Cloning characterisation and expression of a G-protein-couple receptor from Lymnaea stagnalis and identification of a leucokinin-like peptide PSFHSWSamide as its endogenous ligand

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The Lymnaea Cardioexcitatory Peptide (LyCEP) Receptor: A G-Protein–Coupled Receptor for a Novel Member of the RFamide Neuropeptide Family

A novel G-protein–coupled receptor (GRL106) resembling neuropeptide Y and tachykinin receptors was cloned from the molluscLymnaea stagnalis. Application of a peptide extract from the Lymnaea brain to Xenopus oocytes expressing GRL106 activated a calcium-dependent chloride channel. Using this response as a bioassay, we purified the ligand for GRL106,Lymnaea cardioexcitatory peptide (LyCEP), an RFamide-type decapeptide (TPHWRPQGRF-NH2) displaying significant similarity to the Achatina cardioexcitatory peptide (ACEP-1) as well as to the recently identified family of mammalian prolactin-releasing peptides. In the Lymnaeabrain, the cells that produce egg-laying hormone are the predominant site of GRL106 gene expression and appear to be innervated by LyCEP-containing fibers. Indeed, LyCEP application transiently hyperpolarizes isolated egg-laying hormone cells. In theLymnaea pericardium, LyCEP-containing fibers end blindly at the pericardial lumen, and the heart is stimulated by LyCEPin vitro. These data confirm that LyCEP is an RFamide ligand for GRL10

Service Creation in the SPICE Service Platform

Convergence is driving the uptake of new technologies on networking and service layers. The emerging new network infrastructure centered on 3GPP and IMS will have a horizontal layering with a good separation of control functions, media handling, networking issues, and service layers. This is a first step for fast service creations. In addition, we need a good tool chain for service creation as well as a rich execution environment. This paper describes the approach of the IST project SPICE with respect to Service Creation Environment and Service Execution Environment

An adeno-associated viral vector transduces the rat hypothalamus and amygdala more efficient than a lentiviral vector

<p>Abstract</p> <p>Background</p> <p>This study compared the transduction efficiencies of an adeno-associated viral (AAV) vector, which was pseudotyped with an AAV1 capsid and encoded the green fluorescent protein (GFP), with a lentiviral (LV) vector, which was pseudotyped with a VSV-G envelop and encoded the discosoma red fluorescent protein (dsRed), to investigate which viral vector transduced the lateral hypothalamus or the amygdala more efficiently. The LV-dsRed and AAV1-GFP vector were mixed and injected into the lateral hypothalamus or into the amygdala of adult rats. The titers that were injected were 1 × 10⁸or 1 × 10⁹genomic copies of AAV1-GFP and 1 × 10⁵transducing units of LV-dsRed.</p> <p>Results</p> <p>Immunostaining for GFP and dsRed showed that AAV1-GFP transduced significantly more cells than LV-dsRed in both the lateral hypothalamus and the amygdala. In addition, the number of LV particles that were injected can not easily be increased, while the number of AAV1 particles can be increased easily with a factor 100 to 1000. Both viral vectors appear to predominantly transduce neurons.</p> <p>Conclusions</p> <p>This study showed that AAV1 vectors are better tools to overexpress or knockdown genes in the lateral hypothalamus and amygdala of adult rats, since more cells can be transduced with AAV1 than with LV vectors and the titer of AAV1 vectors can easily be increased to transduce the area of interest.</p

Lentivirus-mediated transgene delivery to the hippocampus reveals sub-field specific differences in expression

<p>Abstract</p> <p>Background</p> <p>In the adult hippocampus, the granule cell layer of the dentate gyrus is a heterogeneous structure formed by neurons of different ages, morphologies and electrophysiological properties. Retroviral vectors have been extensively used to transduce cells of the granule cell layer and study their inherent properties in an intact brain environment. In addition, lentivirus-based vectors have been used to deliver transgenes to replicative and non-replicative cells as well, such as post mitotic neurons of the CNS. However, only few studies have been dedicated to address the applicability of these widespread used vectors to hippocampal cells in vivo. Therefore, the aim of this study was to extensively characterize the cell types that are effectively transduced in vivo by VSVg-pseudotyped lentivirus-based vectors in the hippocampus dentate gyrus.</p> <p>Results</p> <p>In the present study we used Vesicular Stomatitis Virus G glycoprotein-pseudotyped lentivirual vectors to express EGFP from three different promoters in the mouse hippocampus. In contrast to lentiviral transduction of pyramidal cells in CA1, we identified sub-region specific differences in transgene expression in the granule cell layer of the dentate gyrus. Furthermore, we characterized the cell types transduced by these lentiviral vectors, showing that they target primarily neuronal progenitor cells and immature neurons present in the sub-granular zone and more immature layers of the granule cell layer.</p> <p>Conclusion</p> <p>Our observations suggest the existence of intrinsic differences in the permissiveness to lentiviral transduction among various hippocampal cell types. In particular, we show for the first time that mature neurons of the granule cell layer do not express lentivirus-delivered transgenes, despite successful expression in other hippocampal cell types. Therefore, amongst hippocampal granule cells, only adult-generated neurons are target for lentivirus-mediated transgene delivery. These properties make lentiviral vectors excellent systems for overexpression or knockdown of genes in neuronal progenitor cells, immature neurons and adult-generated neurons of the mouse hippocampus in vivo.</p

Detection of Molecular Paths Associated with Insulitis and Type 1 Diabetes in Non-Obese Diabetic Mouse

Recent clinical evidence suggests important role of lipid and amino acid metabolism in early pre-autoimmune stages of type 1 diabetes pathogenesis. We study the molecular paths associated with the incidence of insulitis and type 1 diabetes in the Non-Obese Diabetic (NOD) mouse model using available gene expression data from the pancreatic tissue from young pre-diabetic mice. We apply a graph-theoretic approach by using a modified color coding algorithm to detect optimal molecular paths associated with specific phenotypes in an integrated biological network encompassing heterogeneous interaction data types. In agreement with our recent clinical findings, we identified a path downregulated in early insulitis involving dihydroxyacetone phosphate acyltransferase (DHAPAT), a key regulator of ether phospholipid synthesis. The pathway involving serine/threonine-protein phosphatase (PP2A), an upstream regulator of lipid metabolism and insulin secretion, was found upregulated in early insulitis. Our findings provide further evidence for an important role of lipid metabolism in early stages of type 1 diabetes pathogenesis, as well as suggest that such dysregulation of lipids and related increased oxidative stress can be tracked to beta cells

Gene expression differences between stroke-associated and asymptomatic carotid plaques

Atherosclerotic carotid stenosis is an important risk factor for stroke. Carotid plaques (CPs) causing stroke may present a distinct type of molecular pathology compared with transient ischemic attack (TIA)-associated or asymptomatic plaques. We compared the gene expression profiles of CPs from stroke patients (n = 12) and asymptomatic patients (n = 9), both with similar risk factors and severity of carotid stenosis (>70%). Sixty probes showed over 1.5-fold expression difference at 5% false discovery rate. Functional clustering showed enrichment of genes in 51 GO categories and seven pathways, the most significant of which relate to extracellular-matrix interaction, PPAR gamma signaling, scavanger receptor activity, and lysosomal activity. Differential expression of ten genes was confirmed in an extended replication group (n = 43), where the most significant expression differences were found in CD36 (2.1-fold change, p = 0.005), CD163 (1.7-fold change, p = 0.007) and FABP4 (2.2-fold change, p = 0.015). These include four genes not previously linked to plaque destabilization: GLUL (2.2-fold change, p = 0.016), FUCA1 (2.2-fold change, p = 0.025), IL1RN (1.6-fold change, p = 0.034), and S100A8 (2.5-fold change, p = 0.047). Strong correlations were found to plaque ulceration, plaque hemorrhage, and markers of apoptosis and proliferation (activated caspase 3, TUNEL, and Ki67). Protein expression of these genes was confirmed by immunohistochemistry and was found in the atheromatous areas of CPs critical for plaque destabilization. This study presents a comprehensive transcriptional analysis of stroke-associated CPs and demonstrates a significant transcriptome difference between stroke-associated and asymptomatic CPs. Follow-up studies on the identified genes are needed to define whether they could be used as biomarkers of symptomatic CPs or have a role in plaque destabilization