S-nitrosoglutathione-induced toxicity in Drosophila melanogaster: Delayed pupation and induced mild oxidative/nitrosative stress in eclosed flies

The toxicity of the nitric oxide donor S-nitrosoglutathione (GSNO) was tested on the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with GSNO at concentrations of 1.0, 1.5 or 4.0 mM. Food supplementation with GSNO caused a developmental delay in the flies. Biochemical analyses of oxidative stress markers and activities of antioxidant and associated enzymes were carried out on 2-day-old flies that emerged from control larvae and larvae fed on food supplemented with GSNO. Larval exposure to GSNO resulted in lower activities of aconitase in both sexes and also lower activities of catalase and isocitrate dehydrogenase in adult males relative to the control cohort. Larval treatment with GSNO resulted in higher carbonyl protein content and higher activities of glucose-6-phosphate dehydrogenase in males and higher activities of superoxide dismutase and glutathione-S-transferase in both sexes. Among the parameters tested, aconitase activity and developmental end points may be useful early indicators of toxicity caused by GSNO.     

Asymmetric structure and domain binding interfaces of human tyrosyl‐tRNA synthetase studied by molecular dynamics simulations

Human tyrosyl‐tRNA synthetase (HsTyrRS) is composed of two structural modules: N‐terminal catalytic core and an EMAP II‐like C‐terminal domain. The structures of these modules are known, but no crystal structure of the full‐length HsTyrRS is currently available. An all‐atom model of the full‐length HsTyrRS was developed in this work. The structure, dynamics, and domain binding interfaces of HsTyrRS were investigated by extensive molecular dynamics (MD) simulations. Our data suggest that HsTyrRS in solution consists of a number of compact asymmetric conformations, which differ significantly by their rigidity, internal mobility, orientation of C‐terminal modules, and the strength of interdomain binding. Interfaces of domain binding obtained in MD simulations are in perfect agreement with our previous coarse‐grained hierarchical rotations technique simulations. Formation of the hydrogen bonds between R93 residue of the ELR cytokine motif and the residues A340 and E479 in the C‐module was observed. This observation supports the idea that the lack of cytokine activity in the full‐length HsTyrRS is explained by interactions between N‐modules and C‐modules, which block the ELR motif. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

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.     

Identification of CD46 binding sites within the adenovirus serotype 35 fiber knob

Species B human adenoviruses (Ads) are often associated with fatal illnesses in immunocompromised individuals. Recently, species B Ads, most of which use the ubiquitously expressed complement regulatory protein CD46 as a primary attachment receptor, have gained interest for use as gene therapy vectors. In this study, we focused on species B Ad serotype 35 (Ad35), whose trimeric fiber knob domain binds to three CD46 molecules with a K_D (equilibrium dissociation constant) of 15.5 nM. To study the Ad35 knob-CD46 interaction, we generated an expression library of Ad35 knobs with random mutations and screened it for CD46 binding. We identified four critical residues (Phe242, Arg279, Ser282, and Glu302) which, when mutated, ablated Ad35 knob binding to CD46 without affecting knob trimerization. The functional importance of the identified residues was validated in surface plasmon resonance and competition binding studies. To model the Ad35 knob-CD46 interaction, we resolved the Ad35 knob structure at 2-Å resolution by X-ray crystallography and overlaid it onto the existing structure for Ad11-CD46 interaction. According to our model, all identified Ad35 residues are in regions that interact with CD46, whereby one CD46 molecule binds between two knob monomers. This mode of interaction might have potential consequences for CD46 signaling and intracellular trafficking of Ad35. Our findings are also fundamental for better characterization of species B Ads and design of antiviral drugs, as well as for application of species B Ads as in vivo and in vitro gene transfer vectors.     

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.