Accelerated Growth, Gigantism and Likely Sterility in Autotransgenic Triploid Mud Loach Misgurnus mizolepis

Triploidy was induced in two selected lines of autotransgenic mud Ioach Misgurnus mizolepis containing no heterospecific genetic material. Cytological evaluations, including cellular DNA content, chromosome count, and erythrocyte measurement, proved the successful induction of triploidy. Patterns of Southern blot hybridization and tissue distribution of GH mRNA in autotransgenic triploids were similar to those of diploid autotransgenics. The autotransgenic triploids also displayed growth acceleration 22–25 times that of nontransgenic diploids, although the acceleration was relatively modest when compared to their diploid transgenic counterparts (more than a 30-fold increase). Significantly improved feed conversion was observed in both autotransgenic diploids and triploids, ranging from 1.8 to 2.0 times that in nontransgenics. Autotransgenic triploids were sterile at gonadal level, evidenced by significantly retarded and abnormal gametogenesis. With the persistence of accelerated growth and enhanced feed conversion efficiency, the autotransgenic triploid mud loach may offer economic benefits to aquaculture. Total absence of heterospecific genetic material and sterility may also allay public concern regarding food safety and environmental risk of crossbreeding between transgenics and conspecific populations.     

Application of ultrasonic treatment to extraction of collagen from the skins of sea bass Lateolabrax japonicus

This study investigates an efficient method to extract collagen from the skins of sea bass. The ultrasonic treatment (20 kHz with amplitude of 20–80 %) was applied to fish skin for 24 h after the addition of 0.01, 0.05, and 0.1 M acetic acid (1:200 sample/acetic acid, w/v). As a result, the rate (Ki) of the collagen yield increased depending on the amount of acid added, the duration of ultrasonic treatment and the amplitude of the ultrasonic waves. The subunit compositions of the extracted components were analyzed by SDS-PAGE, and the main components were determined as collagen, including the α1 (α3), α2, β, and γ chains. And in addition to collagen, some unknown components were also observed after a longer period of ultrasonic treatment. Therefore, we had to optimize the efficient extraction conditions for pure collagen while minimizing the creation of unknown components. The most effective extraction condition for collagen by ultrasonic treatment was 80 % amplitude with 0.1 M acetic acid for 3 h of treatment. It was found that the component extracted by the ultrasonic treatment was indeed collagen, since there were no changes in the main components of collagen after pepsin treatment.     

Molecular cloning and characterization of LPS-binding protein/bactericidal permeability-increasing protein (LBP/BPI) from olive flounder,Paralichthys olivaceus

A lipopolysaccharide (LPS)-binding protein/bactericidal permeability-increasing protein (LBP/BPI) homolog was isolated from peripheral blood leukocytes cDNA library of olive flounder Paralichthys olivaceus. The isolated LBP/BPI cDNA is 2806 bp in length with a 1419 bp open reading frame (ORF) that encodes a protein of 472 amino acid residues. The LPS-binding domain is well conserved in the N-terminal barrel, showing high sequence identities with other teleost LBP/BPI as well as those of mammals. RT-PCR analysis revealed that mRNA expression of LBP/BPI was significantly elevated in all tested tissues (liver, gill, intestine, head kidney, and spleen) after intraperitoneal injection of the gram-negative bacterium Edwardsiella tarda or the gram-positive bacterium Streptococcus iniae. This expression pattern profile corresponded to that of acute inflammatory cytokines, suggesting that it plays a role in the innate immune response, in particular, the acute phase response.     

Molecular cloning and expression analysis of double-stranded RNA-dependent protein kinase (PKR) in rock bream (Oplegnathus fasciatus)

We have cloned and characterized rock bream double-stranded RNA-dependent protein kinase (PKR), which is a key component of type I IFN inducible innate immune system. Full-length rock bream PKR cDNA consists of 2115 bp ORF encoding 704 amino acids, 124 bp 5′ UTR, and 529 bp 3′ UTR. Conserved domain analysis revealed that rock bream PKR contained two tandem dsRBM and kinase domain consisted of 11 kinase sub-motifs, which are characteristics found in other PKRs. Rock bream PKR was constitutively expressed in the spleen of rock bream and, upon injection of poly I:C, up-regulated not later than 12 h post injection and returned to baseline level at 48 h post injection. Although the eIF2α kinase activity of rock bream PKR was not examined in the present study, dsRNA inducible nature of rock bream PKR implies the possible important role of this gene in innate immune system of rock bream as suggested in zebrafish and flounder.     

Biomarkers for identifying the early phases of osteoarthritis secondary to medial patellar luxation in dogs

The levels of tartrate resistant acid phosphatase (TRAP), matrix metalloproteinase-2 (MMP-2), and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) in synovial fluid (SF) and serum in cases of canine osteoarthritis (OA) were measured. OA was induced by a surgically-created medial patellar luxation in the left stifle of 24 dogs. SF and blood samples were collected at 1.5- and 3-month intervals, respectively. Every 3 months, one dog was euthanatized to collect tissue samples from both stifles. TRAP levels in SF and serum were measured using a spectrophotometer, and TRAP-positive cells in joint tissues were identified by enzyme histochemistry. MMP-2 and TIMP-2 in SF and serum were detected by Western blotting and ELISA, respectively. TRAP in SF from the stifles and serum was significantly increased (p < 0.05) after 3 months. TIMP-2 in SF and serum was significantly decreased (p < 0.05), whereas MMP-2 in SF was significantly increased (p < 0.05) during the progression of OA. Histochemistry revealed an increased number of TRAP-positive cells in tissues from OA-affected joints. Assays measuring TRAP, MMP-2, and TIMP-2 in SF and serum, and methods that detect increased numbers of TRAP-positive cells in the joint tissues can play an important role in identifying the early phases of degenerative changes in canine joint components.     

Expression of estrogen receptor alpha and beta in the uterus and vagina of immature rats treated with 17-ethinyl estradiol

The action of estrogen on target organs has been actively studied with the discovery of estrogen receptor (ER) beta. This study was carried out to examine the expression of ERalpha and ERbeta in the uterus and the vagina of immature Sprague-Dawley rats treated with 17-ethinyl estradiol (EE). Twenty days old rats were subcutaneously treated with EE at the doses of 0 (vehicle control), 0.03, 0.3, 1.0, 3.0, and 10.0 microg/kg/day for three consecutive days. The treatment of EE at the doses of 0.3, 1.0, 3.0 and 10.0 microg/kg/day significantly increased the weights of the uterus and vagina of rats (p<0.01) and retained fluid in the uterus of rats. At the high doses of 3.0 and 10.0 microg/kg/day, the treatment of EE caused an increase in the uterine height, hypertrophy, and a decrease in the expression of ERalpha and ERbeta in the uterine luminal and glandular epithelium. The treatment of EE at the doses of 3.0 and 10.0 microg/kg/day also caused cornification and a decrease in the expression of ERalpha and ERbeta in the vaginal epithelium. These results suggest that the EE treatment decrease the expression of ERalpha and ERbeta in the uterus and vagina of immature rats and that may be associated with the morphological changes such as increase in the uterine height, hypertrophy of the uterine epithelium, and cornification of the vagina.