Toad atrial natriuretic peptide : cDNA cloning and functional analysis in isolated perfused kidneys

Complementary DNA (cDNA) encoding Bufo marinus (toad) preproatrial natriuretic peptide (preproANP) was isolated by reverse-transcription polymerase chain reaction. Sequence analysis of toad preproANP cDNA revealed an open reading frame of 150 amino acid residues, which shared 72% and 66% identity with Rana catesbeiana and Xenopus laevis preproANP, respectively. The deduced amino acid sequence of toad ANP that corresponded to ANP 1–24 of R. catesbeiana and Rana ridibunda was identical, but it differed by four residues from that of X. laevis. ANP mRNA transcripts were also shown to be expressed in the toad kidney. Subsequently, the effect of frog ANP (1–24) on renal function in toad was examined using a perfused kidney preparation. The arterial infusion of frog ANP caused a dose-dependent decrease in the arterial perfusion pressure that was associated with an increase in the glomerular filtration rate (GFR) and a renal natriuresis and diuresis. The renal natriuresis and diuresis resulted predominantly from an increased GFR rather than from direct tubular effects. This study demonstrates that ANP can regulate renal function, which suggests it may be involved in overall fluid volume regulation.

Individual variation in thermal performance curves: swimming burst speed and jumping endurance in wild-caught tropical clawed frogs

The importance of studying individual variation in locomotor performance has long been recognized as it may determine the ability of an organism to escape from predators, catch prey or disperse. In ectotherms, locomotor performance is highly influenced by ambient temperature (Ta), yet several studies have showed that individual differences are usually retained across a Ta gradient. Less is known, however, about individual differences in thermal sensitivity of performance, despite the fact that it could represent adaptive sources of phenotypic variation and/or additional substrate for selection to act upon. We quantified swimming and jumping performance in 18 wild-caught tropical clawed frogs (Xenopus tropicalis) across a Ta gradient. Maximum swimming velocity and acceleration were not repeatable and individuals did not differ in how their swimming performance varied across Ta. By contrast, time and distance jumped until exhaustion were repeatable across the Ta gradient, indicating that individuals that perform best at a given Ta also perform best at another Ta. Moreover, thermal sensitivity of jumping endurance significantly differed among individuals, with individuals of high performance at low Ta displaying the highest sensitivity to Ta. Individual differences in terrestrial performance increased with decreasing Ta, which is opposite to results obtained in lizards at the inter-specific and among-individual levels. To verify the generality of these patterns, we need more studies on individual variation in thermal reaction norms for locomotor performance in lizards and frogs.

Regulation of HbPIP2;3, a latex-abundant water transporter, is associated with latex dilution and yield in the rubber tree (Hevea brasiliensis Muell. Arg.)

Rubber tree (Hevea brasiliensis) latex, the source of natural rubber, is synthesised in the cytoplasm of laticifers. Efficient water inflow into laticifers is crucial for latex flow and production since it is the determinant of the total solid content of latex and its fluidity after tapping. As the mature laticifer vessel rings are devoid of plasmodesmata, water exchange between laticifers and surrounding cells is believed to be governed by plasma membrane intrinsic proteins (PIPs). To identify the most important PIP aquaporin in the water balance of laticifers, the transcriptional profiles of ten-latex-expressed PIPs were analysed. One of the most abundant transcripts, designated HbPIP2;3, was characterised in this study. When tested in Xenopus laevis oocytes HbPIP2;3 showed a high efficiency in increasing plasmalemma water conductance. Expression analysis indicated that the HbPIP2;3 gene was preferentially expressed in latex, and the transcripts were up-regulated by both wounding and exogenously applied Ethrel (a commonly-used ethylene releaser). Although regular tapping up-regulated the expression of HbPIP2;3 during the first few tappings of the virginal rubber trees, the transcriptional kinetics of HbPIP2;3 to Ethrel stimulation in the regularly tapped tree exhibited a similar pattern to that of the previously reported HbPIP2;1 in the virginal rubber trees. Furthermore, the mRNA level of HbPIP2;3 was associated with clonal yield potential and the Ethrel stimulation response. Together, these results have revealed the central regulatory role of HbPIP2;3 in laticifer water balance and ethylene stimulation of latex production in Hevea.

A glycosyl hydrolase family 16 gene is responsible for the endogenous production of β-1,3-glucanases within decapod crustaceans

To identify the gene responsible for the production of a β-1,3-glucanase (laminarinase) within crustacea, a glycosyl hydrolase family 16 (GHF16) gene was sequenced from the midgut glands of the gecarcinid land crab, Gecarcoidea natalis and the freshwater crayfish, Cherax destructor. An open reading frame of 1098bp for G. natalis and 1095bp for C. destructor was sequenced from cDNA. For G. natalis and C. destructor respectively, this encoded putative proteins of 365 and 364 amino acids with molecular masses of 41.4 and 41.5kDa. mRNA for an identical GHF16 protein was also expressed in the haemolymph of C. destructor. These putative proteins contained binding and catalytic domains that are characteristic of a β-1,3-glucanase from glycosyl hydrolase family 16. The amino acid sequences of two short 8-9 amino acid residue peptides from a previously purified β-1,3-glucanase from G. natalis matched exactly that of the putative protein sequence. This plus the molecular masses of the putative proteins matching that of the purified proteins strongly suggests that the sequences obtained encode for a catalytically active β-1,3-glucanase. A glycosyl hydrolase family 16 cDNA was also partially sequenced from the midgut glands of other amphibious (Mictyrisplatycheles and Paragrapsus laevis) and terrestrial decapod species (Coenobita rugosus, Coenobita perlatus, Coenobita brevimanus and Birgus latro) to confirm that the gene is widely expressed within this group. There are three possible hypothesised functions and thus evolutionary routes for the β-1,3-glucanase: 1) a digestive enzyme which hydrolyses β-1,3-glucans, 2) an enzyme which cleaves β-1,3-glycosidic bonds within cell walls to release cell contents or 3) an immune protein which can hydrolyse the cell walls of potentially pathogenic micro-organisms.

Characterisation and vascular expression of nitric oxide synthase 3 in amphibians

In mammals, nitric oxide (NO) produced by nitric oxide synthase 3 (NOS3) localised in vascular endothelial cells is an important vasodilator but the presence of NOS3 in the endothelium of amphibians has been concluded to be absent, based on physiological studies. In this study, a nos3 cDNA was sequenced from the toad, Rhinella marina. The open reading frame of R. marina nos3 encoded an 1170 amino acid protein that showed 81 % sequence identity to the recently cloned Xenopus tropicalis nos3. Rhinella marina nos3 mRNA was expressed in a range of tissues and in the dorsal aorta and pulmonary, mesenteric, iliac and gastrocnemius arteries. Furthermore, nos3 mRNA was expressed in the aorta of Xenopus laevis and X. tropicalis. Quantitative real-time PCR showed that removal of the endothelium of the lateral aorta of R. marina significantly reduced the expression of nos3 mRNA compared to control aorta with the endothelium intact. However, in situ hybridisation was not able to detect any nos3 mRNA in the dorsal aorta of R. marina. Immunohistochemistry using a homologous R. marina NOS3 antibody showed immunoreactivity (IR) within the basal region of many endothelial cells of the dorsal aorta and iliac artery. NOS3-IR was also observed in the proximal tubules and collecting ducts of the kidney but not within the capillaries of the glomeruli. This is the first study to demonstrate that vascular endothelial cells of an amphibian express NOS3.