13 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

    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

    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

    Transcript expression profile of agnathan VPAC receptors and PACAP.

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    <p>Tissue distribution patterns of (A) hfVPACa and (B) hfVPACb; a relative abundance of 1 was set arbitrarily for the mRNA expressed in the brain. (C) The expression level of hfVPAC receptors in the hagfish brain was calculated from respective standard curves. (D) Tissue distribution pattern of jlpPACAP; a relative abundance of 1 was set arbitrarily for the mRNA expressed in the brain. Data are expressed as the mean ± S.E.M. of four experiments performed in duplicates.</p

    <i>In situ</i> hybridization analysis of hfVPAC mRNAs in the brain of <i>E.burgeri</i>.

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    <p>(A) Summary of relative abundance of hfVPACa and hfVPACb receptor expressions in various brain regions: High (+++), moderate (++), low (+), very low (–).The images and schematic diagrams show the distribution of hagfish mRNA in collateral sections of several brain regions (B–G). From left to right of the schematic diagrams, hfVPACa and hfVPACb images inclusive of their negative controls using a 1∶30 ratio of DIG-labeled anti-sense probe and unlabeled anti-sense probe and positive signals using specific complementary probes are shown. Fast green was used for counterstaining. Scale bars, 0.25mm for B and D; 0.15mm for C, E–G.</p

    Functional characterization of hfVPACa and hfVPACb.

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    <p>Intracellular cAMP accumulation in response to 100 nM vertebrate superfamily peptides on COS-7 cells transiently transfected with (A) hfVPACa and (B) hfVPACb. Peptide species: slp, sea lamprey; gf, goldfish; h, human; o, ovine; zf, zebrafish; x, <i>Xenopus</i>; ct, catfish and cp, carp. Data represent the mean ± S.E.M. of at least 4 experiments performed in duplicates, p<0.01 is denoted by “*”. Effects of graded concentrations of (C) agnathan and (D) mammalian VIP and PACAP peptides on COS-7 cells transiently expressing hfVPACa. Data are expressed as the mean ± S.E.M. of at least 6 experiments performed in duplicates. Measurement of intracellular calcium elevation in CHO-K1 cells transiently expressing (E) hfVPACa and (F) hfVPACb in response to graded concentrations of sea lamprey PACAP. Data are expressed as the mean ± S.E.M. of at least 4 experiments. RFU, relative fluorescence units. (G) Shown are representative of confocal fluorescence images of CHO-K1 cells expressing (i) hfVPACa-pEYFP, (ii) hfVPACb-pEYFP and (iii) pEYFP-N1.</p
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