Molecular cloning and expression profiling of procollagen α1 (I) of cultured Pacific bluefin tuna

Type I collagen is widely distributed in most organs in teleosts. It plays a role not only in intercellular adhesion, but also in molecular signaling. In this study, Pacific bluefin tuna (PBT) procollagen α1 (I) cDNA was cloned and characterized. The nine fragments of a procollagen α1 (I) chain cDNA clone were prepared and spliced together to create the complete coding region. The resulting amino acid sequence was homologous with that of other teleosts. The mRNA expression profile of PBT procollagen α1 (I) in various tissues and the phylogenetic analysis with other vertebrate procollagen α1 (I) chains suggest that PBT procollagen α1 (I) could be a precursor form of the PBT type I collagen α1 chain. In addition, its level of expression in PBT larvae and early juveniles gradually increased with somatic growth. This increase was related to the standard length, wet body weight, and protein content of each individual fish. Therefore, the expression profile of procollagen α1 (I) may be a useful indicator for somatic growth in fish larvae and juveniles.     

Teratogenic effects of isolated and combined short‐term hypercapnia and hypoxia on red sea bream (Pagrus major) embryos

Some vertebral anomalies in cultured fish arise from unusual environmental conditions during embryogenesis. We investigated the individual and combined teratogenic effects of short‐term hypercapnia and hypoxia on embryos of red sea bream (RSB), Pagrus major. Ten‐somite stage embryos were exposed to hypercapnia (60 and 120 mg/L dissolved carbon dioxide: DCD) and hypoxia (0%, 10%, 25%, 50%, and 75% dissolved oxygen: DO) independently and concomitantly for seven different periods (0, 30, 60, 90, 120, 150 and 180 min) to examine somitic disturbances at hatching, which are prodromal symptoms of centrum defects. Another experiment examined vertebral anomalies in juveniles raised from eggs exposed to hypercapnia (120 mg/L DCD) and hypoxia (10% DO) independently and concomitantly. Short‐time exposures (30–180 min) to hypercapnia (60 and 120 mg/L) and hypoxia (10% DO or less) independently and additively caused larval somitic disturbances and juvenile centrum defects. The results indicate that short‐term hypercapnia and hypoxia generally have the same teratogenic effect on embryos, although there were some differences in the locations of the somitic disturbances and centrum defects, with additive teratogenicity when the conditions were combined. These results suggest the necessity of maintaining appropriate DCD and DO concentrations during egg incubation and transport and during reproduction in recirculating aquaculture.     

Detection and identification of rumen bacteria constituting a fibrolytic consortium dominated by Fibrobacter succinogenes

A fibrolytic consortium, dominated by the rumen cellulolytic bacterium Fibrobacter succinogenes, was artificially constructed on hay stems to detect and identify rumen bacteria that can potentially interact with F. succinogenes . Consortium-bacterial members were determined by DGGE and sequencing analysis targeted bacterial 16S rDNA. An artificial consortium was formed in a 2-step incubation of hay stems; the first step with group 1, 2 or 3 F. succinogenes strains, the second step with rumen fluid. After consortium formation, morphologically different bacteria were observed in association with F. succinogenes . DGGE exhibited more than 30 bands, the pattern of which depended on the F. succinogenes group. Sequencing suggested that Butyrivibrio fibrisolvens, Pseudobutyrivibrio ruminis , Clostridium sp., F. succinogenes group 2, Prevotella ruminicola and unclassified Bacteroides were prominent in the group 1 consortium and that Treponema bryantii , B. fibrisolvens , Acinetobacter sp, and Wolinella succinogenes were prominent in the group 2 consortium. However, in the group 3 consortium, F. succinogenes -like bacteria were microscopically undetectable, whereas cellulolytic Ruminococcus albus and F. succinogenes group 1 were prominent, suggesting that the group 3 cannot be a core member of this consortium. This study is the first attempt to identify bacterial members of a fibrolytic consortium dominated by a specific bacterium.     

Hepatoprotective action of dietary bluefin tuna skin proteins on CCl4-intoxicated mice

We have shown that dietary bluefin tuna skin (TUS) protects against carbon tetrachloride (CCl₄)-induced hepatic damage in mice. The CCl₄-induced necrotic area was decreased in mice fed a TUS-containing diet. Consistent with the decreased necrotic area, dietary TUS markedly lowered the elevated serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and the thiobarbituric acid-reactive substance (TBARS) formation induced by CCl₄ injection. TUS diets also decreased phosphorylation of inhibitory kappa B-?? and blocked the translocation of nuclear factor-kappa B to the nucleus. TUS is composed mainly (80.7?%) of type I collagen, and our results revealed that dietary tuna collagen peptides (TUCP) attenuated the increased hepatic necrotic area, serum AST and ALT activities, and liver TBARS levels induced by CCl₄, similar to TUS, thus enabling us to attribute the hepatoprotective action of TUS in CCl₄-intoxicated mice to tuna collagen. Therefore, TUS and TUCP may be potential food resources that are capable of alleviating hepatitis symptoms.     

Anti-inflammatory effects of intramammary infusions of glycyrrhizin in lactating cows with mastitis caused by coagulase-negative staphylococci

OBJECTIVE: To determine the anti-inflammatory effects of glycyrrhizin (GL) in lactating cows with mastitis attributable to naturally occurring infection with coagulase-negative staphylococci (CNS). ANIMALS: 12 lactating Holstein cows with mastitis attributable to infection with CNS and 2 healthy cows without mastitis. PROCEDURE: Clinical signs, number of bacteria in milk, somatic cell count (SCC) in milk, concentrations of alpha-lactalbumin and lactoferrin in milk, and concentration of histamine in milk were investigated before and after intramammary infusion of GL (6 cows) or antimicrobials (6 cows). Glands of 2 healthy cows were infused with staphylococcal enterotoxin; milk leukocytes were then harvested and incubated with various doses of GL. RESULTS: In cows infected with CNS that had a low bacterial concentration in milk, infusion of GL alone resulted in significant improvements in swelling, firmness of glands, and number of clots in milk, and it decreased the SCC, but not significantly. Percentage of neutrophils decreased significantly (to < 30%) by 2 days after infusion. Use of lactoferrin as a marker of inflammation in mammary glands revealed a decrease in concentrations, whereas use of alpha-lactalbumin as a marker of recovery for mammary glands revealed significant increases in concentrations in the GL-infused group. Accompanying these anti-inflammatory effects, a decrease in the concentration of histamine in milk was observed in the GL-infused group. Glycyrrhizin decreased histamine production by milk leukocytes in a concentration-dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE: Infusion of GL may regulate intramammary inflammation through modulation of inflammatory mediators such as histamine.     

A morphological study of the thyroid gland in Risso's Dolphin,Grampus griseus

To accumulate histological information of cetaceans and basic information about metabolic systems of marine mammals, the thyroid gland of Risso's dolphins was examined by gross anatomical and light and electron microscopic observations. Gross anatomically, right and left lobes of the thyroid were not clearly discriminated, and no isthmus was observed. By light microscopy, irregular or oval follicular lumens were seen, and surrounded by follicular epithelial cells. By electron microscopy, the rough endoplasmic reticulum (rER) was seen adjacently to mitochondria at the basal and lateral regions of the follicular epithelial cells. RERs at the basal side of the cells sometimes contained flocculent material with the same electron density as the follicular lumen component. Microvilli were poorly developed at the apical surface of the cells. In the apical regions of the cells, there were typical Golgi complexes, multivesicular bodies, and granules with various size and electron density. The parafollicular cells were recognized among the follicular epithelial cells and in the interstitial regions but never protruded into the follicular lumen. These cells were present singly and/or formed clusters among the follicular epithelial cells, and often located adjacent to capillaries. They were obviously discriminated from follicular epithelial cells by higher electron density of their granules. In their cytoplasm, well-developed rERs, primary lysosomes, secondary lysosomes, multivesicular bodies, and phagosomes were recognized.     

Age and growth of the silky shark Carcharhinus falciformis from the Pacific Ocean

ABSTRACT:   The present study estimated the age and growth of the silky shark Carcharhinus falciformis in the Pacific Ocean. Samples and biological data were collected from Japanese tuna longline and purse seine fisheries from 1992 to 1999. Vertebra centra were picked from 145 males and 153 females for age determination. The number of annual rings observed for males and females was 0–8 and 0–13, respectively. Combined sex von Bertalanffy growth equations were obtained as follows: L_t  = 216.4(1 − e^{−0.148( t +1.76)}) where L_t is precaudal length in cm at age t . A mature size for males was considered to be approximately 135–140 cm (precaudal length), with an estimated age of 5–6 years, whereas corresponding values for females were 145–150 cm and 6–7 years, respectively. Birth size ranged from 48 to 60 cm. There was no remarkable difference in growth, birth size and age at maturity between the Pacific and Atlantic Oceans. The life history parameters of the silky shark are approximately the same in both oceans.     

Morphometric and histochemical study of involution in the rat uterus after parturition

Morphological changes to and collagen loss from the rat uterus during postpartum involution were investigated. The expression patterns of collagen type III and myeloperoxidase (MPO) were also determined. Morphological changes were studied on days 1, 3, 5, 10, 15, 20, 22 and 25 postpartum. As a control, diestrus rats’ uterine were used. Specimens from the uterine horn were embedded in paraffin, cut into 8 µm coronal sections, and stained with hematoxylin‐eosin. The thickness of the longitudinal and circular smooth muscle layers and of the endometrium were measured. The collagen content was determined using hydroxyproline analysis. Immunostaining was used to examine the expression of collagen type III on days 1, 3, 5, 10, 15 and 20 postpartum; and MPO on days 1, 3, 5, 10 and 22 postpartum. The thickness of the smooth muscle layers was found to decrease rapidly postpartum: the circular smooth muscle layer returned to that of a non‐pregnant, control uterus by day 5 postpartum and the longitudinal smooth muscle layer by day 15 postpartum. Eosinophilic cells were observed in the endometrial stroma adjacent to the myometrium on days 10, 15 and 20 postpartum, and were confirmed as collagenous cells. Immunostaining identified collagen type III positive cells in the vessel‐rich layer adjacent to the placental site on days 1, 3, 5 and 10 postpartum, and these cells were confirmed to be phagocytic. Postpartum reduction in the weight of the uterus was accompanied by decreases in both the collagen content and the thickness of the longitudinal and circular smooth muscle layers. Furthermore, the phagocytic cells were shown to express MPO during postpartum involution of the uterus.