Nitric oxide-dependent generation of reactive species in sickle cell disease. Actin tyrosine induces defective cytoskeletal polymerization

The intermittent vascular occlusion occurring in sickle cell disease (SCD) leads to ischemia-reperfusion injury and activation of inflammatory processes including enhanced production of reactive oxygen species and increased expression of inducible nitric-oxide synthase (NOS2). Appreciating that impaired nitric oxide-dependent vascular function and the concomitant formation of oxidizing and nitrating species occur in concert with increased rates of tissue reactive oxygen species production, liver and kidney NOS2 expression, tissue 3-nitrotyrosine (NO(2)Tyr) formation and apoptosis were evaluated in human SCD tissues and a murine model of SCD. Liver and kidney NOS2 expression and NO(2)Tyr immunoreactivity were significantly increased in SCD mice and humans, but not in nondiseased tissues. TdT-mediated nick end-label (TUNEL) staining showed apoptotic cells in regions expressing elevated levels of NOS2 and NO(2)Tyr in all SCD tissues. Gas chromatography mass spectrometry analysis revealed increased plasma protein NO(2)Tyr content and increased levels of hepatic and renal protein NO(2)Tyr derivatives in SCD (21.4 +/- 2.6 and 37.5 +/- 7.8 ng/mg) versus wild type mice (8.2 +/- 2.2 and 10 +/- 1.2 ng/mg), respectively. Western blot analysis and immunoprecipitation of SCD mouse liver and kidney proteins revealed one principal NO(2)Tyr-containing protein of 42 kDa, compared with controls. Enzymatic in-gel digestion and MALDI-TOF mass spectrometry identified this nitrated protein as actin. Electrospray ionization and fragment analysis by tandem mass spectrometry revealed that 3 of 15 actin tyrosine residues are nitrated (Tyr(91), Tyr(198), and Tyr(240)) at positions that significantly modify actin assembly. Confocal microscopy of SCD human and mouse tissues revealed that nitration led to morphologically distinct disorganization of filamentous actin. In aggregate, we have observed that the hemoglobin point mutation of sickle cell disease that mediates hemoglobin polymerization defects is translated, via inflammatory oxidant reactions, into defective cytoskeletal polymerization.     

Guidelines for consistent reporting of exchanges/to nature within life cycle inventories (LCI)

Data availability and data quality are still critical factors for successful LCA work. The SETAC-Europe LCA Working Group ‘Data Availability and Data Quality’ has therefore focused on ongoing developments toward a common data exchange format, public databases and accepted quality measures to find science-based solutions than can be widely accepted. A necessary prerequisite for the free flow and exchange of life cycle inventory (LCI) data and the comparability of LCIs is the consistent definition, nomenclature, and use of inventory parameters. This is the main subject of the subgroup ‘Recommended List of Exchanges’ that presents its results and findings here:

Non-warning odors trigger innate color aversions--as long as they are novel

Warning signals made by unpalatable insects to potential predatorscommonly target more than one sense: such signals are "multimodal." Pyrazines are odors produced by warningly colored insects whenattacked, and have been shown to interact with food coloration,biasing avian predators against novel and typically aposematicfood. However, at present it is not known whether this is anadaptation by prey to exploit a general feature of avian psychology,or an evolutionary response by birds to enhance their avoidanceof unpalatable prey. Here we investigate the effect of otherodors on the innate responses of naive domestic chicks (Gallusgallus domesticus) to food that is of novel color, or of acolor that is associated with warning coloration, yellow. Inthe first experiment, we demonstrate that natural and artificialodors that have no association with aposematism in the wildcan produce biases against both novel colored foods and yellowcolored foods. In a second experiment, we also show that odor novelty is vital for eliciting such effects. These results supportthe idea that warning odors have evolved in response to preexistingpsychological biases against novel odors in predators, ratherthan predators evolving specific responses against odors associatedwith unpalatable prey.     

Phosphatidylinositol 3-kinase-dependent,MEK- independent proliferation in response to CaR activation

Although ovarian surface epithelial(OSE) cells are responsible for the majority of ovarian tumors, we knowrelatively little about the pathway(s) that is responsible forregulating their proliferation. We found that phosphatidylinositol3-kinase (PI3K) is activated in OSE cells in response to elevatedextracellular calcium, and the PI3K inhibitors wortmannin and LY-294002inhibited extracellular signal-regulated kinase (ERK) activation by~75%, similar to effects of the mitogen-activated protein kinase/ERK kinase inhibitor PD-98059. However, in assays of proliferation, we found that PD-98059 inhibited proliferation by ~50%, whereas wortmannin inhibited >90% of the proliferative response to elevated calcium. Expression of a dominant negative PI3K totally inhibited ERKactivation in response to calcium. These results demonstrate that ERKactivation cannot account for the full proliferative effect of elevatedcalcium in OSE cells and suggest the presence of an ERK-independent,PI3K-dependent component in the proliferative response.

     

Cell cycle-dependent expression of volume-activated chloride currents in nasopharyngeal carcinoma cells

Patch-clamping and cell imageanalysis techniques were used to study the expression of thevolume-activated Cl current,I_Cl(vol), and regulatory volume decrease (RVD)capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated aCl current with a linear conductance, negligibletime-dependent inactivation, and a reversal potential close to theCl equilibrium potential. The sequence of anionpermeability was I > Br > Cl > gluconate. The Cl channelblockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),and ATP inhibited I_Cl(vol). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by adouble chemical-block (thymidine and hydroxyurea) technique. Theexpression of I_Cl(vol) was cell cycle dependent,being high in G₁ phase, downregulated in S phase, butincreasing again in M phase. Hypotonic solution activated RVD, whichwas cell cycle dependent and inhibited by the Cl channelblockers NPPB, tamoxifen, and ATP. The expression of I_Cl(vol) was closely correlated with the RVDcapacity in the cell cycle, suggesting a functional relationship.Inhibition of I_Cl(vol) by NPPB (100 µM)arrested cells in G₀/G₁. The data also suggest that expression of I_Cl(vol) and RVD capacity areactively modulated during the cell cycle. The volume-activatedCl current associated with RVD may therefore play animportant role during the cell cycle progress.

     

Fetal development of mouse oocytes and zygotes cryopreserved in a nonconventional freezing medium

This study (1) analyzed fetal development of mouse embryos after oocyte cryopreservation in CJ2, a choline-based medium, (2) examined the effect of culture duration in vitro on subsequent fetal development, and (3) compared survival and fetal development of zygotes frozen in embryo transfer freeze medium (ETFM; sodium-based medium) or CJ2. Unfertilized oocytes and zygotes were cryopreserved using a slow-cooling protocol. After thawing, oocytes were inseminated after drilling a hole in their zona, cultured in vitro either to the two-cell or blastocyst stage, and transferred to the oviducts or uterine horns of recipient mice. In parallel experiments, frozen-thawed zygotes were similarly cultured and transferred. Implantation rates for transferred embryos were high (range 66-88%), regardless of whether they had been frozen as oocytes or zygotes and whether they had been transferred to the oviduct or uterus. However, fetal development was significantly higher when two-cell embryos were transferred. With blastocyst transfer, control embryos implanted and produced a greater proportion of fetuses than did oocytes frozen in CJ2, whereas transfer at the two-cell stage resulted in similar proportions of implantation sites and fetuses. Blastocyst transfer of zygotes cryopreserved in ETFM or CJ2 produced similar fetal development rates (23.6% vs 20.0%), but when frozen-thawed zygotes were transferred at the two-cell stage the fetal development rates were higher in the ETFM group (53.3%) than in the CJ2 group (32.0%). A high proportion (46.7%) of oocytes frozen in CJ2 in a nonprogrammable freezer and plunged at -20 degrees C developed into live offspring. This study shows that in the mouse (1) oocytes frozen in CJ2 can develop into viable fetuses, (2) prolonging culture in vitro has a detrimental effect on embryo transfer outcome, and (3) CJ2 offers no advantage for zygote cryopreservation.     

Eye size in birds and the timing of song at dawn

Why do different species of birds start their dawn choruses at different times? We test the hypothesis that the times at which different species start singing at dawn are related to their visual capability at low light intensities. Birds with large eyes can achieve greater pupil diameters and hence, all other things being equal, greater visual sensitivity and resolution than birds with small eyes. We estimated the maximum pupil diameter of passerine birds by measuring the diameter of the exposed eye surface, and measured the times of the first songs at dawn of songbirds present in different bird communities, and the light intensities at these times. Using phylogenetic comparative analyses, we found that songbirds with large eyes started to sing at lower light intensities (and therefore earlier) than species with smaller eyes. These relationships were stronger when differences in body size were controlled for statistically, and were consistent between two phylogenies and when species were treated as independent data points. Our results therefore provide robust support for the hypothesis that visual capability at low light levels influences the times at which birds start to sing at dawn.     

Naked mole-rats: unique opportunities and husbandry challenges

The naked mole-rat is a relative newcomer to biomedical and behavioral studies. The authors review this unusual rodent's uses in research, husbandry, reproduction, and common diseases.     

Critical residues for structure and catalysis in short-chain dehydrogenases/reductases

Short-chain dehydrogenases/reductases form a large, evolutionarily old family of NAD(P)(H)-dependent enzymes with over 60 genes found in the human genome. Despite low levels of sequence identity (often 10-30%), the three-dimensional structures display a highly similar alpha/beta folding pattern. We have analyzed the role of several conserved residues regarding folding, stability, steady-state kinetics, and coenzyme binding using bacterial 3beta/17beta-hydroxysteroid dehydrogenase and selected mutants. Structure determination of the wild-type enzyme at 1.2-A resolution by x-ray crystallography and docking analysis was used to interpret the biochemical data. Enzyme kinetic data from mutagenetic replacements emphasize the critical role of residues Thr-12, Asp-60, Asn-86, Asn-87, and Ala-88 in coenzyme binding and catalysis. The data also demonstrate essential interactions of Asn-111 with active site residues. A general role of its side chain interactions for maintenance of the active site configuration to build up a proton relay system is proposed. This extends the previously recognized catalytic triad of Ser-Tyr-Lys residues to form a tetrad of Asn-Ser-Tyr-Lys in the majority of characterized short-chain dehydrogenases/reductase enzymes.