Replacements of lysine 32 in yeast cytochrome c. Effects on the binding and reactivity with physiological partners

Lysine 32 has been previously implicated by chemical modification and modeling studies as a key component of the domain which controls recognition and binding of cytochrome c to its physiological partners, e.g. cytochrome b2, cytochrome c peroxidase, and cytochrome oxidase. In order to quantitate the importance of this residue, we have investigated the role of Lys-32 in the reactivity of cytochrome c in redox reactions in vitro and in vivo with protein partners by using a series of altered forms of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae in which Lys-32 is replaced by Leu-32, Gln-32, Trp-32, and Tyr-32. Leu-32 and Gln-32 represent substitutions which change charge without seriously affecting the steric bulk of the side chain or the stability of the protein. For the Leu-32- and Gln-32-altered proteins, steady state kinetic studies with cytochrome c peroxidase, cytochrome b2, and cytochrome oxidase showed that neither of the steady state kinetic parameters, Km nor Vmax, were substantially modified by mutation. Studies of single turnover kinetics with a small molecule (ascorbate) or within bound complexes with either cytochrome b5 or cytochrome c peroxidase demonstrated that redox kinetics are only slightly affected by these substitutions. NMR experiments demonstrated that the Gln-32-altered protein can still bind strongly to a physiological partner, cytochrome c peroxidase. Growth in lactate medium demonstrated that the activity in vivo compared with the normal value was reduced to only 85% with the Gln-32- and Leu-32-altered proteins and to 65% with the Trp-32- and Tyr-32-altered proteins. These findings suggest that the evolutionary invariance of Lys-32 reflects only small quantitative changes in the binding and reactivity of cytochrome c.     

Identifying genetic networks underlying myometrial transition to labor

Background  

Early transition to labor remains a major cause of infant mortality, yet the causes are largely unknown. Although several marker genes have been identified, little is known about the underlying global gene expression patterns and pathways that orchestrate these striking changes.     

Virus genome dynamics under different propagation pressures: reconstruction of whole genome haplotypes of west nile viruses from NGS data

Background

Extensive focus is placed on the comparative analyses of consensus genotypes in the study of West Nile virus (WNV) emergence. Few studies account for genetic change in the underlying WNV quasispecies population variants. These variants are not discernable in the consensus genome at the time of emergence, and the maintenance of mutation-selection equilibria of population variants is greatly underestimated. The emergence of lineage 1 WNV strains has been studied extensively, but recent epidemics caused by lineage 2 WNV strains in Hungary, Austria, Greece and Italy emphasizes the increasing importance of this lineage to public health. In this study we explored the quasispecies dynamics of minority variants that contribute to cell-tropism and host determination, i.e. the ability to infect different cell types or cells from different species from Next Generation Sequencing (NGS) data of a historic lineage 2 WNV strain.

Results

Minority variants contributing to host cell membrane association persist in the viral population without contributing to the genetic change in the consensus genome. Minority variants are shown to maintain a stable mutation-selection equilibrium under positive selection, particularly in the capsid gene region.

Conclusions

This study is the first to infer positive selection and the persistence of WNV haplotype variants that contribute to viral fitness without accompanying genetic change in the consensus genotype, documented solely from NGS sequence data. The approach used in this study streamlines the experimental design seeking viral minority variants accurately from NGS data whilst minimizing the influence of associated sequence error.     

Progressive handgrip exercise: evidence of nitric oxide-dependent vasodilation and blood flow regulation in humans

In the peripheral circulation, nitric oxide (NO) is released in response to shear stress across vascular endothelial cells. We sought to assess the degree to which NO contributes to exercise-induced vasodilation in the brachial artery (BA) and to determine the potential of this approach to noninvasively evaluate NO bioavailability. In eight young (25 ± 1 yr) healthy volunteers, we used ultrasound Doppler to examine BA vasodilation in response to handgrip exercise (4, 8, 12, 16, 20, and 24 kg) with and without endothelial NO synthase blockade [intra-arterial N(G)-monomethyl-L-arginine (L-NMMA), 0.48 mg · dl(-1) · min(-1)]. Higher exercise intensities evoked significant BA vasodilation (4-12%) that was positively correlated with the hyperemic stimulus (r = 0.98 ± 0.003, slope = 0.005 ± 0.001). During NO blockade, BA vasodilation at the highest exercise intensity was reduced by ～70% despite similar exercise-induced increases in shear rate (control, +224 ± 30 s(-1); L-NMMA, +259 ± 46 s(-1)). The relationship and slope of BA vasodilation with increasing shear rate was likewise reduced (r = 0.48 ± 0.1, slope = 0.0007 ± 0.0005). We conclude that endothelial NO synthase inhibition with L-NMMA abolishes the relationship between shear stress and BA vasodilation during handgrip exercise, providing clear evidence of NO-dependent vasodilation in this experimental model. These results support this paradigm as a novel and valid approach for a noninvasive assessment of NO-dependent vasodilation in humans.     

The Hawaiian Algal Database: a laboratory LIMS and online resource for biodiversity data

Background  

Organization and presentation of biodiversity data is greatly facilitated by databases that are specially designed to allow easy data entry and organized data display. Such databases also have the capacity to serve as Laboratory Information Management Systems (LIMS). The Hawaiian Algal Database was designed to showcase specimens collected from the Hawaiian Archipelago, enabling users around the world to compare their specimens with our photographs and DNA sequence data, and to provide lab personnel with an organizational tool for storing various biodiversity data types.