The Peculiar Facets of Nitric Oxide as a Cellular Messenger: From Disease-Associated Signaling to the Regulation of Brain Bioenergetics and Neurovascular Coupling

In this review, we address the regulatory and toxic role of ^·NO along several pathways, from the gut to the brain. Initially, we address the role on ^·NO in the regulation of mitochondrial respiration with emphasis on the possible contribution to Parkinson’s disease via mechanisms that involve its interaction with a major dopamine metabolite, DOPAC. In parallel with initial discoveries of the inhibition of mitochondrial respiration by ^·NO, it became clear the potential for toxic ^·NO-mediated mechanisms involving the production of more reactive species and the post-translational modification of mitochondrial proteins. Accordingly, we have proposed a novel mechanism potentially leading to dopaminergic cell death, providing evidence that NO synergistically interact with DOPAC in promoting cell death via mechanisms that involve GSH depletion. The modulatory role of NO will be then briefly discussed as a master regulator on brain energy metabolism. The energy metabolism in the brain is central to the understanding of brain function and disease. The core role of ^·NO in the regulation of brain metabolism and vascular responses is further substantiated by discussing its role as a mediator of neurovascular coupling, the increase in local microvessels blood flow in response to spatially restricted increase of neuronal activity. The many facets of NO as intracellular and intercellular messenger, conveying information associated with its spatial and temporal concentration dynamics, involve not only the discussion of its reactions and potential targets on a defined biological environment but also the regulation of its synthesis by the family of nitric oxide synthases. More recently, a novel pathway, out of control of NOS, has been the subject of a great deal of controversy, the nitrate:nitrite:NO pathway, adding new perspectives to ^·NO biology. Thus, finally, this novel pathway will be addressed in connection with nitrate consumption in the diet and the beneficial effects of protein nitration by reactive nitrogen species.

     

The role of oxygen availability in the embryonation of Heterakis gallinarum eggs.

The importance of oxygen availability in the embryonation of the infective egg stages of the gastrointestinal nematode parasite Heterakis gallinarum was studied in the laboratory. Unembryonated H. gallinarum eggs were kept under either aerobic conditions by gassing with oxygen, or anaerobic conditions by gassing with the inert gas nitrogen, under a range of constant temperatures. Oxygenated eggs embryonated at a rate influenced by temperature. Conversely, eggs treated with nitrogen showed no embryonation although when these eggs were transferred from nitrogen to oxygen gas after 60 days of treatment, embryonation occurred. This demonstrated that oxygen is an essential requirement for H. gallinarum egg development, although undeveloped eggs remain viable, even after 60 days in low oxygen conditions. The effects of climate on the biology of free-living stages studied under constant laboratory conditions cannot be applied directly to the field where climatic factors exhibit daily cycles. The effect of fluctuating temperature on development was investigated by including an additional temperature group in which H. gallinarum eggs were kept under daily temperature cycles between 12 and 22°C. Cycles caused eggs to develop significantly earlier than those in the constant mean cycle temperature, 17°C, but significantly slower than those in constant 22°C suggesting that daily temperature cycles had an accelerating effect on H. gallinarum egg embryonation but did not accelerate to the higher temperature. These results suggest that daily fluctuations in temperature influence development of the free-living stages and so development cannot be accurately predicted on the basis of constant temperature culture.     

Random walks and the gas model: spacing behaviour of Grey-Cheeked Mangabeys

1. Simple mechanical models (random walks and the 'gas model') were used to investigate the movement patterns and intergroup encounter rates of a high- and low-density subpopulation of Grey-Cheeked Mangabeys ( Cercocebus albigena johnstoni, Lydeker) at two sites in the Kibale Forest, Western Uganda.
2. Random walk simulations showed that the presence of conspecific groups could act as 'social barriers' which constrained group movements, and promoted high levels of site attachment to a specific home range area.
3. Encounter rate models showed that in the low-density subpopulation (Kanyawara), intergroup encounters occurred no more frequently than expected if groups were moving randomly and independently of each other. This was in contrast to previous analyses which suggested that Grey-Cheeked Mangabeys employed a social spacing mechanism (mediated by male loud calls) in order to reduce the frequency of encounter to below chance levels.
4. Encounters in the high-density subpopulation (Ngogo) were found to occur less frequently than expected at short range (within 100 m). This was suggested to be due to the operation of a territorial mate defence strategy by males at this site.     

Restoring avian wing digits

The precise identification of the digits of the avian wing is of importance in evolutionary studies. If the digits are numbered two, three and four, this has been taken to suggest that birds are not descended directly from dinosaurs. If the digits are numbered one, two and three, dinosaur origins become more plausible. Studies of the development of the avian wing have failed to resolve this dilemma. However, in some instances, it is possible to deduce information about evolutionary morphologies by manipulating development experimentally. We grafted beads loaded with fibroblast growth factor 4 into the distal tip of chick wing buds at times when the apical ectodermal ridge is regressing. The consequence was that the cartilage structure conventionally labelled ''element 5'' increased dramatically in size and acquired a digit-like morphology in some instances. Corresponding changes in soft tissue morphology were also observed. We conclude that it may be possible to resolve the issue of avian digit homology by the induction of experimental atavisms of this kind.     

Calcium binding mode of gamma-carboxyglutamic acids in conantokins.

Conantokin-T (con-T) and conantokin-G (con-G) are two highly homologous peptide toxins found in Conus venom. The former is a 21-residue peptide with four gamma-carboxyglutamic acid (Gla) residues (at positions 3, 4, 10 and 14), while the latter is a 17-residue peptide with five gamma-carboxyglutamic acid residues (at positions 3, 4, 7, 10 and 14). Despite the apparent similarity in number and relative positions of the gamma-carboxyglutamic acid residues, (113)Cd-NMR studies indicated a distinct metal binding behavior for con-G and con-T. There appears to be four binding sites in con-G in contrast to one metal binding site in con-T. To elucidate the mode of calcium binding by the gamma-carboxyglutamic acid residues in these conantokins, we designed various analogous peptides with their gamma-carboxyglutamic acid replaced by other amino acid residues. (113)Cd-NMR experiments on conantokin analogues reveal that the major difference in the number of metal binding sites between con-G and con-T is due to the residue at position 7. We also performed molecular simulations to calculate the relative binding free energies of several potential binding sites. Based on our theoretical and experimental results, we propose a 'four-site' binding model for conantokin-G and a 'single-site' binding model for conantokin-T.     

Endotoxemia increases relative perfusion to dorsal-caudal lung regions.

Changes in the spatial distribution of perfusion during acute lung injury and their impact on gas exchange are poorly understood. We tested whether endotoxemia caused topographical differences in perfusion and whether these differences caused meaningful changes in regional ventilation-to-perfusion ratios and gas exchange. Regional ventilation and perfusion were measured in anesthetized, mechanically ventilated pigs in the prone position before and during endotoxemia with the use of aerosolized and intravenous fluorescent microspheres. On average, relative perfusion halved in ventral and cranial lung regions, doubled in caudal lung regions, and increased 1.5-fold in dorsal lung regions during endotoxemia. In contrast, there were no topographical differences in perfusion before endotoxemia and no topographical differences in ventilation at any time point. Consequently, endotoxemia increased regional ventilation-to-perfusion ratios in the caudal-to-cranial and dorsal-to-ventral directions, resulting in end-capillary PO2 values that were significantly lower in dorsal-caudal than ventral-cranial regions. We conclude that there are topographical differences in the pulmonary vascular response to endotoxin that may have important consequences for gas exchange in acute lung injury.     

A protocol for micropropagation of Alpinia galanga

Emerging buds of rhizome of Alpinia galanga Willd produced shoots and roots simultaneously when cultured in MS medium supplemented with kinetin 3.0 mg l^-1. Each explanted shoot bud produced 8 shoots in average and roots simultaneously within 8 weeks. Shoot proliferation could be continued even after a year by transferring each divided shoot explant to the same medium. Regenerated plantlets could be sucessfully transferred to soil where they grew well within 10–12 weeks with 80% survivality. This revised version was published online in June 2006 with corrections to the Cover Date.