Hypothermia-induced cardioprotection using extended ischemia and early reperfusion cooling

Optimal timing of therapeutic hypothermia for cardiac ischemia is unknown. Our prior work suggests that ischemia with rapid reperfusion (I/R) in cardiomyocytes can be more damaging than prolonged ischemia alone. Also, these cardiomyocytes demonstrate protein kinase C (PKC) activation and nitric oxide (NO) signaling that confer protection against I/R injury. Thus we hypothesized that hypothermia will protect most using extended ischemia and early reperfusion cooling and is mediated via PKC and NO synthase (NOS). Chick cardiomyocytes were exposed to an established model of 1-h ischemia/3-h reperfusion, and the same field of initially contracting cells was monitored for viability and NO generation. Normothermic I/R resulted in 49.7 +/- 3.4% cell death. Hypothermia induction to 25 degrees C was most protective (14.3 +/- 0.6% death, P < 0.001 vs. I/R control) when instituted during extended ischemia and early reperfusion, compared with induction after reperfusion (22.4 +/- 2.9% death). Protection was completely lost if onset of cooling was delayed by 15 min of reperfusion (45.0 +/- 8.2% death). Extended ischemia/early reperfusion cooling was associated with increased and sustained NO generation at reperfusion and decreased caspase-3 activation. The NOS inhibitor N(omega)-nitro-L-arginine methyl ester (200 microM) reversed these changes and abrogated hypothermia protection. In addition, the PKCepsilon inhibitor myr-PKCepsilon v1-2 (5 microM) also reversed NO production and hypothermia protection. In conclusion, therapeutic hypothermia initiated during extended ischemia/early reperfusion optimally protects cardiomyocytes from I/R injury. Such protection appears to be mediated by increased NO generation via activation of protein kinase Cepsilon; nitric oxide synthase.     

Epsin: inducing membrane curvature

Epsin was originally discovered by virtue of its binding to another accessory protein, Eps15. Members of the epsin family play an important role as accessory proteins in clathrin-mediated endocytosis. Epsin isoforms have been described that differ in intracellular site of action and/or in tissue distribution, although all epsins essentially contribute to membrane deformation. Besides inducing membrane curvature, epsin also plays a key function as adaptor protein, coupling various components of the clathrin-assisted uptake and fulfils an important role in selecting and recognizing cargo. Furthermore, epsin possesses the ability to block vesicle formation during mitosis. To perform all these functions, epsin, apart from interacting with PtdIns(4,5)P2 via its ENTH domain, also engages in several protein interactions with different components of the clathrin-mediated endocytic system. Recently, RNA interference has successfully been exploited to generate a cell line constitutively silencing epsin expression, which can be used to study internalization of multiple ligands.     

Is the Scotia Sea a centre of Antarctic marine diversification? Some evidence of cryptic speciation in the circum-Antarctic bivalve <Emphasis Type="Italic">Lissarca notorcadensis</Emphasis> (Arcoidea: Philobryidae)

The bivalve Lissarca notorcadensis is one of the most abundant species in Antarctic waters and has colonised the entire Antarctic shelf and Scotia Sea Islands. Its brooding reproduction, low dispersal capabilities and epizoic lifestyle predict limited gene flow between geographically isolated populations. Relationships between specimens from seven regions in the Southern Ocean and outgroups were assessed with nuclear 28S rDNA and mitochondrial cytochrome oxidase subunit I (COI) genes. The 28S dataset indicate that while Lissarca appears to be a monophyletic genus, there is polyphyly between the Limopsidae and Philobryidae. Thirteen CO1 haplotypes were found, mostly unique to the sample regions, and two distinct lineages were distinguished. Specimens from the Weddell and Ross Sea form one lineage while individuals from the banks and islands of the Scotia Sea form the other. Within each lineage, further vicariance was observed forming six regionally isolated groups. Our results provide initial evidence for reproductively isolated populations of L. notorcadensis. The islands of the Scotia Sea appear to act as centres of speciation in the Southern Ocean.     

Nitric oxide during ischemia attenuates oxidant stress and cell death during ischemia and reperfusion in cardiomyocytes

Nitric oxide (NO) has been implicated as a cardioprotective agent during ischemia/reperfusion (I/R), but the mechanism of protection is unknown. Oxidant stress contributes to cell death in I/R, so we tested whether NO protects by attenuating oxidant stress. Cardiomyocytes and murine embryonic fibroblasts were administered NO (10-1200 nM) during simulated ischemia, and cell death was assessed during reperfusion without NO. In each case, NO abrogated cell death during reperfusion. Cells overexpressing endothelial NO synthase (NOS) exhibited a similar protection, which was abolished by the NOS inhibitor N(omega)-nitro-l-arginine methyl ester. Protection was not mediated by guanylate cyclase or the mitochondrial K(ATP) channel, as inhibitors of these systems failed to abolish protection. NO did not prevent decreases in mitochondrial potential, but cells protected with NO demonstrated recovery of potential at reperfusion. Measurements using C11-BODIPY reveal that NO attenuates lipid peroxidation during ischemia and reperfusion. Measurements of oxidant stress using the ratiometric redox sensor HSP-FRET demonstrate that NO attenuates protein oxidation during ischemia. These findings reveal that physiological levels of NO during ischemia can attenuate oxidant stress both during ischemia and during reperfusion. This response is associated with a remarkable attenuation of cell death, suggesting that ischemic cell death may be a regulated event.     

The structure of the Caenorhabditis elegans manganese superoxide dismutase MnSOD‐3‐azide complex

C. elegans MnSOD‐3 has been implicated in the longevity pathway and its mechanism of catalysis is relevant to the aging process and carcinogenesis. The structures of MnSOD‐3 provide unique crystallographic evidence of a dynamic region of the tetrameric interface (residues 41–54). We have determined the structure of the MnSOD‐3‐azide complex to 1.77‐Å resolution. Analysis of this complex shows that the substrate analog, azide, binds end‐on to the manganese center as a sixth ligand and that it ligates directly to a third and new solvent molecule also positioned within interacting distance to the His30 and Tyr34 residues of the substrate access funnel. This is the first structure of a eukaryotic MnSOD‐azide complex that demonstrates the extended, uninterrupted hydrogen‐bonded network that forms a proton relay incorporating three outer sphere solvent molecules, the substrate analog, the gateway residues, Gln142, and the solvent ligand. This configuration supports the formation and release of the hydrogen peroxide product in agreement with the 5‐6‐5 catalytic mechanism for MnSOD. The high product dissociation constant k₄ of MnSOD‐3 reflects low product inhibition making this enzyme efficient even at high levels of superoxide.     

Dual target strategy: combining distinct non‐dopaminergic treatments reduces neuronal cell loss and synergistically modulates l‐DOPA‐induced rotational behavior in a rodent model of Parkinson's disease

The glutamate metabotropic receptor 5 (mGluR5) and the adenosine A_2A receptor (A_2AR) represent major non‐dopaminergic therapeutic targets in Parkinson's disease (PD) to improve motor symptoms and slow down/revert disease progression. The 6‐hydroxydopamine rat model of PD was used to determine/compare the neuroprotective and behavioral impacts of single and combined administration of one mGluR5 antagonist, 2‐methyl‐6‐(phenylethynyl)pyridine (MPEP), and two A_2AR antagonists, (E)‐phosphoric acid mono‐[3‐[8‐[2‐(3‐methoxyphenyl)vinyl]‐7‐methyl‐2,6‐dioxo‐1‐prop‐2‐ynyl‐1,2,6,7‐tetrahydropurin‐3‐yl]propyl] (MSX‐3) and 8‐ethoxy‐9‐ethyladenine (ANR 94). Chronic treatment with MPEP or MSX‐3 alone, but not with ANR 94, reduced the toxin‐induced loss of dopaminergic neurons in the substantia nigra pars compacta. Combining MSX‐3 and MPEP further improved the neuroprotective effect of either antagonists. At the behavioral level, ANR 94 and MSX‐3 given alone significantly potentiated l ‐DOPA‐induced turning behavior. Combination of either A_2AR antagonists with MPEP synergistically increased L‐DOPA‐induced turning. This effect was dose‐dependent and required subthreshold drug concentration, which per se had no motor stimulating effect. Our findings suggest that co‐treatment with A_2AR and mGluR5 antagonists provides better therapeutic benefits than those produced by either drug alone. Our study sheds some light on the efficacy and advantages of combined non‐dopaminergic PD treatment using low drug concentration and establishes the basis for in‐depth studies to identify optimal doses at which these drugs reach highest efficacy.

     

Submergence of black ash logs to control emerald ash borer and preserve wood for American Indian basketmaking

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