イシダイのマイトジェン刺激リンパ球によるマクロファージ活性因子の産生と本因子の特性

Spleen lymphocytes were prepared from Japanese parrotfish (Oplegnathus fasciatus) and incubated with 100 mg/ml of Concanavalin A bound to Sepharose 4B beads for 48h, and then the stimulated cell supernatants were collected. Thyoglycolate induced, allogenic, peritoneal macrophages were incubated with or without stimulated cell supernatants. Then, the phagocytic activity against inactivated yeast and peroxidase production were measured. Stimulated cell supernatants increased the phagocytic activity and the peroxidase production of allogenic peritoneal macrophages. The activities of these stimulated cell supernatants were lost completely when they were heated at 56℃ for 30 min or dialyzed against a pH 2 buffer for 24h-similar to IFN-γ. Stimulated cell supernatants did not increase phagocytic activity and peroxidase production of heterogenic mouse peritoneal macrophages.These findings suggest that the spleen lymphocytes of Japanese parrotfish can produce a macrophage activating factor in vitro with Concanavalin A bound to Sepharose 4B beads, and that this factor may be IFN-γ.イシダイの脾臓細胞中のリンパ球をコンカナバリンA（Con A）セファロース4B とともに48時間培養し，その上清によるマクロファージ活性化を検討し，以下の結果を得た。Con A 培養上清で培養したイシダイ腹腔マクロファージの貪食能と細胞内のペルオキシダーゼ活性の両機能は共に亢進していた。イシダイCon A 培養上清をホ乳類でINF-γ 失活処理とされている熱（56℃）あるいは酸（pH2）処理を行ったところ，マクロファージの活性化が消失した。イシダイCon A 培養上清の腹腔マクロファージ活性化の種特異性について，マウス腹腔マクロファージを用いて検討したところ，後者は活性化能を持たず，イシダイCon A培養上清の種特異性が存在した。以上のことより，養殖イシダイ脾臓細胞中のリンパ球は，Con A で培養するとINF-γ を産生すると推察され，イシダイの魚体内でINF-γ，マクロファージの免疫防御機構の存在が示唆された

Influence of Interposition of Pink Muscle Fiber into Dorsal Ordinary Muscle on Increasing Rate of K-value in Carp (Cultured)

In order to clarify the influence of the interposition of pink muscle fiber into the dorsal ordinary muscle on the post-mortem temporal change of K-value, using carp (cultured) Cyprinus carpio, the dorsal muscle was divided into five muscle parts toward depth with the naked eye, as follows: the dark muscle part (P-1), the intermediate muscle part (P-2), and three ordinary muscle parts (P-3, P-4, P-5). The muscle fiber types in these parts were discriminated by the inactivation of actomyosin ATPase activity for both acid and alkaline preincubations, and the temporal changes of ATP related compounds in these parts were then measured at a kept temperature of 32℃. Five muscle parts were organized from the muscle fiber types as follows: P-1 was organized from only red muscle fiber type, P-2 from only pink muscle fiber type in a thin layer and from two muscle fiber types of not only pink muscle fiber, but also white muscle fiber of the Ⅱa or Ⅱb subtype in a region of mosaic pattern, and all of P-3, P-4, and P-5 were from two muscle fiber types of white muscle fiber (Ⅱa or Ⅱb subtype) and pink muscle fiber, respectively. The temporal changes of K-value were remarkably faster in order of the dark muscle part of P-1, the intermediate muscle part of P-2, three ordinary muscle parts of P-3, P-4, and P-5, although its change did not exhibit a remarkable difference among three ordinary muscle parts. On the other hand, the temporal change of K-value among muscle fiber types was considerably different and remarkably faster in the order of: red muscle fiber, pink muscle fiber, and white muscle fiber. From these results, it was considered that the interposition of pink muscle fiber into the dorsal ordinary muscle might accelerate the temporal change of K-value

Dietary supplementation with alkylresorcinols prevents muscle atrophy through a shift of energy supply

It has been reported that phytoextracts that contain alkylresorcinols (ARs) protect against severe myofibrillar degeneration found in isoproterenol-induced myocardial infarction. In this study, we examined the effect of dietary ARs derived from wheat bran extracts on muscle atrophy in denervated mice. The mice were divided into the following four groups: (1) sham-operated (control) mice fed with normal diet (S-ND), (2) denervated mice fed with normal diet (D-ND), (3) control mice fed with ARs-supplemented diet (S-AR) and (4) denervated mice fed with ARs-supplemented diet (D-AR). The intake of ARs prevented the denervation-induced reduction of the weight of the hind limb muscles and the myofiber size. However, the expression of ubiquitin ligases and autophagy-related genes, which is associated with muscle proteolysis, was slightly higher in D-AR than in D-ND. Moreover, the abundance of the autophagy marker p62 was significantly higher in D-AR than in D-ND. Muscle atrophy has been known to be associated with a disturbed energy metabolism. The expression of pyruvate dehydrogenase kinase 4 (PDK4), which is related to fatty acid metabolism, was decreased in D-ND as compared with that in S-ND. In contrast, dietary supplementation with ARs inhibited the decrease of PDK4 expression caused by denervation. Furthermore, the abnormal expression pattern of genes related to the abundance of lipid droplets-coated proteins that was induced by denervation was improved by ARs. These results raise the possibility that dietary supplementation with ARs modifies the disruption of fatty acid metabolism induced by lipid autophagy, resulting in the prevention of muscle atrophy

Astaxanthin Prevents Atrophy in Slow Muscle Fibers by Inhibiting Mitochondrial Reactive Oxygen Species via a Mitochondria-Mediated Apoptosis Pathway

Astaxanthin (AX) is a carotenoid that exerts potent antioxidant activity and acts in the lipid bilayer. This study aimed to investigate the effects of AX on muscle-atrophy-mediated disturbance of mitochondria, which have a lipid bilayer. Tail suspension was used to establish a muscleatrophied mouse model. AX diet fed to tail-suspension mice prevented loss of muscle weight, inhibited the decrease of myofiber size, and restrained the increase of hydrogen peroxide (H2O2) production in the soleus muscle. Additionally, AX improved downregulation of mitochondrial respiratory chain complexes I and III in the soleus muscle after tail suspension. Meanwhile, AX promoted mitochondrial biogenesis by upregulating the expressions of adenosine 5\u27-monophosphate-activated protein kinase (AMPK) α-1, peroxisome proliferator-activated receptor (PPAR)-γ, and creatine kinase in mitochondrial (Ckmt) 2 in the soleus muscle of tail-suspension mice. To confirm the AX phenotype in the soleus muscle, we examined its effects on mitochondria using Sol8 myotubes derived from the soleus muscle. We found that AX was preferentially detected in the mitochondrial fraction; it significantly suppressed mitochondrial reactive oxygen species (ROS) production in Sol8 myotubes. Moreover, AX inhibited the activation of caspase 3 via inhibiting the release of cytochrome c into the cytosol in antimycin A-treated Sol8 myotubes. These results suggested that AX protected the functional stability of mitochondria, alleviated mitochondrial oxidative stress and mitochondriamediated apoptosis, and thus, prevented muscle atrophy

Characteristics of a self-assembled fibrillar gel prepared from red stingray collagen

A translucent collagen gel was formed from a transparent acidic solution of red stingray collagen by adjusting to physiological ionic strength and pH in phosphate buffer and then incubating at 25?37°C. During fibril formation from red stingray collagen, the turbidity increased when the NaCl concentration was increased at constant pH and the rate of fibril formation was accelerated by higher pH or lower NaCl concentration. The T m of red stingray collagen fibrillar gel was estimated as 44.3 ± 3.5°C, which was higher than that of the collagen solution, 33.2°C. In addition, red stingray collagen gel maintained its shape without melting and was suitable for culture of mouse stromal cells at 37°C

A serine proteinase from the sarcoplasmic fraction of red sea bream Pagrus major is possibly derived from blood

Collagen degradation is known to be involved in the post mortem tenderization of fish muscle. A serine proteinase that is assumed to be related to collagen degradation after fish death was purified from the sarcoplasmic fraction of red sea bream Pagrus major by ammonium sulfate fractionation and column chromatography on Sephacryl S-300, Q Sepharose and Phenyl Sepharose CL-4B. The enzyme hydrolyzed gelatin and was obtained as a protein band of approximately 38 kDa upon sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. The N-terminal amino acid sequence of the enzyme was determined for 32 residues. A protein that had the same N-terminal amino acid sequence as the enzyme for ten residues was purified from the serum of red sea bream and showed the same characteristics as the enzyme. Therefore, it is suggested that the serine proteinase migrates from the blood to muscle and degrades muscle proteins after the death of the fish

Development of Plasma Vitellogenin Assay for Estrogenic Effects of Endocrine-Disrupting Chemicals Using Ovariectomized Goldfish (Carassius auratus)

Plasma vitellogenin (VTG) assay was developed using ovariectomized goldfish (Carassius auratus) for determining the estrogenic effects of endocrine-disrupting chemicals. In a laboratory study, we assessed the estrogenic activity of commercial fish diets by using a diet for ornamental carp (CD) and a casein-based formulated fish diet (FD), which was shown to not contain soybean or fish meal in a previous study. In ovariectomized fish, plasma VTG concentrations were significantly higher in the CD-fed group than in the FD-fed group. These results indicate that the estrogen activity of CD may be high enough to cause induction of plasma VTG in ovariectomized goldfish as previously observed in male goldfish. Moreover, the effect of estrogen on plasma VTG induction was confirmed by significant plasma VTG production following the exposure of FD-fed ovariectomized goldfish to a nominal estradiol-17β concentration of 100 μg/l for 31 days. Our data suggest that induction of plasma VTG using ovariectomized goldfish is a good tool for evaluating the estrogenic effects of endocrine-disrupting chemicals