Characterization and ontogenetic development of digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae

The major digestive enzymes in Pacific bluefin tuna Thunnus orientalis larvae were characterized, and the physiological characteristics of the enzymes during early ontogeny were clarified using biochemical and molecular approaches. The maximum activity of trypsin (Try), chymotrypsin (Ct) and amylase (Amy) was observed at pH 6–11, 8–11 and 6–9, respectively. Maximum activity of Try, Ct and Amy occurred at 50 °C, that of lipase (Lip) was at 60 °C and that of pepsin (Pep) was at 40–50 °C. These pH and thermal profiles were similar to those for other fish species but differed from those previously reported for adult bluefin tuna. Enzyme activity for all enzymes assayed was found to decrease at high temperatures (Try, Ct, Amy and Pep: 50 °C; Lip: 40 °C), which is similar to findings for other fish species with one marked exception—increased Try activity was observed at 40 °C. Lip activity appeared to be dependent on bile salts under our assay conditions, resulting in a significant increase in activity in the presence of bile salts. Ontogenetic changes in pancreatic digestive enzymes showed similar gene expression patterns to those of other fish species, whereas marked temporal increases in enzyme activities were observed at 10–12 days post hatching (dph), coinciding with previously reported timing of the development of the pyloric caeca in bluefin tuna larvae. However, complete development of digestive function was indicated by the high pep gene expression from 19 dph, which contradicts the profile of Pep activity and previously reported development timing of the gastric gland. These findings contribute to the general knowledge of bluefin tuna larval digestive system development.     

Cloning of bovine MAIL and its mRNA expression in white blood cells of Holstein cows

Molecule possessing ankyrin-repeats induced by lipopolysaccharide (MAIL) is known as an IkappaB protein induced after administration of bacterial lipopolysaccharide (LPS) to mice. In the present study, we cloned bovine MAIL cDNA and examined its mRNA expression in white blood cells isolated from Holstein cows. Bovine MAIL had more than 80% amino acid identities with murine and human MAILs, highly conserved ankyrin-repeat motifs and PEST-like sequences. Bovine MAIL mRNA was undetectable in isolated peripheral white blood cells, but rapidly induced (<1h) after stimulation by LPS and lipid A in vitro in a dose-dependent manner. The lipid A-induced MAIL mRNA expression was found in polymorphonuclear cells, monocytes/macrophages and total lymphocytes, but not in T-lymphocytes. MAIL mRNA was also induced in vivo in peripheral blood leukocytes of cows after intramammary injection of Escherichia coli derived from coliform mastitis. Thus, bovine MAIL, as rodent MAILs, is induced by inflammatory stimuli in specific immune cells in vitro and in vivo, suggesting a role in inflammatory responses to bacterial infection in cattle.     

Epigenetic assessment of environmental chemicals detected in maternal peripheral and cord blood samples

Epigenetic alteration is an emerging paradigm underlying the long-term effects of chemicals on gene functions. Various chemicals, including organophosphate insecticides and heavy metals, have been detected in the human fetal environment. Epigenetics by DNA methylation and histone modifications, through dynamic chromatin remodeling, is a mechanism for genome stability and gene functions. To investigate whether such environmental chemicals may cause epigenetic alterations, we studied the effects of selected chemicals on morphological changes in heterochromatin and DNA methylation status in mouse ES cells (ESCs). Twenty-five chemicals, including organophosphate insecticides, heavy metals and their metabolites, were assessed for their effect on the epigenetic status of mouse ESCs by monitoring heterochromatin stained with 4￠,6-diamino-2-phenylindole (DAPI). The cells were surveyed after 48 or 96 h of exposure to the chemicals at the serum concentrations of cord blood. The candidates for epigenetic mutagens were examined for the effect on DNA methylation at genic regions. Of the 25 chemicals, five chemicals (diethyl phosphate (DEP), mercury (Hg), cotinine, selenium (Se) and octachlorodipropyl ether (S-421)) caused alterations in nuclear staining, suggesting that they affected heterochromatin conditions. Hg and Se caused aberrant DNA methylation at gene loci. Furthermore, DEP at 0.1 ppb caused irreversible heterochromatin changes in ESCs, and DEP-, Hg- and S-421-exposed cells also exhibited impaired formation of the embryoid body (EB), which is an in vitro model for early embryos. We established a system for assessment of epigenetic mutagens. We identified environmental chemicals that could have effects on the human fetus epigenetic status.     

Fat depot‐specific differences in leptin mRNA expression and its relation to adipocyte size in steers

ABSTRACT This experiment was conducted to investigate leptin mRNA expression, adipocyte size, and their relationship in several adipose tissues of fattening steers. Subcutaneous, perirenal, intermuscular and intramuscular adipose tissues were collected from three crossbred steers (Japanese Black cattle X Holstein) aged 21 months. The mRNA level and adipocyte diameter were determined in these adipose tissues. The intramuscular adipose tissue had a lower leptin mRNA level than the intermuscular and perirenal adipose tissues (P < 0.05). Leptin mRNA level was lower in the subcutaneous depot than in the intermuscular depot (P < 0.05). Adipocyte diameter was larger in the intermuscular adipose tissue than in the subcutaneous and intramuscular adipose tissues (P < 0.05). Leptin mRNA level was positively correlated with adipocyte diameter (r² = 0.81, P < 0.05). These results suggest that the cattle have fat depot‐specific differences in leptin gene expression, which are a result of a difference in adipocyte size.     

Effect of the strain combination of the donor and recipient on the production efficiency of W‐bearing sperm in mixed‐sex germline chimeric chickens

Effect of the strain combination of the donor and recipient on production efficiency of W‐bearing sperm in mixed‐sex chimeric testes was analyzed. The combinations of the donors and recipients were White Leghorn (WL) and Rhode Island Red (RIR), and vice versa. Generated mixed‐sex chimeras that had the male phenotype at sexual maturity were classified into four groups: (1) a female WL donor and a male RIR recipient; (2) a male WL donor and a female RIR recipient; (3) a female RIR donor and a male WL recipient; (4) a male RIR donor and a female WL recipient. The mean number of W‐bearing sperm detected by in situ hybridization among 10 000 sperm observed were 147, 165, 30 and 45 in groups 1, 2, 3 and 4, respectively. The numbers in groups 1 and 2 were both significantly higher than those of groups 3 and 4 (P < 0.05). The combination of a WL donor and a RIR recipient produced W‐bearing sperm more efficiently than the reverse combination.     

Ability of CD8+ T cell anti-feline immunodeficiency virus (FIV) activity and FIV proviral DNA load in mononuclear cells in FIV-infected cats

We investigated the relationship between CD8+ T cell anti-feline immunodeficiency virus (FIV) activity and FIV proviral DNA load integrated in mononuclear cells. The anti-FIV activity and the proviral DNA load were correlated, and the number of proviral DNA copies was high in cats with decreased anti-FIV activity. Particularly, no anti-FIV activity was detected in the cats staged as having an acquired immunodeficiency syndrome (AIDS)-related complex or AIDS, and the number of proviral DNA copies was obviously increased compared to those in the cats in the asymptomatic stage. These results suggest that decreased anti-FIV activity destroys the control of in vivo FIV replication, which leads to an increased proviral DNA load with the progression of the clinical stage of disease.     

Production and characterization of monoclonal antibodies against porcine interleukin-4

Interleukin 4 (IL-4) is an important regulatory cytokine produced by activated T lymphocytes and mast cells, and regulates the growth and differentiation of cells such as B and T lymphocytes. In the present study, recombinant thioredoxin (Trx)-porcine IL-4 (pIL-4) fusion protein was prepared by Escherichia coli (E. coli), and by using this protein as an immunogen, monoclonal antibodies (mAbs) against pIL-4 were produced to establish a basis for a research on immune responses in pigs. Six stable hybridoma cell lines were successfully established and specific binding of each mAb to recombinant pIL-4 produced by E. coli and insect cells infected with recombinant baculovirus was shown by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Isotype analyses of these mAbs revealed that the subclass of 5 out of 6 mAbs was IgG1 and the rest was IgG2b. Further, assessment of their epitopes by competition binding assay indicated that the mAbs obtained in this study bound to 4 different epitopes. The recombinant proteins and mAbs produced in this study will be useful tools for the assessment of porcine immune system.     

Molecular study on chicken tumor necrosis factor receptor-II and tumor necrosis factor receptor-associated factor-5

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) were identified as signal transducers for the tumor necrosis factor receptor (TNFR) superfamily. In this study, we cloned and characterized two genes that encode chicken TNFR-II and TRAF5. The initial cDNA fragments were obtained by suppressive subtractive hybridization (SSH) of chicken spleen cells with or without lipopolysaccharide stimulation (Salmonella typhimurium SL1181 (RE-mutant)). The results showed that chicken TNFR-II is 1518 bp in length with an open reading frame (ORF) of 1386 bp having 31% homology with human TNFR-II. Expression analysis of chicken TNFR-II revealed that it is highly expressed in the spleen and bursa of Fabricius. The chicken cell lines IN24, MSB1 and 1104B express TNFR-II abundantly. The time course analysis of expression in spleen, bursa of Fabricius and IN24 cell line showed that TNFR-II is maximally expressed at 6 h after stimulation in bursa of Fabricius and after 8 h stimulation in the IN24 cell line. With regard to TRAF5, the complete sequence was 1936 bp in length with an ORF of 1671 bp that showed 71.3% homology with human TRAF5. Expression analysis showed that, among the tissues examined, TRAF5 was strongly expressed in spleen and bursa of Fabricius, while among the cell lines examined, it was maximally expressed in IN24. Thus, both genes were expressed in the same tissues and cell line among examined materials. These results suggest that chicken TNFR-II may interact with TRAF5 adaptor protein to complete its signal transduction pathway.