15 research outputs found

    Evolutionary analysis of the osteichthyans secretin GPCR family.

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    <p>The maximum likelihood (ML) optimal tree topology is presented and was constructed with MEGA5. ML bootstrap values higher than 50% are indicated at nodes. To facilitate interpretation, PTHR was used as an outgroup based on the proposed models for secretin GPCR family evolution <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053482#pone.0053482-Cardoso1" target="_blank">[7]</a>. The tree supported the identities of lfGHRHR and xGHRHR<sub>2</sub> as the orthologs of mammalian GHRHR and chicken GHRHR<sub>2</sub> respectively. Accession numbers of the sequences used were listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053482#pone.0053482.s009" target="_blank">Table S2</a>.</p

    Functional characterization of lfGHRHR and xGHRHR<sub>2</sub>.

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    <p>Intracellular cAMP accumulation ([cAMP]<sub>i</sub>) in response to 100 nM of GHRH and related peptides on CHO-K1 cells transfected with (A) lfGHRHR and (D) xGHRHR<sub>2</sub> (*** indicates <i>P</i><0.001, ** indicates <i>P</i><0.01, and * indicates <i>P</i><0.05). Effects of GHRH and related peptides on graded concentrations of peptides on [cAMP]<sub>i</sub> (B) lfGHRHR- and (E) xGHRHR<sub>2</sub>-expressing cells. The intracellular calcium mobilization ([Ca<sup>2+</sup>]<sub>i</sub>) assays of (C) lfGHRHR- and (F) xGHRHR<sub>2</sub>-expressing cells. For [cAMP]<sub>i</sub>, values represent mean ± SEM (n = 4). For ([Ca<sup>2+</sup>]<sub>i</sub>, data were expressed in ΔRFU value (maximum changes in the fluorescence signals from baseline) and converted to percentage of the maximum of xGHRH-induced [Ca<sup>2+</sup>]<sub>i</sub> elevation. Results are expressed as mean ± SEM from at least 10 independent experiments, cell number = 20 to 50. Peptide species: h, human; x, <i>X. laevis</i>, zf, zebrafish <i>D. rerio</i>; gf, goldfish <i>C. auratus</i>.</p

    Gene linkage comparisons of GHRHRs in the Osteichthyes lineage.

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    <p>Genes in vicinity of GHRHR were mapped, and syntenic genes were linked by straight lines. Size of the chromosomal region analyzed was given underneath based on the current edition of Ensembl databases. Syntenic genes encoding other secretin GPCR receptors were drawn in grey boxes, and the conserved flanking genes of GHRHR were drawn in closed boxes. (A) Gene environment of GHRHR in the Sarcopterygii lineage represented by human, mouse, lizard, chicken, frog and coelacanth was compared. Despite the syntenic genomic locations of GHRHR and neighbouring genes from human to avians, GHRHR was not located in frog. (B) Gene environment of GHRHR in the Actinopterygii lineage represented by fugu, tetraodon, stickleback, medaka, and zebrafish. Apart from the less conserved genomic region of zebrafish GHRHR, genes in proximity of other teleost GHRHRs were highly syntenic. However, they displayed an entirely different gene environment when compared to the sarcopterygian GHRHRs. (C) Genomic location analysis of xGHRHR characterized in present work and GHRHR<sub>2</sub> in zebrafish and chicken. Gene synteny could neither be identified inter-species nor between the two GHRHR genes in the same species. The figures were not drawn to scale.</p

    Amino acid alignment of VIP and PACAP-27 peptide sequences and C-terminal processing sites from various chordates.

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    <p>The amino acid alignment of (A) VIP and (B) PACAP-27 were generated using the default settings of Mega 5.0 software. Predicted sequences are denoted by “*” and retrieved from Ensembl or PreEnsembl online genome databases. The tribasic processing sites are boxed. Identical residues to that of human VIP and PACAP are denoted by “.”. Residues indicated important for selectivity and interaction based on mammalian studies are denoted by “–” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044691#pone.0044691-Nicole1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044691#pone.0044691-Chakder1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044691#pone.0044691-Vandermeers1" target="_blank">[46]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044691#pone.0044691-ODonnell1" target="_blank">[61]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044691#pone.0044691-Bourgault2" target="_blank">[72]</a>.</p

    Phylogenetic analysis of vertebrate VIP/PACAP receptors (VPAC<sub>1</sub>, VPAC<sub>2</sub> and PAC<sub>1</sub>).

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    <p>The tree was constructed based on the PAM Matrix (Dayhoff) model by Maximum-Likelihood method, MEGA 5.0 software. The monophyletic groups are indicated on the right. Cloned receptor sequences from this study are boldfaced. Predicted sequences from the Ensembl genome database are denoted by “*”. The numbers above each branch indicate the percentage of bootstrap replications in which that branch was found based on 500 replications. Glucagon, GLP-1, GLP-2 and GIP receptor sequences were used as the outgroup.</p

    An evolutionary scheme of the VIP/PACAP ligands and receptors in vertebrates.

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    <p>The boxes denote exons for the ligands and genes for the receptors. Unknown or unclear events are denoted by dotted lines or question marks. The phylogenetic timeline for the events are not to scale.</p

    Phylogenetic analysis of PHI/VIP and PRP/PACAP hormone precursors.

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    <p>The tree was constructed by the PAM Matrix (Dayhoff) model by Maximum-Likelihood method, MEGA 5.0 software. Predicted sequences from the Ensembl genome database are denoted by “*”. The deduced sequences from this study are boldfaced. The numbers above each branch indicate the percentage of bootstrap replications in which that branch was found based on 500 replications. Proglucagon sequences were used as the outgroup.</p

    ERRα upregulates SCT expression in N-42 cells.

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    <p>(A) The 5′ upstream sequence of the <i>SCT</i> gene contains a ERE-half site. The nucleotide sequences (180 bp) upstream of the start codon of the mouse, human and rat <i>SCT</i> gene are shown. The first nucleotide of the start codon (ATG) is assigned as +1. The consensus sequences of the putative ERE-half site are highlighted in red and underlined. The mutation sites are indicated by the boxes (B) Effects of over-expressing ERRs in N-42 on the mouse <i>SCT</i> promoter. mSCTP (0.5 µg) and various amounts of the 3 isoforms of ERR/pcDNA3 (0, 0.5, 1.0 and 2.0 µg) were cotransfected into N-42 cells. Total DNA was adjusted to 2.5 µg by pKS<sup>+</sup>. *p<0.05; **p<0.001, compared with mSCTP (0.5 µg). (C) Effects of over-expressing ERRα on the mouse SCT mRNA levels in N-42 cells. The mRNA levels of mouse <i>SCT</i> measured by real-time PCR were normalized with mouse <i>GAPDH</i> levels. *p<0.05; **p<0.001, compared with control (ERRα – 0 µg). (D–E) Effects of endogenous silencing of mouse ERRα on (D) mouse SCT promoter and (E) mRNA levels in N-42 cells. The mSCTP (1.0 µg) was co-transfected with various amounts of siERRα-1 and siERRα-2 (1.0 and 2.0 µg), pSilencer or siControl into N-42 cells. The mRNA levels of mouse <i>SCT</i> measured by real-time PCR were normalized with mouse <i>GAPDH</i> levels. Data represent the mean ± SEM of three experiments performed in duplicates. *p<0.05; **p<0.001, compared with mSCTP – 1.0 µg. (B) *p<0.05; **p<0.001, compared with control (pSilencer – 2.0 µg). (F) Western blot analysis of ERRα protein in N-42 cells transfected with (1) pSilencer (2.0 µg), (2) siERRα-S1, (3) siERRα-S2 and (4) siControl (2.0 µg). The GAPDH western blot was used as the loading control. (G) Mutation analysis of ERE-half site. Four mutants (M1–M4, 0.5 µg) were cotransfected with pKS<sup>+</sup> or ERRα expression vector (2.0 µg). ⧫p<0.05; ERRα cotransfected promoter compared with the same construct that transfected with pKS<sup>+</sup> (0.5 µg).</p

    Effects of water deprivation, saline dinking and central ANGII administration on mouse <i>ERRα</i> expressions in mouse brain.

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    <p>Mice were water deprived, provided with hypertonic saline (2%) for 1 or 5 d, or centrally injected with ANGII peptide. The mRNA levels of mouse <i>ERR</i>α in (A) the whole mouse brain or (C) the isolated osmosentitive brain regions SFO, OVLT, MnPO and PVN, were measured by real-time PCR were normalized with mouse <i>GAPDH</i> levels. Data are expressed as the mean ± SEM (n = 10/group). *p<0.05; **p<0.001, compared with control. (B) Immunohistochemical staining showing ERRα immunoreactivities in the SFO, MnPO, OVLT and PVN of the WT mouse brain. Negative control was done by using 1× PBS instead of the primary anti-ERRα antibody. Bars, 6 µm.</p
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