Neuronostatin encoded by the somatostatin gene regulates neuronal, cardiovascular, and metabolic functions.

Somatostatin is important in the regulation of diverse neuroendocrine functions. Based on bioinformatic analyses of evolutionarily conserved sequences, we predicted another peptide hormone in pro-somatostatin and named it neuronostatin. Immuno-affinity purification allowed the sequencing of an amidated neuronostatin peptide of 13 residues from porcine tissues. In vivo treatment with neuronostatin induced c-Fos expression in gastrointestinal tissues, anterior pituitary, cerebellum, and hippocampus. In vitro treatment with neuronostatin promoted the migration of cerebellar granule cells and elicited direct depolarizing actions on paraventricular neurons in hypothalamic slices. In a gastric tumor cell line, neuronostatin induced c-Fos expression, stimulated SRE reporter activity, and promoted cell proliferation. Furthermore, intracerebroventricular treatment with neuronostatin increased blood pressure but suppressed food intake and water drinking. Our findings demonstrate diverse neuronal, neuroendocrine, and cardiovascular actions of a somatostatin gene-encoded hormone and provide the basis to investigate the physiological roles of this endogenously produced brain/gut peptide.

The involvement of calcium in the regulation of GPX1 expression.

Detrimental effects of salinity on plants are known to be partially alleviated by external Ca(2+). Previously we demonstrated that in citrus cells, phospholipid hydroperoxide glutathione peroxidase (GPX1) is induced by salt and its activation can be monitored by pGPX1::GUS fusion in transformed tobacco cells. In this paper we further characterized the induction of GPX1 by additional treatments, which are known to affect Ca(2+) transport. Omission of Ca(2+) changed the pattern of the transient salt-induced expression of GPX1 and chelation of Ca(2+) by EGTA, or treatment with caffeine, abolished the salt-induced GPX1 transcript. On the other hand, La(3+) was found to be as potent as NaCl in inducing GPX1 transcription and the combined effect of La(3+) and NaCl seemed to be additive. Pharmacological perturbation of either external or internal Ca(2+) pools by La(3+), EGTA, caffeine, Ca(2+) channel blockers, or a Ca(2+)-ATPase inhibitor rendered the imposed salt stress more severe. Except for La(3+), all these Ca(2+) effectors had no effect on their own. In addition, the fluidizer benzyl alcohol dramatically increased the NaCl-induced GPX1 transcription. Taken together, our results show that: 1) the mode of action of La(3+) on GPX1 expression differs from its established role as a Ca(2+) channel blocker, 2) membrane integrity has an important role in the perception of salt stress, and 3) internal stores of Ca(2+) are involved in activating GPX1 expression in response to salt stress. We propose that the common basis for these effects lies in the membrane bound Ca(2+).

Matching receptome genes with their ligands for surveying paracrine/autocrine signaling systems.

Sequencing of genomes from diverse organisms facilitates studies on the repertoire of genes involved in intercellular signaling. Extending previous efforts to annotate most human plasma membrane receptors in the Human Plasma Membrane Receptome database, we matched cognate ligands with individual receptors by surveying the published literature. In the updated online database we called "liganded receptome," users can search for individual ligands or receptors to reveal their pairing partners and browse through receptor or ligand families to identify relationships between ligands and receptors in their respective families. Because local signaling systems are prevalent in diverse normal and diseased tissues, we used the liganded receptome knowledgebase to interrogate DNA microarray datasets for genome-wide analyses of potential paracrine/autocrine signaling systems. In addition to viewing ligand-receptor coexpression based on precomputed DNA microarray data, users can submit their own microarray data to perform online genome-wide searches for putative paracrine/autocrine signaling systems. Investigation of transcriptome data based on liganded receptome allows the discovery of paracrine/autocrine signaling for known ligand-receptor pairs in previously uncharacterized tissues or developmental stages. The present annotation of ligand-receptor pairs also identifies orphan receptors and ligands without known interacting partners in select families. Because hormonal ligands within the same family usually interact with paralogous receptors, this genomic approach could also facilitate matching of orphan receptors and ligands. The liganded receptome is accessible at http://receptome.stanford.edu.

Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on food intake.

Ghrelin, a circulating appetite-inducing hormone, is derived from a prohormone by posttranslational processing. On the basis of the bioinformatic prediction that another peptide also derived from proghrelin exists, we isolated a hormone from rat stomach and named it obestatin-a contraction of obese, from the Latin "obedere," meaning to devour, and "statin," denoting suppression. Contrary to the appetite-stimulating effects of ghrelin, treatment of rats with obestatin suppressed food intake, inhibited jejunal contraction, and decreased body-weight gain. Obestatin bound to the orphan G protein-coupled receptor GPR39. Thus, two peptide hormones with opposing action in weight regulation are derived from the same ghrelin gene. After differential modification, these hormones activate distinct receptors.

The salt-stress signal transduction pathway that activates the gpx1 promoter is mediated by intracellular H2O2, different from the pathway induced by extracellular H2O2.

Several genes encoding putative glutathione peroxidase have been isolated from a variety of plants, all of which show the highest homology to the phospholipid hydroperoxide isoform. Several observations suggest that the proteins are involved in biotic and abiotic stress responses. Previous studies on the regulation of gpx1, the Citrus sinensis gene encoding phospholipid hydroperoxide isoform, led to the conclusion that salt-induced expression of gpx1 transcript and its encoded protein is mediated by oxidative stress. In this paper, we describe the induction of gpx1 promoter:uidA fusions in stable transformants of tobacco (Nicotiana tabacum) cultured cells and plants. We show that the induction of gpx1 by salt and oxidative stress occurs at the transcriptional level. gpx1 promoter analysis confirmed our previous assumption that the salt signal is transduced via oxidative stress. We used induction of the fusion construct to achieve better insight into, and to monitor salt-induced oxidative stress. The gpx1 promoter responded preferentially to oxidative stress in the form of hydrogen peroxide, rather than to superoxide-generating agents. Antioxidants abolished the salt-induced expression of gpx1 promoter, but were unable to eliminate the induction by H2O2. The commonly employed NADPH-oxidase inhibitor diphenyleneiodonium chloride and catalase inhibited the H2O2-induced expression of gpx1 promoter, but did not affect its induction by salt. Our results led us to conclude that salt induces oxidative stress in the form of H2O2, its production occurs in the intracellular space, and its signal transduction pathway activating the gpx1 promoter is different from the pathway induced by extracellular H2O2.

Protein related to DAN and cerberus is a bone morphogenetic protein antagonist that participates in ovarian paracrine regulation.

Bone morphogenetic proteins (BMPs) are important for body patterning and morphogenesis, whereas several BMP antagonists regulate the functions of BMPs during embryonic development and tissue differentiation. Protein related to DAN and cerberus (PRDC) is a secreted protein with a cystine knot structure identified by gene trapping in embryonic stem cells. Although PRDC shows sequence homology with proteins of the BMP antagonist family, its biological activity and physiological functions are unclear. We generated recombinant PRDC and its paralog, gremlin, and tested their ability to suppress actions initiated by diverse BMP proteins. Similar to the known BMP antagonist, gremlin, PRDC blocked ligand signaling induced by BMP2 and BMP4 but had minimal effects on reporter gene activation induced by GDF-9, activin, or transforming growth factor-beta. Co-precipitation assays further demonstrated the direct protein-protein interactions between PRDC and BMP2 or BMP4. Reverse transcriptase-PCR analyses indicated that PRDC transcripts are widely expressed showing higher levels in ovary, brain, and spleen. In mouse ovary, PRDC transcripts were increased following gonadotropin treatment. In situ hybridization analyses further indicated that ovarian PRDC transcripts are localized in granulosa cells of selective follicles. In addition, co-treatment with PRDC antagonized the inhibitory effects of BMP4 on the follicle-stimulating hormone stimulation of progesterone production by cultured rat granulosa cells. Thus, PRDC is a potent BMP antagonist with a wide tissue expression pattern, and ovarian PRDC expressed in granulosa cells could be involved in follicular development by antagonizing the actions of theca cell-derived BMPs.

Comparative genomic analysis of the eight-membered ring cystine knot-containing bone morphogenetic protein antagonists.

TGF-beta family proteins with a cystine knot motif serve as ligands for diverse families of plasma membrane receptors. Bone morphogenetic protein (BMP) antagonists represent a subgroup of these proteins, some of which bind BMPs and antagonize their actions during development and morphogenesis. Availability of completed genome sequences from diverse organisms allows bioinformatic analysis of the evolution of BMP antagonists and facilitates their classification. Using a regular expression algorithm (http://BioRegEx.stanford.edu), an exhaustive search of the human genome identified all cystine knot-containing BMP antagonists. Based on the size of the cystine ring, these proteins were divided into three subfamilies: CAN (eight-membered ring), twisted gastrulation (nine-membered ring), as well as chordin and noggin (10-membered ring). The CAN family can be divided further into four subgroups based on a conserved arrangement of additional cysteine residues-gremlin and PRDC, cerberus and coco, and DAN, together with USAG-1 and sclerostin. We searched for orthologs of human BMP antagonists in the genomes of model organisms and analyzed their phylogenetic relationship. New human paralogs were identified together with the verification of orthologous relationships of known genes. We also discuss the physiological roles of the CAN subfamily of BMP antagonists and the associated genetic defects. Based on the known three-dimensional structure of key cystine knot proteins, we postulated disulfide bondings for eight-membered ring BMP antagonists to predict their potential folding and dimerization.

Indole acetic acid distribution coincides with vascular differentiation pattern during Arabidopsis leaf ontogeny.

We used an anti-indole acetic acid (IAA or auxin) monoclonal antibody-based immunocytochemical procedure to monitor IAA level in Arabidopsis tissues. Using immunocytochemistry and the IAA-driven beta-glucuronidase (GUS) activity of Aux/IAA promoter::GUS constructs to detect IAA distribution, we investigated the role of polar auxin transport in vascular differentiation during leaf development in Arabidopsis. We found that shoot apical cells contain high levels of IAA and that IAA decreases as leaf primordia expand. However, seedlings grown in the presence of IAA transport inhibitors showed very low IAA signal in the shoot apical meristem (SAM) and the youngest pair of leaf primordia. Older leaf primordia accumulate IAA in the leaf tip in the presence or absence of IAA transport inhibition. We propose that the IAA in the SAM and the youngest pair of leaf primordia is transported from outside sources, perhaps the cotyledons, which accumulate more IAA in the presence than in the absence of transport inhibition. The temporal and spatial pattern of IAA localization in the shoot apex indicates a change in IAA source during leaf ontogeny that would influence flow direction and, consequently, the direction of vascular differentiation. The IAA production and transport pattern suggested by our results could explain the venation pattern, and the vascular hypertrophy caused by IAA transport inhibition. An outside IAA source for the SAM supports the notion that IAA transport and procambium differentiation dictate phyllotaxy and organogenesis.