Frog albumin is expressed in skin and characterized as a novel potent trypsin inhibitor

A novel potent trypsin inhibitor was purified and characterized from frog Bombina maxima skin. A full-length cDNA encoding the protein was obtained from a cDNA library constructed from the skin. Sequence analysis established that the protein actually comprises three conserved albumin domains. B. maxima serum albumin was subsequently purified, and its coding cDNA was further obtained by PCR-based cloning from the frog liver. Only two amino acid variations were found in the albumin sequences from the skin and the serum. However, the skin protein is distinct from the serum protein by binding of a haem b (0.95 mol/mol protein). Different from bovine serum albumin, B. maxima albumin potently inhibited trypsin. It bound tightly with trypsin in a 1: 1 molar ratio. The equilibrium dissociation constants (K-D) obtained for the skin and the serum proteins were 1.92 x 10(-9) M and 1.55 x 10(-9) M, respectively. B. maxima albumin formed a noncovalent complex with trypsin through an exposed loop formed by a disulfide bond (Cys(53)-Cys(62)), which comprises the scissile bond Arg(58)(P-1)-His(59)(P-1'). No inhibitory effects on thrombin, chymotrypsin, elastase, and subtilisin were observed under the assay conditions. Immunohistochemical study showed that B. maxima albumin is widely distributed around the membranes of epithelial layer cells and within the stratum spongiosum of dermis in the skin, suggesting that it plays important roles in skin physiological functions, such as water economy, metabolite exchange, and osmoregulation.

Effects of salinity on food intake, growth, and survival of pufferfish (Fugu obscurus)

The food intake, growth, food conversion ratio and survival of yearling pufferfish, Fugu obscurus Abe, were investigated under different water salinity conditions over a 54-day period. Within the salinity regimes of 0 (freshwater), 8, 18, and 35parts per thousand, the food intake levels were 0.97%, 1.43%, 1.19% and 1.01%, respectively; food conversion ratios were 1.31, 1.93, 1.61 and 1.36, respectively; and specific growth rates were 0.41%, 1.15%, 0.84%, and 0.35%, respectively. The three data series were reduced with increasing salinity. However, the survival rates did not show the same tendencies, which were 80%, 100%, 100%, and 67%, respectively. There were significant differences among the treatments. In conclusion, the yearling pufferfish optimum culture salinity condition was about 8parts per thousand.

Osmoregulatory actions of growth hormone in juvenile tilapia (Oreochromis niloticus)

Growth hormone (GH) effectively promotes seawater (SW) adaptation in salmonids, but little is known of its effect in tilapias. Experiments were performed to investigate the effects of recombinant eel GH (reGH) on osmoregulatory actions and ultrastructural features of gill chloride cells in juvenile tilapia, Oreochromis niloticus. Tilapia showed a markedly improved SW survival, when directly transferred from freshwater (FW) to 62.5% SW 24h after a single reGH injection (0.25 or 2.5 mu g g(-1)) or 3 reGH injections (0.25 mu g g(-1) every other day). Plasma Na+ and Mg2+ levels were significantly reduced by reGH (0.25 and 2.5 mu g g(-1)) compared with saline injections; Ca2+ concentrations were reduced significantly by high dose of reGH (2.5 mu g g(-1)) after SW transfer. However, fish failed to survive more than 24h when directly transferred to 70 % SW, although the fish treated with reGH could survive longer than the controls. When examined by electron microscopy, the chloride cells were identified as mitochondrion-rich and an extensive tubular system was induced by GH treatment. The results of the present study suggest that, similar to its effect on salmonids, GH also exerts acute osmoregulatory actions and enhances SW adaptation in juvenile tilapia. GH also stimulates the differentiation of chloride cells toward SW adaptation.