Antisense reduction of thylakoidal ascorbate peroxidase in <Emphasis Type="Italic">Arabidopsis</Emphasis> enhances Paraquat-induced photooxidative stress and Nitric Oxide-induced cell death

The production and characterization of Arabidopsis plants containing a transgene in which the Arabidopsis tAPX is inserted in antisense orientation, is described. tAPX activity in these transgenic tAPX plants is around 50% of control level. The tAPX antisense plants are phenotypically indistinguishable from control plants under normal growth conditions; they show, however, enhanced sensitivity to the O₂^–-generating herbicide, Paraquat. Interestingly, the tAPX antisense plants show enhanced symptoms of damage when cell death is triggered through treatment with the nitric oxide-donor, SNP. These results are in accordance with the ones recently obtained with transgenic plants overexpressing tAPX; altogether, they suggest that tAPX, besides the known ROS scavenging role, is also involved in the fine changes of H₂O₂ concentration during signaling events.     

Beringian paleoecology inferred from permafrost-preserved fungal DNA

The diversity of fungi in permanently frozen soil from northeastern Siberia was studied by culture-independent PCR amplification of diverse environmental 18S rRNA genes. Elaborate protocols to avoid contamination during drilling, sampling, and amplification were used. A broad diversity of eukaryotic DNA sequences that were 510 bp long, including sequences of various fungi, plants, and invertebrates, could be obtained reproducibly from samples that were up to 300,000 to 400,000 years old. The sequences revealed that ancient fungal communities included a diversity of cold-adapted yeasts, dark-pigmented fungi, plant-parasitic fungi, and lichen mycobionts. DNA traces of tree-associated macrofungi in a modern tundra sample indicated that there was a shift in fungal diversity following the last ice age and supported recent results showing that there was a severe change in the plant composition in northeastern Siberia during this period. Interestingly, DNA sequences with high homology to sequences of coprophilic and keratinophilic fungi indicated that feces, hair, skin, and nails could have been sources of ancient megafauna DNA recently reported to be present in small amounts of Siberian permafrost sediments.     

Activation of glucose transport during simulated ischemia in H9c2 cardiac myoblasts is mediated by protein kinase C isoforms

Glucose transport into cells may be regulated by a variety of conditions, including ischemia. We investigated whether some enzymes frequently involved in the metabolic adaptation to ischemia are also required for glucose transport activation. Ischemia was simulated by incubating during 3 h H9c2 cardiomyoblasts in a serum- and glucose-free medium in hypoxia. Under these conditions 2-deoxy-d-[2,6-³H]-glucose uptake was increased (57% above control levels, p < 0.0001) consistently with GLUT1 and GLUT4 translocation to sarcolemma. Tyrosine kinases inhibition via tyrphostin had no effect on glucose transport up-regulation induced by simulated ischemia. On the other hand, chelerythrine, a broad range inhibitor of protein kinase C isoforms, and rottlerin, an inhibitor of protein kinase C delta, completely prevented the stimulation of the transport rate. A lower activation of hexose uptake (19%, p < 0.001) followed also treatment with Gö6976, an inhibitor of conventional protein kinases C. Finally, PD98059-mediated inhibition of the phosphorylation of ERK 1/2, a downstream mitogen-activated protein kinase (MAPK), only partially reduced the activation of glucose transport induced by simulated ischemia (31%, p < 0.01), while SB203580, an inhibitor of p38 MAPK, did not exert any effect. These results indicate that stimulation of protein kinase C delta is strongly related to the up-regulation of glucose transport induced by simulated ischemia in cultured cardiomyoblasts and that conventional protein kinases C and ERK 1/2 are partially involved in the signalling pathways mediating this process.     

Inhibition of nitric oxide synthesis blocks the inhibitory response to capsaicin in intestinal circular muscle preparations from different species

Moderate concentrations of the sensory stimulant drug capsaicin caused relaxation in human and animal intestinal circular muscle preparations (guinea-pig proximal, mouse distal colon, human small intestine and appendix) in vitro. With the exception of the guinea-pig colon, the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine (L-NOARG; 10(-4) M) strongly inhibited the relaxant effect of capsaicin. Tetrodotoxin, an inhibitor of voltage-sensitive Na+ channels failed to significantly reduce the inhibitory effect of capsaicin in the guinea-pig colon, human ileum and appendix; it caused an approximately 50% reduction in the mouse colon. The relaxant effect of capsaicin was strongly reduced in colonic preparations from transient receptor potential vanilloid type (TRPV1) receptor knockout mice as compared to their wildtype controls. It is concluded that nitric oxide, possibly of sensory origin, is involved in the relaxant action of capsaicin in the circular muscle of the mouse and human intestine.     

Copepod and microzooplankton grazing in mesocosms fertilised with different Si:N ratios: no overlap between food spectra and Si:N influence on zooplankton trophic level

We hypothesized that the trophic level of marine copepods should depend on the composition of the protist community. To test this hypothesis, we manipulated the phytoplankton composition in mesocosms and measured grazing rates of copepods and mesozooplankton in those mesocosms. Twelve mesocosms with Northeast Atlantic phytoplankton were fertilised with different Si:N ratios from 0:1 to 1:1. After 1 week, ten of the mesocosms were filled with natural densities of mesozooplankton, mainly calanoid copepods, while two remained as mesozooplankton-free controls. Both before and after the addition of copepods there was a positive correlation of diatom dominance with Si:N ratios. During the second phase of the experiment, copepod and microzooplankton grazing rates on different phytoplankton species were assessed by a modification of the Landry-Hassett dilution technique, where the bottles containing the different dilution treatments were replaced by dialysis bags incubated in situ. The results indicated no overlap in the food spectrum of microzooplankton (mainly ciliates) and copepods. Ciliates fed on nanoplankton, while copepods fed on large or chain-forming diatoms, naked dinoflagellates, and ciliates. The calculated trophic level of copepods showed a significantly negative but weak correlation with Si:N ratios. The strength of this response was strongly dependent on the trophic levels assumed for ciliates and mixotrophic dinoflagellates.     

Nociceptin/orphanin FQ prevents ethanol-induced gastric lesions in the rat

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the NOP receptor, exerts a variety of effects on the gastrointestinal tract. The present study was aimed at evaluating the possible implication of N/OFQ in the maintenance of gastric mucosal integrity. N/OFQ was given either centrally or peripherally 30 min prior to intragastric administration (i.g.) of 1 ml/rat of ethanol (either 25% or 50%, v/v), which produces macroscopically visible gastric lesions. Intracerebroventricular (i.c.v.) injection of 2 microg/rat of N/OFQ significantly reduced lesions caused by 50% ethanol, while 1 microg/rat was enough to significantly reduce lesions caused by 25% ethanol. Intracerebroventricular injection of 5 microg/rat of the selective NOP receptor antagonist, UFP-101, completely reversed the protective effect of N/OFQ, 1 or 4 microg/rat against 25% or 50% ethanol, respectively. The intraperitoneal (i.p.) injection of N/OFQ produced a significant reduction of lesions induced by 50% ethanol, the peak effect being observed at 10 microg/kg. Intraperitoneal pretreatment with UFP-101, 120 microg/kg, completely abolished the protective effect of peripherally injected N/OFQ. Therefore, N/OFQ acts both centrally and peripherally as a protective agent against ethanol-induced gastric lesions, and its effect is mediated by NOP receptors.     

Experimental demonstration that mammalian oocytes are not selective towards X- or Y-bearing sperm

Mammalian oocytes are thought to be neutral as for X- or Y-bearing sperm selection is concerned, and penetration of an oocyte by an X- or a Y-bearing sperm is considered a random event. This assumption is mainly based on a posteriori evidences of a nearly equal sex ratio at birth, but it has never been experimentally demonstrated. We have designed a simple experiment, which allowed the penetration of an oocyte by more than one sperm and the further sexing by PCR of each single pronucleus present within the ooplasm. For the first time, we provide experimental evidence that mammalian oocytes do not play a selecting role since a single oocyte may be simultaneously fertilised by both X- and Y-bearing sperm.     

International Union of Physiological Sciences teaching workshop, April 7-10, 2005, Pali Mountain, California

Fifty-six physiologists from fourteen countries participated in the sixth International Union of Physiological Sciences Teaching Workshop. The four-day program included a poster session, a debate on integrative versus disciplinary curricula, and interactive lectures on evidence-based education, inquiry laboratories, and student preparation for physiology courses. Participants worked in small groups on one of four topics: developing free web-based laboratory resources, information technology, curriculum planning and design, and other issues in classroom teaching.