Protein abundance of Rhesus glycoproteins in the gills of <i>Protopterus annectens</i>.

<p>Protein abundance of (a) Rhesus blood group-associated glycoprotein (Rhag), (b) Rhesus family B glycoprotein (Rhbg) and (c) Rhesus family C glycoprotein (Rhcg) in the gills of <i>Protopterus annectens</i> kept in fresh water on day 0 (FW; control), or after 6 days (d; the induction phase) or 6 months (mon; the maintenance phase) of aestivation, or after 1 or 3 d of arousal (Ar; the arousal phase) from 6 mon of aestivation. (i) Examples of immunoblot of Rhag, Rhbg or Rhcg. (ii) The protein abundance of Rhag, Rhbg or Rhcg expressed as arbitrary densitometric units per 100 μg, 100 μg or 50 μg protein, respectively. Results represent mean ± S.E.M. (<i>N</i> = 4). Means not sharing the same letter are significantly different (<i>P</i><0.05).</p

Molecular characterization of Rhesus family C glycoprotein (Rhcg) from the gills of <i>Protopterus annectens</i>.

<p>A multiple amino acid alignment of Rhcg from <i>Protopterus annectens</i> with <i>Callorhinchus milii</i> Rhcg (AFO96383.1), <i>Anabas testudineus</i> Rhcg1 (AIC81183.1) and Rhcg2 (AIC81184.1), <i>Xenopus laevis</i> Rhcg (NP_001088553.1), <i>Mus musculus</i> RhCG (AAF19373.1), <i>Homo sapiens</i> RhCG (AAF19372.1) and <i>Escherichia coli</i> ammonia transporter (AmtB; NP_414985.1). Identical amino acid residues are indicated by asterisks, strongly similar amino acids are indicated by colons and weakly similar amino acids are indicated by periods. Residues involved in NH₄⁺ binding and deprotonation of NH₄⁺ for NH₃ conduction are indicated by red and blue triangles, respectively. The phenylalanine gate is indicated by open diamonds. The π cation binding sites of <i>E</i>. <i>coli</i> AmtB are indicated by “π”. Potential <i>N</i>-glycosylation and phosphorylation sites are indicated by open and shaded arrows, respectively. The predicted transmembrane domains (TM1‒12) of Rhcg of <i>P</i>. <i>annectens</i> are indicated by open boxes and were predicted using MEMSATS and MEMSAT-SVA provided by PSIPRED protein structure prediction server.</p

Validation of anti-Rhesus glycoproteins antibodies from <i>Protopterus annectens</i>.

<p>Immunoblots incubated with (a) anti-Rhesus blood group-associated glycoprotein (Rhag), (b) anti-Rhesus family B glycoprotein (Rhbg) and (c) anti-Rhesus family C glycoprotein (Rhcg) antibodies. Recombinant proteins of Rhag, Rhbg, Rhcg and the protein from the gills of <i>Protopterus annectens</i> kept in fresh water on day 0 (FW) were loaded for gel separation.</p

Molecular characterization of aquaporin 1aa (Aqp1aa) from the gills of <i>Anabas testudineus</i>.

<p>Multiple amino acid alignment of Aqp1aa from the gills of <i>A. testudineus</i>, with five other known Aqp1/Aqp1a from <i>Sparus aurata</i> (seabream Aqp1a; ABM26907.1), <i>Takifugu obscurus</i> (pufferfish Aqp1; ADG86337.1), <i>Protopterus annectens</i> (lungfish Aqp1; BAI48049.1), <i>Xenopus laevis</i> (frog AQP1; NP_001085391.1), and <i>Homo sapiens</i> (human AQP1; CAQ51480.2). Identical amino acids are indicated by shaded residues. Substrate discrimination sites at the aromatic/arginine (ar/R) constriction are indicated with arrows. Central pore-lining residues are indicated with open triangles. The binding site for AQP1-inhibitor HgCl₂ is indicated by an asterisk. The Asn-Pro-Ala (NPA) motifs are underlined. P denotes phosphorylation sites and N denotes <i>N</i>-glycosylation sites. The predicted transmembrane domains (TM) are underlined. The transmembrane domains of Aqp1 of <i>A. testudineus</i> were predicted using MEMSATS & MEMSAT-SVA provided by PSIPRED protein structure prediction server.</p

<i>Aquaporin 1aa</i> (<i>aqp1aa</i>) mRNA expression in <i>Anabas testudineus</i> kept in freshwater or exposed to ammonia.

<p>Absolute quantification (×10² copies of transcript per ng cDNA; <i>N</i> = 5) of <i>aqp1aa</i> mRNA expression from (A) the gills, (B) the anterior gut, (C) the posterior gut, (D) the kidney and (E) the skin of <i>A. testudineus</i> kept in freshwater (FW) or exposed to 100 mmol l⁻¹ NH₄Cl for 1 or 6 days. Results represent means ± S.E.M. Means not sharing the same letter are significantly different (<i>P</i><0.05).</p

Tissue expression of <i>aquaporin 1aa</i> (<i>aqp1aa</i>) of <i>Anabas testudineus</i> in freshwater.

<p>Tissue expression of <i>aqp1aa</i> was examined in gills, accessory breathing organs (ABO), brain, liver, kidney, anterior gut (AG), posterior gut (PG) and skin of <i>A. testudineus</i> kept in freshwater.</p

The percentage sequence identity, arranged in a descending order of similarity, between the deduced amino acid sequence of aquaporin 1aa (Aqp1aa) of <i>Anabas testudineus</i> and Aqp sequences of other fish species obtained from GenBank (accession numbers in brackets).

<p>The percentage sequence identity, arranged in a descending order of similarity, between the deduced amino acid sequence of aquaporin 1aa (Aqp1aa) of <i>Anabas testudineus</i> and Aqp sequences of other fish species obtained from GenBank (accession numbers in brackets).</p

Phylogenetic analysis of aquaporin 1aa (Aqp1aa) of <i>Anabas testudineus</i>.

<p>The phylogenetic tree illustrates the relationship between Aqp1aa of <i>A. testudineus</i> and AQP1/Aqp1 of selected vertebrates.</p

<i>Aquaporin 1aa</i> (<i>aqp1aa</i>) mRNA expression in <i>Anabas testudineus</i> kept in freshwater or seawater.

<p>Absolute quantification (×10² copies of transcript per ng cDNA; <i>N</i> = 5) of <i>aqp1aa</i> mRNA expression from (A) the gills, (B) the anterior gut, (C) the posterior gut, (D) the kidney and (E) the skin of <i>A. testudineus</i> kept in freshwater (FW) or exposed to seawater (SW; salinity 30) for 1 or 6 days after a progressive increase in salinity. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061163#s3" target="_blank">Results</a> represent means ± S.E.M. Means not sharing the same letter are significantly different (<i>P</i><0.05).</p

<i>Aquaporin 1aa</i> (<i>aqp1aa</i>) mRNA expression in <i>Anabas testudineus</i> kept in freshwater or exposed to terrestrial conditions.

<p>Absolute quantification (×10² copies of transcript per ng cDNA; <i>N</i> = 5) of <i>aqp1aa</i> mRNA expression from (A) the gills, (B) the anterior gut, (C) the posterior gut, (D) the kidney and (E) the skin of <i>A. testudineus</i> kept in freshwater (FW) or exposed to terrestrial conditions for 1 day. Results represent means ± S.E.M. ^*Significantly different from the FW control (<i>P</i><0.05).</p