Thiol oxidation causes pulmonary vasodilation by activating K+ channels and inhibiting store-operated Ca2+ channels

Cellular redox change regulates pulmonary vascular tone by affecting function of membrane and cytoplasmic proteins, enzymes, and second messengers. This study was designed to test the hypothesis that functional modulation of ion channels by thiol oxidation contributes to regulation of excitation-contraction coupling in isolated pulmonary artery (PA) rings. Acute treatment with the thiol oxidant diamide produced a dose-dependent relaxation in PA rings; the IC50 was 335 and 58 microM for 40 mM K+ - and 2 microM phenylephrine-induced PA contraction, respectively. The diamide-mediated pulmonary vasodilation was affected by neither functional removal of endothelium nor 8-bromoguanosine-3'-5'-cyclic monophosphate (50 microM) and HA-1004 (30 microM). A rise in extracellular K+ concentration (from 20 to 80 mM) attenuated the thiol oxidant-induced PA relaxation. Passive store depletion by cyclopiazonic acid (50 microM) and active store depletion by phenylephrine (in the absence of external Ca2+ both induced PA contraction due to capacitative Ca2+ entry. Thiol oxidation by diamide significantly attenuated capacitative Ca2+ entry-induced PA contraction due to active and passive store depletion. The PA rings isolated from left and right PA branches appeared to respond differently to store depletion. Although the active tension induced by passive store depletion was comparable, the active tension induced by active store depletion was 3.5-fold greater in right branches than in left branches. These data indicate that thiol oxidation causes pulmonary vasodilation by activating K+ channels and inhibiting store-operated Ca2+ channels, which subsequently attenuate Ca2+ influx and decrease cytosolic free Ca2+ concentration in pulmonary artery smooth muscle cells. The mechanisms involved in thiol oxidation-mediated pulmonary vasodilation or activation of K+ channels and inhibition of store-operated Ca2+ channels appear to be independent of functional endothelium and of the cGMP-dependent protein kinase pathway.     

A correlation between long-term in vitro dynamic calcification and abnormal flow patterns past bioprosthetic heart valves

In this paper, a long-term in vitro dynamic calcification of three porcine aortic heart valves was investigated using a combined approach that involved accelerated wear testing of the valves in the rapid calcification solution, hydrodynamic assessment of the progressive change of effective orifice area (EOA) along with the transaortic pressure gradient, and quantitative visualization of the flow. The motivation for this study was developing a standardized, economical, and feasible in vitro testing methodology for bioprosthetic heart valve calcification, which would address both the hydrodynamic performance of the valves as well as the subsequent changes in the flow field. The results revealed the failure of the test valves at 40 million cycles mark, associated with the critical decrease in the EOA, followed by the increase in the maximum value of viscous shear stress of up to 52%, compared to the values measured at the beginning of the study. The decrease in the EOA was subsequently followed by the rapid increase in the maximum streamwise velocity of the central orifice jet up to the value of about 2.8 m/s, compared to the initial value of 2 m/s, and to the value of 2.2 m/s in the case of a control valve along with progressive narrowing of the velocity profile for two test valves. The significance of the current work is in demonstrating a correlation between calcification of the aortic valve and spatial as well as the temporal development of abnormal flow features.     

Spermatozoa production by triploid males in the New Zealand freshwater snail Potamopyrgus antipodarum

Asexual lineages derived from dioecious taxa are typically assumed to be all female. Even so, asexual females from a variety of animal taxa occasionally produce males. The existence of these males sets the stage for potential gene flow across asexual lineages as well as between sexual and asexual lineages. A recent study showed that asexual triploid female Potamopyrgus antipodarum, a New Zealand freshwater snail often used as a model to study sexual reproduction, occasionally produce triploid male offspring. Here, we show that these triploid male P. antipodarum (1) have testes that produce morphologically normal sperm, (2) make larger sperm cells that contain more nuclear DNA than the sperm produced by diploid sexual males, and (3) produce sperm that range in DNA content from haploid to diploid, and are often aneuploid. Analysis of meiotic chromosomes of triploid males showed that aberrant pairing during prophase I probably accounts for the high variation in DNA content among sperm. These results indicate that triploid male P. antipodarum produce sperm, but the extent to which these sperm are able to fertilize female ova remains unclear. Our results also suggest that the general assumption of sterility in triploid males should be more closely examined in other species in which such males are occasionally produced. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 227–234.     

Oligomeric Yeast Frataxin Drives Assembly of Core Machinery for Mitochondrial Iron-Sulfur Cluster Synthesis

Mitochondrial biosynthesis of iron-sulfur clusters (ISCs) is a vital process involving the delivery of elemental iron and sulfur to a scaffold protein via molecular interactions that are still poorly defined. Analysis of highly conserved components of the yeast ISC assembly machinery shows that the iron-chaperone, Yfh1, and the sulfur-donor complex, Nfs1-Isd11, directly bind to each other. This interaction is mediated by direct Yfh1-Isd11 contacts. Moreover, both Yfh1 and Nfs1-Isd11 can directly bind to the scaffold, Isu1. Binding of Yfh1 to Nfs1-Isd11 or Isu1 requires oligomerization of Yfh1 and can occur in an iron-independent manner. However, more stable contacts are formed when Yfh1 oligomerization is normally coupled with the binding and oxidation of Fe²⁺. Our observations challenge the view that iron delivery for ISC synthesis is mediated by Fe²⁺-loaded monomeric Yfh1. Rather, we find that the iron oxidation-driven oligomerization of Yfh1 promotes the assembly of stable multicomponent complexes in which the iron donor and the sulfur donor simultaneously interact with each other as well as with the scaffold. Moreover, the ability to store ferric iron enables oligomeric Yfh1 to adjust iron release depending on the presence of Isu1 and the availability of elemental sulfur and reducing equivalents. In contrast, the use of anaerobic conditions that prevent Yfh1 oligomerization results in inhibition of ISC assembly on Isu1. These findings suggest that iron-dependent oligomerization is a mechanism by which the iron donor promotes assembly of the core machinery for mitochondrial ISC synthesis.ISC³ biosynthesis is an essential function that eukaryotic cells initiate in mitochondria and probably other cellular compartments using three core components: a sulfur donor, an iron donor, and an ISC assembly scaffold (1, 2). In yeast mitochondria, the cysteine-desulfurase, Nfs1, and the iron-chaperone, Yfh1, are believed to provide sulfur and iron, respectively, for ISC assembly on the Isu1 scaffold (1), whereas the Nfs1-binding protein, Isd11, has been shown to stabilize Nfs1 (3). These components are highly conserved and the human orthologues of Yfh1 (frataxin), Isu1 (ISCU), and Isd11 (ISD11) are implicated in the etiology of severe disorders including Friedreich ataxia and mitochondrial myopathy (4).Previous studies have underscored the complexity of the interactions among eukaryotic ISC assembly components as well as their metal dependence. Supplementation of mitochondrial lysates with Fe²⁺ under aerobic conditions led to co-isolation of Yfh1 and Isu1 along with Nfs1 and Isd11 by pulldown or immunoprecipitation assays (5–7). Furthermore, aerobic preincubation of histidine-tagged Yfh1 monomer with Fe²⁺ enabled Isu1 to be pulled down by Yfh1 in the absence of other proteins (5). These studies have led to the current view that iron delivery for yeast ISC synthesis involves direct contacts between iron-loaded monomeric Yfh1 and Isu1 (5–7). Although Yfh1 oligomerization is normally coupled with iron binding, oxidation, and storage (5, 8), the possibility that Isu1 might also interact with oligomeric Yfh1 has remained largely unexplored.Similar to Yfh1, human frataxin was found to interact with multiple ISC assembly components in human cells; however, in this case immunoprecipitation data suggested that frataxin binds to ISCU indirectly, via nickel-dependent contacts with ISD11 (9). Whether direct interactions occur between Yfh1 and Isd11 has not yet been examined.While previous studies focused primarily on Yfh1-Isu1 and frataxin-ISD11 interactions, it is likely that the coordinate delivery of potentially toxic sulfur and iron to Isu1/ISCU involves multiple close interactions whereby the sulfur donor and the iron donor simultaneously interact with each other and with the ISC scaffold, as proposed for prokaryotic ISC assembly (10). However, it is currently unknown whether monomeric Yfh1/frataxin may form direct contacts with more than one partner, and the structure of the eukaryotic ISC assembly machinery is completely undefined. We show that iron oxidation-dependent oligomerization enables Yfh1 to have simultaneous direct interactions with Nfs1-Isd11 and Isu1. Our data provide insights about the sequence of events and the molecular architecture required for the initial step in mitochondrial ISC assembly.     

Small-angle neutron and X-ray scattering analysis of the supramolecular organization of rhodopsin in photoreceptor membrane

The supramolecular organization of the visual pigment rhodopsin in the photoreceptor membrane remains contentious. Specifically, whether this G protein-coupled receptor functions as a monomer or dimer remains unknown, as does the presence or absence of ordered packing of rhodopsin molecules in the photoreceptor membrane. Completely opposite opinions have been expressed on both issues. Herein, using small-angle neutron and X-ray scattering approaches, we performed a comparative analysis of the structural characteristics of the photoreceptor membrane samples in buffer, both in the outer segment of photoreceptor cells, and in the free photoreceptor disks. The average distance between the centers of two neighboring rhodopsin molecules was found to be ~5.8 nm in both cases. The results indicate an unusually high packing density of rhodopsin molecules in the photoreceptor membrane, but molecules appear to be randomly distributed in the membrane without any regular ordering.     

Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown Clades

Inference of evolutionary relationships between nematodes is severely hampered by their conserved morphology, the high frequency of homoplasy, and the scarcity of phylum-wide molecular data. To study the origin of nematode radiation and to unravel the phylogenetic relationships between distantly related species, 339 nearly full-length small-subunit rDNA sequences were analyzed from a diverse range of nematodes. Bayesian inference revealed a backbone comprising 12 consecutive dichotomies that subdivided the phylum Nematoda into 12 clades. The most basal clade is dominated by the subclass Enoplia, and members of the order Triplonchida occupy positions most close to the common ancestor of the nematodes. Crown Clades 8-12, a group formerly indicated as "Secernentea" that includes Caenorhabditis elegans and virtually all major plant and animal parasites, show significantly higher nucleotide substitution rates than the more basal Clades 1-7. Accelerated substitution rates are associated with parasitic lifestyles (Clades 8 and 12) or short generation times (Clades 9-11). The relatively high substitution rates in the distal clades resulted in numerous autapomorphies that allow in most cases DNA barcode-based species identification. Teratocephalus, a genus comprising terrestrial bacterivores, was shown to be most close to the starting point of Secernentean radiation. Notably, fungal feeding nematodes were exclusively found basal to or as sister taxon next to the 3 groups of plant parasitic nematodes, namely, Trichodoridae, Longidoridae, and Tylenchomorpha. The exclusive common presence of fungivorous and plant parasitic nematodes supports a long-standing hypothesis that states that plant parasitic nematodes arose from fungivorous ancestors.     

Cleavage site selection within a folded substrate by the ATP-dependent lon protease

Mechanistic studies of ATP-dependent proteolysis demonstrate that substrate unfolding is a prerequisite for processive peptide bond hydrolysis. We show that mitochondrial Lon also degrades folded proteins and initiates substrate cleavage non-processively. Two mitochondrial substrates with known or homology-derived three-dimensional structures were used: the mitochondrial processing peptidase alpha-subunit (MPPalpha) and the steroidogenic acute regulatory protein (StAR). Peptides generated during a time course of Lon-mediated proteolysis were identified and mapped within the primary, secondary, and tertiary structure of the substrate. Initiating cleavages occurred preferentially between hydrophobic amino acids located within highly charged environments at the surface of the folded protein. Subsequent cleavages proceeded sequentially along the primary polypeptide sequence. We propose that Lon recognizes specific surface determinants or folds, initiates proteolysis at solvent-accessible sites, and generates unfolded polypeptides that are then processively degraded.     

Binding of Imidazole to the Heme of Cytochrome c1 and Inhibition of the bc1 Complex from Rhodobacter sphaeroides: II. KINETICS AND MECHANISM OF BINDING*

The kinetics of imidazole (Im) and N-methylimidazole (MeIm) binding to oxidized cytochrome (cyt) c₁ of detergent-solubilized bc₁ complex from Rhodobacter sphaeroides are described. The rate of formation of the cyt c₁-Im complex exhibited three separated regions of dependence on the concentration of imidazole: (i) below 8 mm Im, the rate increased with concentration in a parabolic manner; (ii) above 20 mm, the rate leveled off, indicating a rate-limiting conformational step with lifetime ∼1 s; and (iii) at Im concentrations above 100 mm, the rate substantially increased again, also parabolically. In contrast, binding of MeIm followed a simple hyperbolic concentration dependence. The temperature dependences of the binding and release kinetics of Im and MeIm were also measured and revealed very large activation parameters for all reactions. The complex concentration dependence of the Im binding rate is not consistent with the popular model for soluble c-type cytochromes in which exogenous ligand binding is preceded by spontaneous opening of the heme cleft, which becomes rate-limiting at high ligand concentrations. Instead, binding of ligand to the heme is explained by a model in which an initial and superficial binding facilitates access to the heme by disruption of hydrogen-bonded structures in the heme domain. For imidazole, two separate pathways of heme access are indicated by the distinct kinetics at low and high concentration. The structural basis for ligand entry to the heme cleft is discussed.     

Control of Neuronal Synchrony by Nonlinear Delayed Feedback

We present nonlinear delayed feedback stimulation as a technique for effective desynchronization. This method is intriguingly robust with respect to system and stimulation parameter variations. We demonstrate its broad applicability by applying it to different generic oscillator networks as well as to a population of bursting neurons. Nonlinear delayed feedback specifically counteracts abnormal interactions and, thus, restores the natural frequencies of the individual oscillatory units. Nevertheless, nonlinear delayed feedback enables to strongly detune the macroscopic frequency of the collective oscillation. We propose nonlinear delayed feedback stimulation for the therapy of neurological diseases characterized by abnormal synchrony.