The Effects of Nitroxyl (HNO) on Soluble Guanylate Cyclase Activity: INTERACTIONS AT FERROUS HEME AND CYSTEINE THIOLS

It has been previously proposed that nitric oxide (NO) is the only biologically relevant nitrogen oxide capable of activating the enzyme soluble guanylate cyclase (sGC). However, recent reports implicate HNO as another possible activator of sGC. Herein, we examine the affect of HNO donors on the activity of purified bovine lung sGC and find that, indeed, HNO is capable of activating this enzyme. Like NO, HNO activation appears to occur via interaction with the regulatory ferrous heme on sGC. Somewhat unexpectedly, HNO does not activate the ferric form of the enzyme. Finally, HNO-mediated cysteine thiol modification appears to also affect enzyme activity leading to inhibition. Thus, sGC activity can be regulated by HNO via interactions at both the regulatory heme and cysteine thiols.Nitric oxide (NO)² is the most studied of the endogenously generated nitrogen oxides and is well known to mediate many aspects of cardiovascular function including the regulation of vascular tone and platelet aggregation (for example, see Ref. 1). These responses are in large part due to the interaction of NO with its most established endogenous receptor, soluble guanylate cyclase (sGC) (2). This 150-kDa heterodimeric heme protein catalyzes the production of the second messenger molecule cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP) (3). The basal activity of sGC is enhanced several hundred fold upon binding of NO to the single regulatory heme site. This stimulation of activity is a result of a conformational change induced by cleavage of the proximal histidine heme ligand upon formation of the ferrous nitrosyl complex, which is preferentially pentacoordinate (4). In addition to heme site regulation of sGC, there are numerous reports indicating that oxidation of cysteine thiol residues on this protein can also alter/regulate both the basal activity and the degree of NO-mediated activation (5–10).Recently, the one-electron reduced and protonated congener of NO, nitroxyl (HNO) has received significant interest as a cardiovascular agent whose actions are independent of NO formation (11). For example, a study by Ellis and co-workers (12) suggests that HNO is a vital component of endothelium-derived relaxing factor along with NO in rat aorta. HNO is also able to mediate murine aorta vasorelaxation even in the presence of NO scavengers (13). Furthermore, the vasodilation produced by HNO was inhibited by the sGC heme site inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one implicating sGC activation in this HNO-mediated effect. In addition to its effects on large conduit vessels like the aorta, HNO also dilates rat small mesenteric resistance-like arteries through sGC-dependent and voltage-dependent K⁺ channel-dependent mechanisms (14). Nitroxyl (derived from the HNO-donor Angeli''s salt) is also a potent dilator of feline pulmonary vasculature equal to that of the NO donors SPER/NO, DETA/NO, and SULFI/NO (15). Most recently, HNO was found to be a potent dilator of rat coronary arteries through an sGC-mediated mechanism (16). The evidence presented in these studies suggests that HNO is able to modulate cGMP levels through an interaction with sGC, an idea in conflict with a previous report showing that NO is the only nitrogen oxide capable of directly activating sGC (17).HNO forms a stable adduct with the ferrous heme of deoxymyoglobin (18, 19) providing precedence for a possible interaction between HNO and sGC that is akin to the interaction of NO with ferrous sGC. In light of all the reports indicating possible HNO-mediated activation of sGC, an examination of the direct interaction of HNO with purified sGC was carried out to evaluate the possibility that HNO may be capable of directly interacting with sGC to elicit activation. Moreover, due to the previously reported thiol redox regulation of sGC (see above) and the known thiophilicity of HNO (20), we also examined the effects of HNO-mediated thiol modification on enzyme activity.     

A mathematical model for the design of fibrin microcapsules with skin cells

The use of fibrin in tissue engineering has greatly increased over the last 10 years. The aim of this research was to develop a mathematical model to relate the microcapsule-size and cell-load to growth and oxygen depletion. Keratinocytes were isolated from rat skins and microencapsulated dropping fibrinogen and thrombin solutions. The cell growth was measured with MTT-assay and confirmed using histochemical technique. The oxygen was evaluated using a Clark sensor. It was found that Fick–Monod model explained the cell growth for the first 48 h, but overestimated the same thereafter. It was necessary to add a logistic equation to reach valid results. In relation to the preferred implant alternative, when considering large initial cell loads, the possibility to implant small loads of fast-growing cells arises from the simulations. In relation to the microcapsule size, it was found that a critical diameter could be established from which cell growth velocity is about the same.     

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 August 2009-30 September 2009

This article documents the addition of 238 microsatellite marker loci and 72 pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Adelges tsugae, Artemisia tridentata, Astroides calycularis, Azorella selago, Botryllus schlosseri, Botrylloides violaceus, Cardiocrinum cordatum var. glehnii, Campylopterus curvipennis, Colocasia esculenta, Cynomys ludovicianus, Cynomys leucurus, Cynomys gunnisoni, Epinephelus coioides, Eunicella singularis, Gammarus pulex, Homoeosoma nebulella, Hyla squirella, Lateolabrax japonicus, Mastomys erythroleucus, Pararge aegeria, Pardosa sierra, Phoenicopterus ruber ruber and Silene latifolia. These loci were cross-tested on the following species: Adelges abietis, Adelges cooleyi, Adelges piceae, Pineus pini, Pineus strobi, Tubastrea micrantha, three other Tubastrea species, Botrylloides fuscus, Botrylloides simodensis, Campylopterus hemileucurus, Campylopterus rufus, Campylopterus largipennis, Campylopterus villaviscensio, Phaethornis longuemareus, Florisuga mellivora, Lampornis amethystinus, Amazilia cyanocephala, Archilochus colubris, Epinephelus lanceolatus, Epinephelus fuscoguttatus, Symbiodinium temperate-A clade, Gammarus fossarum, Gammarus roeselii, Dikerogammarus villosus and Limnomysis benedeni. This article also documents the addition of 72 sequencing primer pairs and 52 allele specific primers for Neophocaena phocaenoides.     

Current patterns of macroalgal diversity and biomass in northern hemisphere rocky shores

Latitudinal gradients in species abundance and diversity have been postulated for nearshore taxa but few analyses have been done over sufficiently broad geographic scales incorporating various nearshore depth strata to empirically test these gradients. Typically, gradients are based on literature reviews and species lists and have focused on alpha diversity across the entire nearshore zone. No studies have used a standardized protocol in the field to examine species density among sites across a large spatial scale while also focusing on particular depth strata. The present research used field collected samples in the northern hemisphere to explore the relationships between macroalgal species density and biomass along intertidal heights and subtidal depths and latitude. Results indicated no overall correlations between either estimates of species density or biomass with latitude, although the highest numbers of both were found at mid-latitudes. However, when strata were examined separately, significant positive correlations were found for both species numbers and biomass at particular strata, namely the intertidal ones. While the data presented in this paper have some limitations, we show that latitudinal macroalgal trends in species density and biomass do exist for some strata in the northern hemisphere with more taxa and biomass at higher latitudes.     

Effect of 6-nonadecyl salicylic acid and its methyl ester on the induction of micronuclei in polychromatic erythrocytes in mouse peripheral blood

The bark of Amphipterygium adstringens is widely used in the traditional Mexican medicine for treating ailments such as gastric ulcers, gastritis and stomach cancer. The 6-nonadecyl salicylic acid (anacardic acid) was isolated from the bark of this species. In previous papers have been informed that the anacardic acids possess anti-tumour, antimicrobial, antiacne, antibacterial and many others medicinal properties. Now we describe cytotoxic and genotoxic effects of this compound and its methyl ester. The cytotoxic and genotoxic effects of 6-nonadecyl salicylic acid (6NDSA) and its methyl ester (ME6NDSA) on CD1 male mice were determined with micronucleus assay at 24, 48 and 72h after oral administration of doses of 0.75, 2.5, 5.0 and 10.0mg/kg. Peripheral blood samples were drawn from the caudal vein and analyzed by Giemsa-stained technique. The results obtained showed that the ratios of polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE) in mice treated with 10mg/kg of 6NDSA were statistically lower after 24h compared with its negative control animals, and that after 72h, PCE/NCE ratios were reduced in animals treated with 6NDSA at all tested dose levels. The methyl ester ME6NDSA showed no such cytotoxic activity. Neither of the test compounds increased the frequency of micronucleated polychromatic erythrocytes from which it appears that administration of 6NDSA and ME6NDSA may not lead to chromosome damage at the evaluated doses.     

Effect of inspiratory muscle fatigue on breathing pattern during inspiratory resistive loading

The purpose of this study was to determine whether induction of either inspiratory muscle fatigue (expt 1) or diaphragmatic fatigue (expt 2) would alter the breathing pattern response to large inspiratory resistive loads. In particular, we wondered whether induction of fatigue would result in rapid shallow breathing during inspiratory resistive loading. The breathing pattern during inspiratory resistive loading was measured for 5 min in the absence of fatigue (control) and immediately after induction of either inspiratory muscle fatigue or diaphragmatic fatigue. Data were separately analyzed for the 1st and 5th min of resistive loading to distinguish between immediate and sustained effects. Fatigue was achieved by having the subjects breathe against an inspiratory threshold load while generating a predetermined fraction of either the maximal mouth pressure or maximal transdiaphragmatic pressure until they could no longer reach the target pressure. Compared with control, there were no significant alterations in breathing pattern after induction of fatigue during either the 1st or 5th min of resistive loading, regardless of whether fatigue was induced in the majority of the inspiratory muscles or just in the diaphragm. We conclude that the development of inspiratory muscle fatigue does not alter the breathing pattern response to large inspiratory resistive loads.     

The glucose transport activity of human erythrocyte membranes. Reconstitution in phospholipid liposomes and fractionation by molecular sieve and ion exchange chromatography

Human erythrocyte membranes, at a protein concentration of 1–2 g/l, were solubilized with 0.12 M cholate in the presence of 0.06 M phospholipid (egg yolk phospholipids or phosphatidylcholine). More than 40% of the protein was solubilized. Cholate was removed by molecular sieve chromatography, whereby liposomes formed. These liposomes exchanged D-glucose faster than L-glucose. The recovery of glucose transport activity in the reconstituted system was estimated to be higher than 16%.The liposomes were heterogeneous in size, as shown by molecular sieve chromatography on Sepharose 4B, and small liposomes predominated. In liposomes formed with phosphatidylcholine, the distribution of glucose transport activity did not parallel the distribution of protein or phospholipid, and the activity was found mainly in the smallest liposomes. The proteins were incorporated mainly into the liposomes that eluted at the lowest ionic strength upon ion exchange chromatography.The glucose transport activity separated into three main peaks upon ion exchange chromatography of egg yolk phospholipid liposomes. The activity eluted at low ionic strength. The liposomes contained proteins mainly from the 3- and 4.5-regions (nomenclature according to Steck, T.L. (1974) J. Cell Biol. 62, 1–19). The activity peaks were highest in the first part of the chromatogram. The protein distribution did not coincide with the variation in activity over each peak. Therefore, it cannot be excluded that a minor component not seen in the electrophoretic analyses might be responsible for the glucose transport activity.     

Biochemical and immunological characterization of α-amylase isoenzymes of Araucaria araucana

Seven α-amylase isoenzymes present in quiescent seeds of the South American conifer Araucaria araucana were purified by affinity chromatography and partially characterized. The molecular masses of these isoenzymes were 45.7, 47.0, 50.2, 51.2, 52.0, 53.5 and 55.2 kDa. The two main isoforms were separated from each other and from the rest of the isoenzymes by anion-exchange chromatography using a linear gradient of 0 to 0.6 M NaCl and slightly different CaCl₂ concentrations. All isoenzyme bands stained with periodic acid/dansylhydrazine, suggesting that they are glycoproteins. Electroblotting of the isoenzymes onto polyvinylidene difluoride membranes allowed determination of the amino acid composition and NH₂-terminal sequence of the 53.5-, 50.2-and 47.0-kDa isoenzymes. Amino acid compositional analysis demonstrated that these enzymes are rich in glycine, aspartic acid/asparagine, alanine, serine, proline and glutamic acid/glutamine. The NH₂-terminal sequences of the three isoenzymes are identical. Comparison of the amino acid compositions and the NH₂-terminal sequence of these isoenzymes with the cereal and Vigna radiata α-amylases demonstrated that there is no relation between them. However, polyclonal antibodies generated against barley α-amylase cross-reacted with all the A . araucana α-amylases. Peptide mapping analysis of the isoenzymes using cyanogen bromide suggests that there are genetic differences between them.