Molecular phylogenetic relationship of Tetraodon pufferfish based on mitochondrial DNA analysis

Pufferfishes belonging to the genus Tetraodon form one of the largest groups in the family Tetraodontidae. This group consists of more than 20 species distributed across freshwater, as well as brackish and coastal waters, of Africa and Southeast Asia. However, their phylogenetic and evolutionary relationships are still ambiguous. In the present study, mitochondrial genes encoding 16S rRNA and cytochrome b were sequenced for 17 Tetraodon species collected from various areas mentioned above. Supermatrix analysis based on the complete sequences of the two genes from Tetraodon species was performed using a tree with 50 tetraodontid species (including 7 Tetraodon species) as a backbone. The obtained phylogenetic tree classified Tetraodon species into three groups, correlating well with their habitats, such as Asian freshwater, Asian brackish water, and African freshwater. We also showed that salinity tolerance of Asian freshwater species T. cochinchinensis was apparently lower than that of Asian brackish water species T. nigroviridis, suggesting that the speciation of Tetraodon species in the Asian freshwater group was caused by the molecular evolution associated with osmotic regulation.     

Identification of alfonsino, Beryx mollis and B. splendens collected in Japan, based on the mitochondrial cytochrome b gene, and their comparison with those collected in New Caledonia

ABSTRACT:   The sequences spanning 307 bp of the mitochondrial cytochrome b gene were determined for 45 sepcimens of Beryx splendens and seven specimens of B. mollis collected in Japan, resulting in identification of 11 and three haplotypes in the two species, respectively. The parsimony tree was constructed from the determined sequences and those registered into the GenBank database as species A and W of B. splendens collected in New Caledonia, featuring with two clades. The first clade comprised species W from New Caledonia and B. mollis in the present study, whereas the second one contained species A from New Caledonia and B. splendens in the present study. These results demonstrate a large geographic distribution for both B. splendens and B. mollis. Some of the haplotypes found in Japan were identical to those of New Caledonia for both B. mollis and B. splendens , suggesting levels of gene flow at the trans -oceanic scale.     

Changes in enzymatic and structural properties of grass carp fast skeletal myosin induced by the laboratory-conditioned thermal acclimation and seasonal acclimatization

ABSTRACT:   Myosins were prepared from fast skeletal muscles of grass carp thermally acclimated to 10, 20 and 30°C in the laboratory as well as from those seasonally acclimatized and collected in January (winter) 2003 and May (spring), August (summer) and November (autumn) 2002. The maximal initial velocities ( V _max) of actin-activated Mg²⁺-ATPase activity for myosins from the 10°C-acclimated and winter grass carp were 1.7–1.8-fold as high as those from the 30°C-acclimated and summer fish. The inactivation rate constant ( K _D) of Ca²⁺-ATPase for myosin from the 10°C-acclimated grass carp was three to fourfold higher than those for myosins from the fish acclimated to 20°C and 30°C, whereas myosin from winter grass carp was about sevenfold as high as that for myosin from summer fish. Myosins from spring and autumn fish showed K _D values comparable to those of the fish acclimated to 30°C and 10°C, respectively. In differential scanning calorimetry analysis, the transition temperature ( T _m) was observed near 38°C and 45–46°C with most myosins. However, the lowest T _m at 32–33°C was given as one of the major endotherms in myosins from the 10°C-acclimated, autumn and winter fish. These responses of grass carp to changed environmental temperatures were almost similar to those for common carp reported previously.     

Alterations of lymphocyte subpopulations in healthy dogs with aging and in dogs with cancer

Changes in an individual's immune status are considered major contributing factors towards the morbidity of cancer and mortality of aging. To evaluate age-related changes in the immune status of dogs, the immunophenotypes (CD3, CD4, CD8 and CD21) of peripheral blood lymphocytes were measured in 160 healthy dogs aged from 1 to 17 years, and in 365 dogs with various tumors and at various stages. In healthy dogs, the absolute numbers of white blood cells, lymphocytes, and CD3(+), CD4(+) and CD21(+) lymphocytes decreased significantly with age. The relative percentages of lymphocytes and CD4(+) cells decreased significantly, while CD8(+) cells increased significantly with age. The CD4:CD8 ratio showed a significant age-related decrease. In contrast, dogs with tumors possessed significantly lower absolute numbers and relative percentages of all lymphocyte phenotypes, while the CD4:CD8 ratio was significantly higher than in age-matched controls. The relative percentages of CD3(+) and CD8(+) lymphocytes were significantly lower in dogs with distant metastases compared with dogs without metastases, and the CD4:CD8 ratio increased with advanced stage. These observations illustrate the significant changes in immune status with age and the presence of marked immunological defects in a large-scale study of dogs with advanced tumors.     

Structural and thermodynamic characterization of tropomyosin from fast skeletal muscle of bluefin tuna

ABSTRACT:   Tuna tropomyosin is a mixture of nearly equimolar amounts of two isoforms (designated α and β). cDNA encoding the α form was cloned from bluefin tuna Thunnus thynnus fast skeletal muscle. The full-length cDNA contained 1220 bp, comprising an open reading frame of 855 bp encoding 284 amino acid residues, flanked by 5'-untranslational regions (156 bp) and 3'-untranslational regions (209 bp). The deduced amino acid sequence showed considerably high homology in a range of 93.7–98.6% to those of other vertebrate α-type tropomyosins. In phylogenetic analysis, bluefin tuna tropomyosin showed the closest relationship with the white croaker counterpart. The predicted mass was 32 919 Da, and isoelectric point was 4.50, assuming acetylation of the N-terminus. By differential scanning calorimetry, bluefin tuna tropomyosin gave two major endothermic peaks at 29.3 and 41.5°C, probably caused by the presence of two isoforms. Circular dichroism spectra supported such a unique denaturation profile.     

Production of recombinant goldfish gonadotropins by baculovirus in silkworm larvae

Recombinant gonadotropins (GTH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) of goldfish Carassius auratus were produced by baculovirus in silkworm larvae. Hemolymph containing recombinant FSH (rFSH) or LH (rLH) was collected from silkworm larvae, and its biological activity was examined in vivo using male goldfish and female bitterling Rhodeus ocellatus ocellatus. Injection of hemolymph containing rFSH or rLH induced milt production in male goldfish, whereas only rLH induced ovulation in female bitterling. These results suggest, that biologically active goldfish recombinant GTH could be applied for the induction of gonadal development in aquaculture fishes as a substitute of pituitary extract, if a method of large scale production is established.     

cDNA cloning of goldfish Hsp27 and its chaperone activity

A cDNA fragment encoding goldfish Hsp27 was amplified by polymerase chain reaction and its nucleotide sequence was determined. The deduced amino-acid sequence of goldfish Hsp27 showed 58–62% identity to Hsp27 from other vertebrates. Three serine residues reported to be phosphorylated in human Hsp27 were conserved in goldfish Hsp27. The Hsp27 gene was transcribed in goldfish culture cells after temperature shift from 20°C to 40°C, but not from 20°C to 35°C. Recombinant goldfish Hsp27 was examined for its in vitro chaperone activity and compared to those of Hsp30 from the same fish and human recombinant Hsp27. Goldfish Hsp27 and Hsp30 showed a similar activity at 10 μM, which was significantly lower than that of human Hsp27. At 1 and 5 μM concentrations, however, goldfish Hsp27 showed slightly lower activity than goldfish Hsp30, but the activities of both goldfish proteins still remained significantly lower than that of human Hsp27. Meanwhile, goldfish Hsp27 formed oligomer, which was slightly smaller than that of Hsp30. These results suggest that goldfish Hsp27 contributes as a molecular chaperone in association with Hsp30 to compensate for stress resulting from rapid temperature fluctuations. The nucleotide sequence reported in this paper has been registered into the DDBJ/EMBL/GenBank databases with accession number AB239443 for goldfish Hsp27.