Improvement in diastolic intraventricular pressure gradients in patients with HOCM after ethanol septal reduction

We sought to validate measurement of intraventricular pressure gradients (IVPG) and analyze their change in patients with hypertrophic obstructive cardiomyopathy (HOCM) after ethanol septal reduction (ESR). Quantitative analysis of color M-mode Doppler (CMM) images may be used to estimate diastolic IVPG noninvasively. Noninvasive IVPG measurement was validated in 10 patients undergoing surgical myectomy. Echocardiograms were then analyzed in 19 patients at baseline and after ESR. Pulsed Doppler data through the mitral valve and pulmonary venous flow were obtained. CMM was used to obtain the flow propagation velocity (Vp) and to calculate IVPG off-line. Left atrial pressure was estimated with the use of previously validated Doppler equations. Data were compared before and after ESR. CMM-derived IVPG correlated well with invasive measurements obtained before and after surgical myectomy [r = 0.8, P < 0.01, Delta(CMM - invasive IVPG) = 0.09 +/- 0.45 mmHg]. ESR resulted in a decrease of resting LVOT systolic gradient from 62 +/- 10 to 29 +/- 5 mmHg (P < 0.001). There was a significant increase in the Vp and IVPG (from 48 +/- 5to 74 +/- 7 cm/s and from 1.5 +/- 0.2 to 2.6 +/- 0.3 mmHg, respectively, P < 0.001 for both). Estimated left atrial pressure decreased from 16.2 +/- 1.1 to 11.5 +/- 0.9 mmHg (P < 0.001). The increase in IVPG correlated with the reduction in the LVOT gradient (r = 0.6, P < 0.01). Reduction of LVOT obstruction after ESR is associated with an improvement in diastolic suction force. Noninvasive measurements of IVPG may be used as an indicator of diastolic function improvement in HOCM.     

Multiple steps of phosphorylation of activated rhodopsin can account for the reproducibility of vertebrate rod single-photon responses

Single-photon responses (SPRs) in vertebrate rods are considerably less variable than expected if isomerized rhodopsin (R*) inactivated in a single, memoryless step, and no other variability-reducing mechanisms were available. We present a new stochastic model, the core of which is the successive ratcheting down of R* activity, and a concomitant increase in the probability of quenching of R* by arrestin (Arr), with each phosphorylation of R* (Gibson, S.K., J.H. Parkes, and P.A. Liebman. 2000. Biochemistry. 39:5738-5749.). We evaluated the model by means of Monte-Carlo simulations of dim-flash responses, and compared the response statistics derived from them with those obtained from empirical dim-flash data (Whitlock, G.G., and T.D. Lamb. 1999. Neuron. 23:337-351.). The model accounts for four quantitative measures of SPR reproducibility. It also reproduces qualitative features of rod responses obtained with altered nucleotide levels, and thus contradicts the conclusion that such responses imply that phosphorylation cannot dominate R* inactivation (Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836-1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733-747.). Moreover, the model is able to reproduce the salient qualitative features of SPRs obtained from mouse rods that had been genetically modified with specific pathways of R* inactivation or Ca2+ feedback disabled. We present a theoretical analysis showing that the variability of the area under the SPR estimates the variability of integrated R* activity, and can provide a valid gauge of the number of R* inactivation steps. We show that there is a heretofore unappreciated tradeoff between variability of SPR amplitude and SPR duration that depends critically on the kinetics of inactivation of R* relative to the net kinetics of the downstream reactions in the cascade. Because of this dependence, neither the variability of SPR amplitude nor duration provides a reliable estimate of the underlying variability of integrated R* activity, and cannot be used to estimate the minimum number of R* inactivation steps. We conclude that multiple phosphorylation-dependent decrements in R* activity (with Arr-quench) can confer the observed reproducibility of rod SPRs; there is no compelling need to invoke a long series of non-phosphorylation dependent state changes in R* (as in Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836-1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733-747.). Our analyses, plus data and modeling of others (Rieke, F., and D.A. Baylor. 1998a. Biophys. J. 75:1836-1857; Field, G.D., and F. Rieke. 2002. Neuron. 35:733-747.), also argue strongly against either feedback (including Ca2+-feedback) or depletion of any molecular species downstream to R* as the dominant cause of SPR reproducibility.     

Structure-function analysis of recombinant substrate protein 22 kDa (SP-22). A mitochondrial 2-CYS peroxiredoxin organized as a decameric toroid

Bovine mitochondrial SP-22 is a member of the peroxiredoxin family of peroxidases. It belongs to the peroxiredoxin 2-Cys subgroup containing three cysteines at positions 47, 66, and 168. The cloning and overexpression in Escherichia coli of recombinant wild type SP-22 and its three cysteine mutants (C47S, C66S, and C168S) are reported. Purified His-tagged SP-22 was fully active with Cys-47 being confirmed as the catalytic residue. The enzyme forms a stable decameric toroid consisting of five basic dimeric units containing intermolecular disulfide bonds linking the catalytically active Cys-47 of one subunit and Cys-168 of the adjacent monomer. The disulfide bonds are not required for overall structural integrity. The toroidal units have average external and internal diameters of 15 and 7 nm, respectively, and can form stacks in a lateral arrangement of two or three rings. C47S had a pronounced tendency to stack in long tubular structures containing up to 60 rings. Further unusual structural features are the presence of radial spikes projecting from the external surface and ordered electron-dense material within the central cavity of the toroid.     

Sensitivity analysis of a spatially-explicit stochastic simulation model of the evolution of resistance in Helicoverpa zea (Lepidoptera: Noctuidae) to Bt transgenic corn and cotton

The sensitivities of a model simulating the evolution of resistance in Helicoverpa zea to Bt toxins in transgenic crops were investigated by examining effects of each of the model parameters on the frequency of resistance alleles after 8 yr. The functional dominance of resistance alleles and the initial frequency of those alleles had a major impact on resistance evolution. The survival of susceptible insects on the transgenic crops and the population dynamics of the insect, driven by winter survival and reproductive rates, were also important. In addition, agricultural practices including the proportion of the acreage planted to corn, and the larval threshold for spraying cotton fields affected the R-allele frequency. Many of these important parameters are inherently variable or cannot be measured with accuracy, so model output cannot be interpreted as being a forecast. However, this analysis is useful in focusing empirical research on those aspects of the insects' life system that have the largest effects on resistance development, and indicates ways in which to improve products and agricultural practices to increase the expected time to resistance. The model can thus be used as a scientific basis for devising a robust resistance management strategy for Bt crops.     

A spatially explicit model simulating western corn rootworm (Coleoptera: Chrysomelidae) adaptation to insect-resistant maize

A stochastic spatially explicit computer model is described that simulates the adaptation by western corn rootworm, Diabrotica virgifera virgifera LeConte, to rootworm-resistance traits in maize. The model reflects the ecology of the rootworm in much of the corn belt of the United States. It includes functions for crop development, egg and larval mortality, adult emergence, mating, egg laying, mortality and dispersal, and alternative methods of rootworm control, to simulate the population dynamics of the rootworm. Adaptation to the resistance trait is assumed to be controlled by a monogenic diallelic locus, whereby the allele for adaptation varies from incompletely recessive to incompletely dominant, depending on the efficacy of the resistance trait. The model was used to compare the rate at which the adaptation allele spread through the population under different nonresistant maize refuge deployment scenarios, and under different levels of crop resistance. For a given refuge size, the model indicated that placing the nonresistant refuge in a block within a rootworm-resistant field would be likely to delay rootworm adaptation rather longer than planting the refuge in separate fields in varying locations. If a portion of the refuge were to be planted in the same fields or in-field blocks each year, rootworm adaptation would be delayed substantially. Rootworm adaptation rates are also predicted to be greatly affected by the level of crop resistance, because of the expectation of dependence of functional dominance on dose. If the dose of the insecticidal protein in the maize is sufficiently high to kill >90% of heterozygotes and approximately 100% of susceptible homozygotes, the trait is predicted to be much more durable than if the dose is lower. A partial sensitivity analysis showed that parameters relating to adult dispersal affected the rate of pest adaptation. Partial validation of the model was achieved by comparing output of the model with field data on population dynamics, and with field data documenting rootworm adaptation to cyclodienes and organophosphates.     

Prediction of tyrosine sulfation sites in animal viruses

Post-translational modification of proteins by tyrosine sulfation enhances the affinity of extracellular ligand-receptor interactions important in the immune response and other biological processes in animals. For example, sulfated tyrosines in polyomavirus and varicella-zoster virus may help modulate host cell recognition and facilitate viral attachment and entry. Using a Position-Specific-Scoring-Matrix with an accuracy of 96.43%, we analyzed the possibility of tyrosine sulfation in all 1517 animal viruses available in the Swiss-Prot database. From a total of 97,729 tyrosines, we predicted 5091 sulfated tyrosine sites from 1024 viruses. Our site predictions in hemagglutinin of influenza A, VP4 of rotavirus, and US28 of cytomegalovirus strongly suggest an important link between tyrosine sulfation and viral disease mechanisms. In each of these three viral proteins, we observed highly conserved amino acid sequences surrounding predicted sulfated tyrosine sites. Tyrosine sulfation appears to be much more common in animal viruses than is currently recognized.     

Complex interactions of carbon monoxide with reduced cytochrome cbb3 oxidase from Pseudomonas stutzeri

Cytochrome cbb(3) oxidase, from Pseudomonas stutzeri, contains a total of five hemes, two of which, a b-type heme in the active site and a hexacoordinate c-type heme, can bind CO in the reduced state. By comparing the cbb(3) oxidase complex and the isolated CcoP subunit, which contains the ligand binding bishistidine-coordinated c-type heme, we have deconvoluted the contribution made by each center to CO binding. A combination of rapid mixing and flash photolysis experiments, coupled with computer simulations, reveals the kinetics of the reaction of c-type heme with CO to be complex as a result of the need to displace an endogenous axial ligand, a property shared with nonsymbiotic plant hemoglobins and some heme-based gas sensing domains. The recombination of CO with heme b(3), unlike all other heme-copper oxidases, including mitochondrial cytochrome c oxidase, is independent of ligand concentration. This observation suggests a very differently organized dinuclear center in which CO exchange between Cu(B) and heme b(3) is significantly enhanced, perhaps reflecting an important determinant of substrate affinity.