Computer Simulation of Assembly and Co-operativity of Hexameric AAA ATPases

AAA ATPases form a functionally diverse superfamily of proteins. Most members form homo-hexameric ring complexes, are catalytically active only in the fully assembled state, and show co-operativity among the six subunits. The mutual dependence among the subunits is clearly evidenced by the fact that incorporation of mutated, inactive subunits can decrease the activity of the remaining wild type subunits. For the first time, we develop here models to describe this form of allostery, evaluate them in a simulation study, and test them on experimental data. We show that it is important to consider the assembly reactions in the kinetic model, and to define a formal inhibition scheme. We simulate three inhibition scenarios explicitly, and demonstrate that they result in differing outcomes. Finally, we deduce fitting formulas, and test them on real and simulated data. A non-competitive inhibition formula fitted experimental and simulated data best. To our knowledge, our study is the first one that derives and tests formal allosteric schemes to explain the inhibitory effects of mutant subunits on oligomeric enzymes.     

Vitronectin and its fragments purified as serum inhibitors of Staphylococcus aureus gamma-hemolysin and leukocidin, and their specific binding to the hlg2 and the LukS components of the toxins.

Staphylococcal gamma-hemolysin and leukocidin are bi-component cytolysins, consisting of LukF (or Hlg1)/Hlg2 and LukF/LukS, respectively. Here, we purified serum inhibitors of gamma-hemolysin and leukocidin from human plasma. Protein sequencing showed that the purified inhibitors of 62, 57, 50 and 38 kDa were the vitronectin fragments with truncation(s) of the C-terminal or both N- and C-terminal regions. The purified vitronectin fragments specifically bound to the Hlg2 component of gamma-hemolysin and the LukS component of leukocidin to form high-molecular-weight complexes with them, leading to inhibition of the toxin-induced lysis of human erythrocytes and human polymorphonuclear leukocytes, respectively. Intact vitronectin also showed inhibitory activity to the toxins. The ability of gamma-hemolysin and leukocidin to bind vitronectin and its fragments is a novel function of the pore-forming cytolysins.     

Anesthetic effects on pulmonary allergic responses in rats: changes in sensitivity to serotonin.

Subsequent to observations that pulmonary responses to antigen challenge are of different magnitudes in sensitized rats that are anesthetized with different drugs, we conducted studies to test whether the alterations in responses were due to changes in airway responsiveness to cholinergic or serotonergic challenge, opioid-receptor mediated events, or changes in mast cell mediator release. Immunoglobulin E-sensitized rats anesthetized with ketamine/urethan had larger changes in lung resistance and plasma histamine after pulmonary antigen challenge compared with rats anesthetized with fentanyl-droperidol. Blockade of opioid receptors with naloxone did not affect the responses. In unsensitized rats, airway responses to aerosolized methacholine were similar for the two anesthetics, indicating unchanged smooth muscle responsiveness; however, airway responses to intravenous serotonin were enhanced by ketamine and ablated by droperidol. We conclude that ketamine- and droperidol-induced alterations of pulmonary allergic responses are due to changes in sensitivity to serotonin and in mast cell mediator release. We speculate that mast cell mediator release may be modulated by a serotonin receptor-linked mechanism.     

Bone density parathyroid hormone and 25-hydroxyvitamin D concentrations in middle aged women.

OBJECTIVE--To examine the relation between bone density and indices of calcium metabolism including parathyroid hormone and 25-hydroxyvitamin D concentrations in middle aged women. DESIGN--A cross sectional study. SETTING AND SUBJECTS--138 women volunteers aged 45-65 with no known osteoporosis and unselected for disease status recruited for a dietary assessment study from the community using general practice registers. Volunteer rate was 20%. MAIN OUTCOME MEASURE--Bone mineral density measured with dual energy x ray absorptiometry. RESULTS--Bone density at the lumbar spine and neck and trochanteric regions of the femur was inversely related to serum intact parathyroid hormone concentrations and positively related to serum 25-hydroxyvitamin D concentrations. These associations were independent of possible confounding factors, including age, body mass index, cigarette smoking habit, menopausal status, and use of diuretics and postmenopausal hormone replacement therapy. These associations were apparent throughout the whole distribution of bone density and 25-hydroxyvitamin D and parathyroid hormone concentrations within the normal range, suggesting a physiological relation. CONCLUSIONS--The findings are consistent with the hypothesis that parathyroid hormone and 25-hydroxyvitamin D concentrations influence bone density in middle aged women. Findings from this study together with other work suggest that the role of vitamin D in osteoporosis should not be neglected. The associations with parathyroid hormone also indicate plausible biological mechanisms. The roughly 5-10% difference in bone density between top and bottom tertiles of serum 25-hydroxyvitamin D concentrations, though not large in magnitude, may have considerable public health implications in terms of prevention of osteoporosis and its sequelae, fractures.     

An implantable transducer for measuring tension in an anterior cruciate ligament graft.

The goal of this study was to develop a new implantable transducer for measuring anterior cruciate ligament (ACL) graft tension postoperatively in patients who have undergone ACL reconstructive surgery. A unique approach was taken of integrating the transducer into a femoral fixation device. To devise a practical in vivo calibration protocol for the fixation device transducer (FDT), several hypotheses were investigated: (1) The use of a cable versus the actual graft as the means for applying load to the FDT during calibration has no significant effect on the accuracy of the FDT tension measurements; (2) the number of flexion angles at which the device is calibrated has no significant effect on the accuracy of the FDT measurements; (3) the friction between the graft and femoral tunnel has no significant effect on measurement accuracy. To provide data for testing these hypotheses, the FDT was first calibrated with both a cable and a graft over the full range of flexion. Then graft tension was measured simultaneously with both the FDT on the femoral side and load cells, which were connected to the graft on the tibial side, as five cadaver knees were loaded externally. Measurements were made with both standard and overdrilled tunnels. The error in the FDT tension measurements was the difference between the graft tension measured by the FDT and the load cells. Results of the statistical analyses showed that neither the means of applying the calibration load, the number of flexion angles used for calibration, nor the tunnel size had a significant effect on the accuracy of the FDT. Thus a cable may be used instead of the graft to transmit loads to the FDT during calibration, thus simplifying the procedure. Accurate calibration requires data from just three flexion angles of 0, 45, and 90 deg and a curve fit to obtain a calibration curve over a continuous range of flexion within the limits of this angle group. Since friction did not adversely affect the measurement accuracy of the FDT, the femoral tunnel can be drilled to match the diameter of the graft and does not need to be overdrilled. Following these procedures, the error in measuring graft tension with the FDT averages less than 10 percent relative to a full-scale load of 257 N.