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      Transcriptomics and proteomics analyses of the PACAP38 influenced ischemic brain in permanent middle cerebral artery occlusion model mice

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          Abstract

          Introduction

          The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is considered to be a potential therapeutic agent for prevention of cerebral ischemia. Ischemia is a most common cause of death after heart attack and cancer causing major negative social and economic consequences. This study was designed to investigate the effect of PACAP38 injection intracerebroventrically in a mouse model of permanent middle cerebral artery occlusion (PMCAO) along with corresponding SHAM control that used 0.9% saline injection.

          Methods

          Ischemic and non-ischemic brain tissues were sampled at 6 and 24 hours post-treatment. Following behavioral analyses to confirm whether the ischemia has occurred, we investigated the genome-wide changes in gene and protein expression using DNA microarray chip (4x44K, Agilent) and two-dimensional gel electrophoresis (2-DGE) coupled with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), respectively. Western blotting and immunofluorescent staining were also used to further examine the identified protein factor.

          Results

          Our results revealed numerous changes in the transcriptome of ischemic hemisphere (ipsilateral) treated with PACAP38 compared to the saline-injected SHAM control hemisphere (contralateral). Previously known (such as the interleukin family) and novel ( Gabra6, Crtam) genes were identified under PACAP influence. In parallel, 2-DGE analysis revealed a highly expressed protein spot in the ischemic hemisphere that was identified as dihydropyrimidinase-related protein 2 (DPYL2). The DPYL2, also known as Crmp2, is a marker for the axonal growth and nerve development. Interestingly, PACAP treatment slightly increased its abundance (by 2-DGE and immunostaining) at 6 h but not at 24 h in the ischemic hemisphere, suggesting PACAP activates neuronal defense mechanism early on.

          Conclusions

          This study provides a detailed inventory of PACAP influenced gene expressions and protein targets in mice ischemic brain, and suggests new targets for thereaupetic interventions.

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          Most cited references37

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          Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells.

          A novel neuropeptide which stimulates adenylate cyclase in rat anterior pituitary cell cultures was isolated from ovine hypothalamic tissues. Its amino acid sequence was revealed as: His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln- Met-Ala- Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-Asn-Lys - NH2. The N-terminal sequence shows 68% homology with vasoactive intestinal polypeptide (VIP) but its adenylate cyclase stimulating activity was at least 1000 times greater than that of VIP. It increased release of growth hormone (GH), prolactin (PRL), corticotropin (ACTH) and luteinizing hormone (LH) from superfused rat pituitary cells at as small a dose as 10(-10)M (GH, PRL, ACTH) or 10(-9)M (LH). Whether these hypophysiotropic effects are the primary actions of the peptide or what physiological action in the pituitary is linked with the stimulation of adenylate cyclase by this peptide remains to be determined.
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            Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38).

            A novel neuropeptide with 38 residues (PACAP38) was isolated from ovine hypothalamic tissues using the pituitary adenylate cyclase activation in rat pituitary cell cultures as a parameter of the biological activity (Miyata et al, Biochem. Biophys. Res. Commun. 164, 567-574, 1989). From the side fractions obtained during the purification of PACAP38, a shorter form peptide with 27 residues corresponding to the N-terminal 27 amino acids of PACAP38 and amidated C-terminus was isolated and named as PACAP27. Synthetic PACAP27 showed a biological activity of adenylate cyclase stimulation comparable to PACAP38. Moreover PACAP27 which shows a considerable homology with vasoactive intestinal polypeptide (VIP) demonstrated a similar vasodepressor activity as VIP, but the adenylate cyclase stimulating activity was about 1000 times greater than VIP.
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              • Article: not found

              Maintenance and modulation of T cell polarity.

              As T cells move through the lymphatics and tissues, chemokine receptors, adhesion molecules, costimulatory molecules and antigen receptors engage their ligands in the microenvironment and contribute to establishing and maintaining cell polarity. Cytoskeletal assemblies, surface proteins and vesicle traffic are essential components of polarity and probably stabilize the activity of lymphocytes that must negotiate their 'noisy' environment. An additional component of polarity is a family of polarity proteins in T cells that includes Dlg, Scrib and Lgl, as well as a complex of partitioning-defective proteins. Ultimately, the strength of a T cell response may rely on correct T cell polarization. Therefore, loss of polarity regulators or guidance cues may interfere with T cell activation.
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                Author and article information

                Journal
                J Neuroinflammation
                J Neuroinflammation
                Journal of Neuroinflammation
                BioMed Central
                1742-2094
                2012
                23 November 2012
                : 9
                : 256
                Affiliations
                [1 ]Department of Forensic Medicine and Molecular Pathology, School of Medicine, Kyoto University, Kyoto, 606-8315, Japan
                [2 ]Department of Anatomy I, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan
                [3 ]Department of Center for Biotechnology, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan
                [4 ]Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, 305-8572, Japan
                Article
                1742-2094-9-256
                10.1186/1742-2094-9-256
                3526409
                23176072
                699e0856-a50b-4c8e-a701-496ea42e74d1
                Copyright ©2012 Hori et al.; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 20 June 2012
                : 19 October 2012
                Categories
                Research

                Neurosciences
                neuroprotection,gabra6,crmp2,crtam,dna microarray,pacap,il6,ischemia
                Neurosciences
                neuroprotection, gabra6, crmp2, crtam, dna microarray, pacap, il6, ischemia

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