Vasopressin-dependent inhibition of the C-type natriuretic peptide receptor,NPR-B/GC-B,requires elevated intracellular calcium concentrations

Natriuretic peptides bind their cognate cell surface guanylyl cyclase receptors and elevate intracellular cGMP concentrations. In vascular smooth muscle cells, this results in the activation of the type I cGMP-dependent protein kinase and vasorelaxation. In contrast, pressor hormones like arginine-vasopressin, angiotensin II, and endothelin bind serpentine receptors that interact with G(q) and activate phospholipase Cbeta. The products of this enzyme, diacylglycerol and inositol trisphosphate, activate the conventional and novel forms of protein kinase C (PKC) and elevate intracellular calcium concentrations, respectively. The latter response results in vasoconstriction, which opposes the actions of natriuretic peptides. Previous reports have shown that pressor hormones inhibit natriuretic peptide receptors NPR-A or NPR-B in a variety of different cell types. Although the mechanism for this inhibition remains unknown, it has been universally accepted that PKC is an obligatory component of this pathway primarily because pharmacologic activators of PKC mimic the inhibitory effects of these hormones. Here, we show that in A10 vascular smooth muscle cells, neither chronic PKC down-regulation nor specific PKC inhibitors block the AVP-dependent desensitization of NPR-B even though both processes block PKC-dependent desensitization. In contrast, the cell-permeable calcium chelator, BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester), abrogates the AVP-dependent desensitization of NPR-B, and ionomycin, a calcium ionophore, mimics the AVP effect. These data show that the inositol trisphosphate/calcium arm of the phospholipase C pathway mediates the desensitization of a natriuretic peptide receptor in A10 cells. In addition, we report that CNP attenuates AVP-dependent elevations in intracellular calcium concentrations. Together, these data reveal a dominant role for intracellular calcium in the reciprocal regulation of these two important vasoactive signaling systems.     

The atrial natriuretic peptide receptor (NPR-A/GC-A) is dephosphorylated by distinct microcystin-sensitive and magnesium-dependent protein phosphatases

Natriuretic peptide receptor (NPR)-A is the primary signaling receptor for atrial natriuretic peptide and brain natriuretic peptide. Ligand binding to NPR-A rapidly activates its guanylyl cyclase domain, but its rate of cGMP synthesis declines with time. This waning of activity is called homologous desensitization and is mediated in part by receptor dephosphorylation. Here, we characterize two distinct NPR-A phosphatase activities. The serine/threonine protein phosphatase inhibitor, microcystin, inhibited the desensitization of NPR-A in membrane guanylyl cyclase assays in the absence of magnesium. EDTA also inhibited the desensitization, whereas MgCl(2) stimulated the desensitization. Because the effects of microcystin and EDTA were additive, and microcystin did not block the magnesium-dependent desensitization, the targets for these agents appear to be distinct. Incubation of membranes at 37 degrees C stimulated the dephosphorylation of NPR-A, and microcystin blocked the temperature-dependent dephosphorylation. The addition of MgCl(2) or MnCl(2), but not CaCl(2), further stimulated the dephosphorylation of NPR-A, and microcystin failed to inhibit this process. The desensitization required changes in the phosphorylation state of NPR-A because the guanylyl cyclase activity of a receptor variant containing glutamate substitutions at all six phosphorylation sites was unaffected by MgCl(2), EDTA, or microcystin. Together, these data indicate that NPR-A is regulated by two distinct phosphatases, possibly including a member of the protein phosphatase 2C family. Finally, we observed that the desensitization of NPR-A in membranes from mouse kidneys and NIH3T3 cells was increased by prior exposure to atrial natriuretic peptide, suggesting that hormone binding enhances receptor dephosphorylation.     

Gramene: development and integration of trait and gene ontologies for rice

Gramene (http://www.gramene.org/) is a comparative genome database for cereal crops and a community resource for rice. We are populating and curating Gramene with annotated rice (Oryza sativa) genomic sequence data and associated biological information including molecular markers, mutants, phenotypes, polymorphisms and Quantitative Trait Loci (QTL). In order to support queries across various data sets as well as across external databases, Gramene will employ three related controlled vocabularies. The specific goal of Gramene is, first to provide a Trait Ontology (TO) that can be used across the cereal crops to facilitate phenotypic comparisons both within and between the genera. Second, a vocabulary for plant anatomy terms, the Plant Ontology (PO) will facilitate the curation of morphological and anatomical feature information with respect to expression, localization of genes and gene products and the affected plant parts in a phenotype. The TO and PO are both in the early stages of development in collaboration with the International Rice Research Institute, TAIR and MaizeDB as part of the Plant Ontology Consortium. Finally, as part of another consortium comprising macromolecular databases from other model organisms, the Gene Ontology Consortium, we are annotating the confirmed and predicted protein entries from rice using both electronic and manual curation.     

Torpor-associated fluctuations in surfactant activity in Gould's wattled bat

The primary function of pulmonary surfactant is to reduce the surface tension (ST) created at the air-liquid interface in the lung. Surfactant is a complex mixture of lipids and proteins and its function is influenced by physiological parameters such as metabolic rate, body temperature and breathing. In the microchiropteran bat Chalinolobus gouldii these parameters fluctuate throughout a 24 h period. Here we examine the surface activity of surfactant from warm-active and torpid bats at both 24 degrees C and 37 degrees C to establish whether alterations in surfactant composition correlate with changes in surface activity. Bats were housed in a specially constructed bat room at Adelaide University, at 24 degrees C and on a 8:16 h light:dark cycle. Surfactant was collected from bats sampled during torpor (2535 degrees C). Alterations in the lipid composition of surfactant occur with changes in the activity cycle. Most notable is an increase in surfactant cholesterol (Chol) with decreases in body temperature [Codd et al., Physiol. Biochem. Zool. 73 (2000) 605-612]. Surfactant from active bats was more surface active at higher temperatures, indicated by lower ST(min) and less film area compression required to reach ST(min) at 37 degrees C than at 24 degrees C. Conversely, surfactant from torpid bats was more active at lower temperatures, indicated by lower ST(min) and less area compression required to reach ST(min) at 24 degrees C than at 37 degrees C. Alterations in the Chol content of bat surfactant appear to be crucial to allow it to achieve low STs during torpor.     

Expression and contribution of endogenous IL-13 in an experimental model of sepsis

IL-13 has been shown to exert potent anti-inflammatory properties. In this study, we elucidated the functional role of endogenous IL-13 in a murine model of septic peritonitis induced by cecal ligation and puncture (CLP). Initial studies demonstrated that the level of IL-13 increased in tissues including liver, lung, and kidney, whereas no considerable increase was found in either peritoneal fluid or serum after CLP. Immunohistochemically, IL-13-positive cells were Kupffer cells in liver, alveolar macrophages in lung, and epithelial cells of urinary tubules in kidney. IL-13 blockade with anti-IL-13 Abs significantly decreased the survival rate of mice after CLP from 53% to 14% on day 7 compared with control. To determine the potential mechanisms whereby IL-13 exerted a protective role in this model, the effects of anti-IL-13 Abs on both local and systemic inflammation were investigated. Administration of anti-IL-13 Abs did not alter the leukocyte infiltration and bacterial load in the peritoneum after CLP but dramatically increased the neutrophil influx in tissues after CLP, an effect that was accompanied by significant increases in the serum levels of aspartate transaminase, alanine transaminase, blood urea nitrogen, and creatinine. Tissue injury caused by IL-13 blockade was associated with increases in mRNA and the protein levels of CXC chemokines macrophage inflammatory protein-2 and KC as well as the CC chemokine macrophage inflammatory protein-1alpha and the proinflammatory cytokine TNF-alpha. Collectively, these results suggest that endogenous IL-13 protected mice from CLP-induced lethality by modulating inflammatory responses via suppression of overzealous production of inflammatory cytokines/chemokines in tissues.     

Microwave oven and boiling waterbath extraction of hepatotoxins from cyanobacterial cells

Low-cost, straightforward methods for the extraction of microcystins and nodularins from cyanobacterial cells were developed using a microwave oven and boiling waterbath. The use of organic solvents, such as methanol, which can interfere with sensitive analytical procedures, e.g. immunoassays, can thus be avoided. Analysis by protein phosphatase inhibition assay and high performance liquid chromatography indicated that purified microcystin-LR was unaffected by the microwave oven and boiling waterbath treatments. Four microcystins of differing hydrophobicities were successfully extracted from Microcystis PCC 7813 by both treatments at yields equivalent to those obtained by longer protocols using methanol. Assessment of the microwave oven and boiling waterbath extraction methods with laboratory strains and environmental samples of cyanobacteria showed good correlation with results from lyophilisation and methanol extraction, when extracts were analysed by high performance liquid chromatography with diode array detection (R(2)>/=0.92). The microwave and boiling waterbath extraction methods also sterilised the environmental bloom samples, as evidenced by the abolition of heterotrophic bacterial growth.     

Iron overload in paediatrics undergoing cardiopulmonary bypass

Pathological changes in iron status are known to occur during bypass and will be superimposed upon physiological abnormalities in iron distribution, characteristic of the neonatal period. We have sought to define the severity of iron overload in these patients. Plasma samples from 65 paediatric patients undergoing cardiopulmonary bypass (CPB) were analysed for non-haem iron, total iron binding capacity, transferrin and bleomycin-detectable iron. Patients were divided into four age groups for analysis. Within each age group, patients who were in iron overload at any time point were statistically compared to those who were not. The most significant changes in iron chemistry were seen in the plasma of neonates, with 25% in a state of plasma iron overload. 18.5% of infants and 14.3% of children at 1-5 years were also in iron overload at some time point during CPB. No children over 5 years, however, went into iron overload. Increased iron saturation of transferrin eliminates its ability to bind reactive forms of iron and to act as an antioxidant. When transferrin is fully saturated with iron, reactive forms of iron are present in the plasma which can stimulate iron-driven oxidative reactions. Our data suggest that paediatric patients are at greater risk of iron overload during CPB, and that some form of iron chelation therapy may be advantageous to decrease oxidative stress.     

A new species of Sanicula L. (Umbelliferae/Apiaceae) endemic to Baja California, Mexico

A new species of the genusSanicula (sect.Sanicoria) endemic to the southern Sierra Juárez of Baja California, Mexico, is described and illustrated.Sanicula moranii resemblesS. deserticola andS. bipinnatifida, but has thicker basal leaves, with broader petioles and rachises, and shorter fruit prickles that are confined to the apical part of the mericarps. The taxonomic relationships of these species are discussed, along with aspects of their distribution and habitat.

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Resumen  Una neuva especie del géneroSanicula (secciónSanicoria) endémica de la Sierra Juárez de Baja California, México, es descrita e ilustrada.Sanicula moranii presenta similitud conS. deserticola yS. bipinnatifida, no obstante tiene gruesas hojas basales con pecíolos y raquis anchos, y espínulas del fruto más cortas que se distribuyen únicamente en la parte apical de los mericarpos. Además, se comentan las relaciones taxonómicas de las tres especies, así como algunos aspectos de sus distribuciones y hábitats.