confidence interval 1.05 to 1.72; p = 0.02. For the 125 mg/day dose, the hazard ratio was 1.32, 95% confidence interval 1.07 to 1.62; p = 0.01. Patients with a history of either ischemic heart disease or congestive heart failure had a significantly greater treatment effect from azimilide than those without it. Torsades de Pointes occurred in 0.9% of patients receiving either of the 2 effective doses. Thus, doses of azimilide <100 mg/day are not effective for control of AF, whereas doses of 100 and 125 mg/day are effective with an acceptable risk of serious toxicity.

Antiarrhythmic effects of azimilide in atrial fibrillation: efficacy and dose-response. Azimilide Supraventricular Arrhythmia Program 3 (SVA-3) Investigators.

OBJECTIVES: The purpose of this study was to assess the effectiveness of azimilide, a class III antiarrhythmic drug, in reducing the frequency of symptomatic arrhythmia recurrences in patients with atrial fibrillation, atrial flutter or both. BACKGROUND: Atrial fibrillation is an increasingly common disorder of the heart rhythm, and most patients with this problem are identified because they have symptoms associated with their arrhythmia. New antiarrhythmic therapies are needed to treat patients with this problem. METHODS: A total of 384 patients with a history of atrial fibrillation, atrial flutter or both were randomly assigned to receive once daily doses of placebo or azimilide; recurrent symptomatic arrhythmias were documented using transtelephonic electrocardiogram (ECG) recording. Azimilide 50 mg, 100 mg or 125 mg was tested; the primary efficacy analysis compared the time to first symptomatic recurrence in the combined azimilide 100 mg and 125 mg dose groups with that in the placebo group using the log-rank test. RESULTS: In the primary efficacy analysis, the time to first symptomatic arrhythmia recurrence was significantly prolonged in the combined azimilide 100 mg and 125 mg daily dose group compared with the placebo group (chi-square 7.96, p = 0.005); the hazard ratio (placebo: azimilide) for this comparison was 1.58 (95% confidence interval [CI] = 1.15, 2.16). In comparisons between individual doses and placebo, the hazard ratio for the 50 mg daily dose was 1.17 (95% CI = 0.83, 1.66; p = 0.37); for the 100 mg group, dose was 1.38 (95% CI = 0.96, 1.98; p = 0.08), and for the 125 mg group, dose was 1.83 (95% CI = 1.24, 2.70; p = 0.002). CONCLUSIONS: Azimilide significantly lengthened the symptomatic arrhythmia-free interval in patients with a history of atrial fibrillation, atrial flutter or both.

Mechanism of protein import across the chloroplast envelope.

The development and maintenance of chloroplasts relies on the contribution of protein subunits from both plastid and nuclear genomes. Most chloroplast proteins are encoded by nuclear genes and are post-translationally imported into the organelle across the double membrane of the chloroplast envelope. Protein import into the chloroplast consists of two essential elements: the specific recognition of the targeting signals (transit sequences) of cytoplasmic preproteins by receptors at the outer envelope membrane and the subsequent translocation of preproteins simultaneously across the double membrane of the envelope. These processes are mediated via the co-ordinate action of protein translocon complexes in the outer (Toc apparatus) and inner (Tic apparatus) envelope membranes.

The transit sequence of ferredoxin contains different domains for translocation across the outer and inner membrane of the chloroplast envelope.

Deletion mutants in the transit sequence of preferredoxin were used in label transfer cross-linking assays to map the interactions of the transit sequence with the import machinery. The deletion mutants gave distinct cross-linking patterns to the Toc and Tic components of the import machinery, consistent with the binding and import properties obtained in in vitro import assays. The cross-linking results revealed two separate properties of the transit peptide: first the presentation of specific binding domains for the initial interaction with outer membrane components, and second the presence of different domains for interaction with the outer and inner membrane components of the transport machinery for full envelope translocation. The N-terminal Delta6-14 deletion blocked import of the precursor at the Toc components, whereas the more internal deletion Delta15-25 blocked import at the Tic components. The information for association with the outer and inner membrane components therefore resides in two separate but partly overlapping domains in the first 25 amino acids of the transit sequence.

Initial binding of preproteins involving the Toc159 receptor can be bypassed during protein import into chloroplasts.

Two integral outer envelope GTPases, Toc34 and Toc86, are proposed to regulate the recognition and translocation of nuclear-encoded preproteins during the early stages of protein import into chloroplasts. Defining the precise roles of Toc86 and Toc34 has been complicated by the inability to distinguish their GTPase activities. Furthermore, the assignment of Toc86 function is rendered equivocal by recent reports suggesting that the standard protocol for the isolation of chloroplasts results in significant proteolysis of Toc86 (B. Bolter, T. May, J. Soll [1998] FEBS Lett 441: 59-62; G. Schatz [1998] Nature 395: 439-440). We demonstrate that Toc86 corresponds to a native protein of 159 kD in pea (Pisum sativum), designated Toc159. We take advantage of the proteolytic sensitivity of Toc159 to selectively remove its 100-kD cytoplasmic GTPase domain and thereby distinguish its activities from other import components. Proteolysis eliminates detectable binding of preproteins at the chloroplast surface, which is consistent with the proposed role of Toc159 as a receptor component. Remarkably, preprotein translocation across the outer membrane can occur in the absence of the Toc159 cytoplasmic domain, suggesting that binding can be bypassed. Translocation remains sensitive to GTP analogs in the absence of the Toc159 GTP-binding domain, providing evidence that Toc34 plays a key role in the regulation of translocation by GTP.

Functions and origins of the chloroplast protein-import machinery.

The vast majority of chloroplast proteins are nuclear-encoded and are imported into the organelle after synthesis in the cytoplasm. Targeting to chloroplasts is mediated by a variety of intrinsic targeting signals that direct the preprotein to its proper organelle subcompartment. Translocation at the envelope membrane is directed by the interactions of an N-terminal transit sequences on the preprotein and a general import machinery composed of the outer-membrane Toc machinery and the inner-membrane Tic machinery. The Toc and Tic components interact to bypass the intermembrane space and provide direct transport of preproteins from the cytoplasm to the stroma. There are at least four targeting pathways to the thylakoid membrane, the cpSec pathway, the delta pH pathway, the cpSRP pathway and the spontaneous pathway. These pathways require distinct intrinsic targeting signals, and apparently evolved to accommodate the translocation of classes of proteins with particular characteristics. Proteins similar to some components of the envelope and thylakoid translocation pathways are found in bacterial systems. However, a number of components do not have bacterial counterparts and are unique to the chloroplast pathways. It therefore appears that the chloroplast translocation systems have evolved from membrane-transport systems that were present in the original endosymbiont by incorporating proteins necessary to adapt to the constraints of endosymbiosis.

Tic22 is targeted to the intermembrane space of chloroplasts by a novel pathway.

Tic22 previously was identified as a component of the general import machinery that functions in the import of nuclear-encoded proteins into the chloroplast. Tic22 is peripherally associated with the outer face of the inner chloroplast envelope membrane, making it the first known resident of the intermembrane space of the envelope. We have investigated the import of Tic22 into isolated chloroplasts to define the requirements for targeting of proteins to the intermembrane space. Tic22 is nuclear-endoded and synthesized as a preprotein with a 50-amino acid N-terminal presequence. The analysis of deletion mutants and chimerical proteins indicates that the precursor of Tic22 (preTic22) presequence is necessary and sufficient for targeting to the intermembrane space. Import of preTic22 was stimulated by ATP and required the presence of protease-sensitive components on the chloroplast surface. PreTic22 import was not competed by an excess of an authentic stromal preprotein, indicating that targeting to the intermembrane space does not involve the general import pathway utilized by stromal preproteins. On the basis of these observations, we conclude that preTic22 is targeted to the intermembrane space of chloroplasts by a novel import pathway that is distinct from known pathways that target proteins to other chloroplast subcompartments.

Protein import into chloroplasts.

Chloroplasts have evolved an elaborate system of membrane and soluble subcompartments to organize and regulate photosynthesis and essential aspects of amino acid and lipid metabolism. The biogenesis and maintenance of organellar architecture rely on protein subunits encoded by both nuclear and plastid genomes. Import of nuclear-encoded proteins is mediated by interactions between the intrinsic N-terminal transit sequence of the nuclear-encoded preprotein and a common import machinery at the chloroplast envelope. Recent investigations have shown that there are two unique membrane-bound translocation systems, in the outer and inner envelope membranes, which physically associate during import to transport preproteins from the cytoplasm to the internal stromal compartment. This review discusses current understanding of these translocation systems and models for the way in which they might function.

Tic20 and Tic22 are new components of the protein import apparatus at the chloroplast inner envelope membrane.

Two components of the chloroplast envelope, Tic20 and Tic22, were previously identified as candidates for components of the general protein import machinery by their ability to covalently cross-link to nuclear-encoded preproteins trapped at an intermediate stage in import across the envelope (Kouranov, A., and D.J. Schnell. 1997. J. Cell Biol. 139:1677-1685). We have determined the primary structures of Tic20 and Tic22 and investigated their localization and association within the chloroplast envelope. Tic20 is a 20-kD integral membrane component of the inner envelope membrane. In contrast, Tic22 is a 22-kD protein that is located in the intermembrane space between the outer and inner envelope membranes and is peripherally associated with the outer face of the inner membrane. Tic20, Tic22, and a third inner membrane import component, Tic110, associate with import components of the outer envelope membrane. Preprotein import intermediates quantitatively associate with this outer/inner membrane supercomplex, providing evidence that the complex corresponds to envelope contact sites that mediate direct transport of preproteins from the cytoplasm to the stromal compartment. On the basis of these results, we propose that Tic20 and Tic22 are core components of the protein translocon of the inner envelope membrane of chloroplasts.

Insertion of the 34-kDa chloroplast protein import component, IAP34, into the chloroplast outer membrane is dependent on its intrinsic GTP-binding capacity.

IAP34 is a 34-kDa component of the outer membrane complex that mediates the initial stages of protein import into chloroplasts (Seedorf, M., Waegemann, K., and Soll, J. (1995) Plant J. 7, 401-411; Kessler, F., Blobel, G., Patel, H. A., and Schnell, D. J. (1994) Science 266, 1035-1039). We have investigated the targeting and insertion of IAP34 at the outer envelope membrane. The analyses of IAP34 deletion mutants and hybrid proteins (consisting of regions of IAP34 fused to the soluble IgG-binding domain of staphylococcal protein A) suggest that the transmembrane domain and C-terminal tail of IAP34 contain information essential but not sufficient for targeting to the outer membrane. Treatment of chloroplasts with exogenous proteases does not affect IAP34 insertion, indicating that targeting does not require surface-exposed receptors at the envelope. GTP or GDP is required for maximal integration of IAP34 into the outer membrane. The GTP/GDP requirement is attributed to the intrinsic GTP binding activity of IAP34 because GTP/GDP binding-deficient mutants are defective in outer membrane insertion. On the basis of these observations, we propose that IAP34 is targeted to the chloroplast by a C-terminal signal and efficiently integrated into the outer membrane by conformation-induced insertion upon GTP/GDP binding.

Analysis of the interactions of preproteins with the import machinery over the course of protein import into chloroplasts.

We have investigated the interactions of two nuclear-encoded preproteins with the chloroplast protein import machinery at three stages in import using a label-transfer crosslinking approach. During energy-independent binding at the outer envelope membrane, preproteins interact with three known components of the outer membrane translocon complex, Toc34, Toc75, and Toc86. Although Toc75 and Toc86 are known to associate with preproteins during import, a role for Toc34 in preprotein binding previously had not been observed. The interaction of Toc34 with preproteins is regulated by the binding, but not hydrolysis of GTP. These data provide the first evidence for a direct role for Toc34 in import, and provide insights into the function of GTP as a regulator of preprotein recognition. Toc75 and Toc86 are the major targets of cross-linking upon insertion of preproteins across the outer envelope membrane, supporting the proposal that both proteins function in translocation at the outer membrane as well as preprotein recognition. The inner membrane proteins, Tic(21) and Tic22, and a previously unidentified protein of 14 kD are the major targets of crosslinking during the late stages in import. These data provide additional support for the roles of these components during protein translocation across the inner membrane. Our results suggest a defined sequence of molecular interactions that result in the transport of nuclear-encoded preproteins from the cytoplasm into the stroma of chloroplasts.

Sources of HIV information among injecting drug users: association with gender, ethnicity, and risk behaviour. AIDS Community Demonstration Projects.

Sources of HIV information were examined for 774 male and female injecting drug users (IDUs). The majority (80.7%) had received HIV information from one or more sources in the prior 3 months. The most frequently mentioned sources were television (39.9%) and friends or family (22.2%). There were few differences in source of HIV information with regard to gender, ethnicity, or age. Differences were more frequently observed between cities. The relationship of information source and subject characteristics with HIV knowledge, perceived risk, drug-related and sexual practices was examined using logistic regression. For men, exposure to mass media sources (OR = 1.48) and small media materials (OR = 2.03) were related to HIV knowledge. Small media and interpersonal information were related to HIV testing for men (OR = 1.95 and 1.85, respectively) and women (OR = 2.25 and 2.54). Interpersonal sources of information were also associated with increased sharing of injection equipment (OR = 2.04) and bleach use (OR = 2.23) among female IDUs. Significant differences in HIV knowledge and risk-related practices were also observed for ethnicity, city, men who have sex with men, and women who had traded sex for money or drugs. Implications for targeting HIV prevention efforts for IDUs are discussed.

Two components of the chloroplast protein import apparatus, IAP86 and IAP75, interact with the transit sequence during the recognition and translocation of precursor proteins at the outer envelope.

The interactions of precursor proteins with components of the chloroplast envelope were investigated during the early stages of protein import using a chemical cross-linking strategy. In the absence of energy, two components of the outer envelope import machinery, IAP86 and IAP75, cross-linked to the transit sequence of the precursor to the small subunit of ribulose-1, 5-bisphosphate carboxylase (pS) in a precursor binding assay. In the presence of concentrations of ATP or GTP that support maximal precursor binding to the envelope, cross-linking to the transit sequence occurred predominantly with IAP75 and a previously unidentified 21-kD polypeptide of the inner membrane, indicating that the transit sequence had inserted across the outer membrane. Cross-linking of envelope components to sequences in the mature portion of a second precursor, preferredoxin, was detected in the presence of ATP or GTP, suggesting that sequences distant from the transit sequence were brought into the vicinity of the outer membrane under these conditions. IAP75 and a third import component, IAP34, were coimmunoprecipitated with IAP86 antibodies from solubilized envelope membranes, indicating that these three proteins form a stable complex in the outer membrane. On the basis of these observations, we propose that IAP86 and IAP75 act as components of a multisubunit complex to mediate energy-independent recognition of the transit sequence and subsequent nucleoside triphosphate-induced insertion of the transit sequence across the outer membrane.

Measuring the adoption of consistent use of condoms using the stages of change model. AIDS Community Demonstration Projects.

The stages of behavior change model has been used to understand a variety of health behaviors. Since consistent condom use has been promoted as a risk-reduction behavior for prevention of human immunodeficiency virus (HIV) infection, an algorithm for staging the adoption of consistent condom use during vaginal sex was empirically developed using three considerations: HIV prevention efficacy, analogy with work on staging other health-related behaviors, and condom use data from groups at high risk for HIV infection. This algorithm suggests that the adoption of consistent condom use among persons at high risk can be meaningfully measured with the model. However, variations in the algorithm details affect both the interpretation of stages and apportionment of persons across stages.

A regression method for analysing ordinal data from intervention trials.

We consider the case of a community intervention trial evaluated with a series of cross-sectional surveys and having outcomes measured on an ordinal scale. We propose a modelling procedure that combines ridit analysis and linear regression methods. We use the multinomial distribution as the basis for variance estimation of the mean ridits and then use simple regression models to estimate differences (for example, between intervention and comparison areas) among the ridits. We illustrate this procedure with data from a community intervention trial promoting condom use, with the adoption of consistent condom use measured on a 5-point ordinal scale.