Predictors of revascularization method and long-term outcome of percutaneous coronary intervention or repeat coronary bypass surgery in patients with multivessel coronary disease and previous coronary bypass surgery.

AIMS: The optimal revascularization strategy in patients with symptomatic multivessel coronary artery disease (CAD) and previous coronary artery bypass grafting (CABG) remains unknown. METHODS AND RESULTS: We evaluated all patients with previous CABG undergoing isolated, non-emergency multivessel revascularization between 1 January 1995 and 31 December 2000. The analysis concentrated on the independent predictors of the revascularization method, as well as on long-term mortality and its predictors, after calculating a propensity score for the method of revascularization. There were 2191 patients (1487 with reoperation and 704 with percutaneous coronary intervention, PCI) in the study. The most important factors in choosing reoperation were presence of more diseased or occluded grafts, previous infarction, lower ejection fraction (EF), longer interval from first CABG, and more total occlusions of native arteries, as well as absence of a patent mammary graft. The distribution of the propensity score was skewed towards the two extremes. At 5 years, the unadjusted cumulative survival was 79.5% for CABG and 75.3% for PCI, P=0.008. After adjustment for the propensity score for PCI vs. CABG, PCI was associated with a hazard ratio of 1.47 (0.94-2.28), P=0.09. The most powerful predictors of mortality were higher age and lower EF. CONCLUSION: The choice of the revascularization method in patients with previous CABG is dictated mostly by anatomical considerations and less by clinical characteristics. In contrast, clinical characteristics predominantly affect long-term outcome, whereas the method of revascularization has a limited effect. A randomized clinical trial addressing this important segment of the population with ischaemic heart disease is warranted.     

Electron transfer precedes ATP hydrolysis during nitrogenase catalysis

The biological reduction of N₂ to NH₃ catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: k_ATP = 70 s⁻¹, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (k_ET = 140 s⁻¹, 25 °C), (ii) ATP hydrolysis (k_ATP = 70 s⁻¹, 25 °C), (iii) Phosphate release (k_Pi = 16 s⁻¹, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (k_diss = 6 s⁻¹, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Fe^ox(ADP)₂ protein and the reduced MoFe protein.Biological nitrogen fixation catalyzed by the Mo-dependent nitrogenase has a limiting reaction stoichiometry shown in Eq. 1 (1, 2):The ATP-driven reduction of one N₂ with evolution of one H₂ requires a minimum of 8 e⁻ and the hydrolysis of 16 ATP molecules in a complex cascade of events in which electron transfer (ET) from the nitrogenase Fe protein to the catalytic MoFe protein is coupled to the hydrolysis of two ATP molecules (1, 3, 4). The Fe protein is a homodimer with a single [4Fe–4S] cluster and two nucleotide binding sites, one in each subunit (5). The MoFe protein is an α₂β₂-tetramer, with each αβ-pair functioning as a catalytic unit that binds an Fe protein (6). Each αβ-unit contains an [8Fe–7S] cluster (abbreviated as P cluster) and a [7Fe–9S–Mo–C–R-homocitrate] cluster (abbreviated as FeMo cofactor or M cluster) (6–10). In each ET event, the Fe protein, in the reduced (1+) state with two bound ATP, first associates with the MoFe protein (Fig. 1). In a recent model, termed “deficit spending,” it is proposed that this association triggers a two-step ET event (11, 12). The first ET step occurs inside the MoFe protein, involving ET from the P cluster resting state (P^N) to the resting FeMo cofactor (M^N), resulting in an oxidized P cluster (P¹⁺) and a reduced FeMo cofactor (M^R) (12). This ET event is conformationally gated (11) with an apparent first-order rate constant (k_ET) between 100 and 140 s⁻¹ (11, 12). In the second ET step, an electron is transferred from the Fe protein [4Fe–4S] cluster to the oxidized P¹⁺ cluster, resulting in the return of the P cluster to the resting oxidation state (P^N) and an oxidized [4Fe–4S]²⁺ cluster in the Fe protein (12). This second step is fast, having a rate constant greater than 1,700 s⁻¹ (12).Open in a separate windowFig. 1.Order of events in nitrogenase complex. (A) Fe protein subunits are shown as two blue ovals (Left) with an ATP bound in each subunit and the [4Fe–4S] cluster (green cubane). (Right) MoFe protein α-subunit (orange) and β-subunit (green), with the P^N cluster shown as a gray box and the FeMo cofactor (M^N) shown as a gray diamond. (B) From left to right, order of events in the nitrogenase ET is shown with rate constants (s⁻¹) displayed where known.Transfer of one electron from the Fe protein to an αβ-unit of MoFe protein is known to be coupled to the hydrolysis of the two ATP molecules bound to the Fe protein, yielding two ADP and two Pi (2). Following the hydrolysis reaction, the two phosphates (Pi) are released from the protein complex with a first-order rate constant (k_Pi) of 22 s⁻¹ at 23 °C (13). The last event in the cycle is the release of the oxidized Fe protein with two ADP bound [Fe^ox(ADP)₂] from the MoFe protein with a rate constant (k_diss) of ∼6 s⁻¹, the rate-limiting step in catalysis at high electron flux (14). After dissociation from the MoFe protein, the [Fe^ox(ADP)₂] protein is prepared for a second round of electron delivery by one-electron reduction to [Fe^red(ADP)₂] and replacement of the two MgADP by MgATP. This cycle is repeated until enough electrons are transferred to the MoFe protein to achieve substrate reduction (15).Although the energetic coupling between ET and ATP hydrolysis is firmly established (1, 3, 4, 16), the nature of this coupling has remained unresolved: does ATP hydrolysis itself provide the principal energy input for the conformational change(s) that drive ET from Fe protein to the MoFe protein, or, does the bound ATP induce the formation of a reactive, “activated” conformation of the complex, with ET being driven by the free energy of ATP-activated protein–protein binding? These alternatives are characterized by different orders of ET and ATP hydrolysis, but the order has never been established. Some studies have indicated that ATP hydrolysis occurs after ET (13, 17, 18), whereas other studies have suggested just the opposite, namely that ATP hydrolysis occurs before ET (15, 16, 19, 20). One of the reasons for this lack of clarity in the order of these key events is the absence of direct measurement of ATP hydrolysis rates by nitrogenase within a single catalytic cycle. The rate constant for Pi release during one cycle has been measured, thereby establishing a lower limit on the rate constant for ATP hydrolysis (13). However, the rate constant for ATP hydrolysis could be much faster than Pi release, and could be faster than the rate constant for ET.Here, we have directly measured the rate constant for ATP hydrolysis for a single nitrogenase turnover cycle, as well as measuring the rate constants for each of the other key steps under the same conditions, thereby allowing an unequivocal assignment of the order of events in a single electron-transfer cycle. Establishing the order of events allows a full thermodynamic Fe–protein cycle to be constructed.     

Alcohol outlets and youth alcohol use: Exposure in suburban areas

The purpose of this study was to explore how exposure to alcohol outlets (around home and school) influenced alcohol use among 242 high-school students (mean age 16.4, 48.8% male, 93.4% White). Results found no relationship between alcohol outlet exposure, using a measure of both distance to and density around students’ homes and schools, and alcohol use. This study suggests that outlet exposure may not influence alcohol use among mostly White, middle-class, and suburban youth. However, the lack of association may also reflect the lower level of alcohol outlets present in low-density residential environments as well as differences in accessibility.