マイクロプレート オ モチイタ オス シバヤギ ケッショウ チュウ テストステロン ノ コウソ メンエキ ソクテイ ホウ ノ ケントウ

Testosterone-3(E)-carboxymethyloxime-BSAを抗原として作成した抗体,ならびに酵素標識ホルモンとしてTestosterone-3(E)-carboxymethyloxime-peroxidaseを使用し,雄シバヤギにおける血漿中テストステロン濃度の酵素免疫測定法(EIA)を検討した。本実験では高い測定感度が得られる二抗体法を用い,作成した抗血清は,350,000倍に希釈しても使用可能な高い力価を有していた。血漿に10～100pgのテストステロンを添加した添加回収試験では,回収率が平均102.8±2.8%となった。測定内変動係数(N=6)は雄シバヤギ頸静脈血漿で5.08%,精索静脈血漿では7.32%,測定間変動係数(N=6)は,頸静脈血漿で5.74%,精索静脈血漿で6.13%であった。以上の結果から,本方法によって雄シバヤギ血漿中テストステロンの測定が可能となった。Enzymeimmunoassay (EIA) of testosterone was examined in which an individual antibody, and testosterone-peroxidase-conjugate were used for that, was measured in male Shiba-goat plasma. An anti-testosterone-3(E)-carboxymethyloxime-BSA antibody was used as an anti-serum, and testosterone-3(E)-carboxymethyloxime-peroxidase was used as a steroid-enzyme conjugate. The anti-serum was diluted by using 2nd antibody method which could get high measurement sensitivity of 350,000 times. Recovery rates of testosterone for each concentration with the addition of 10～100pg to Shiba-goat plasma were 102.8±2.8% of the averages. Inter-assay coefficient of variation (C.V.) for testosterone level from jugular and testicular vein blood plasma samples were 5.08% and 7.32% respectively, as for intra-assay, they became 5.74% and 6.13%. These results suggest that the EIA method is extremely suitable to measure testosterone concentration in blood plasma of the male Shiba-goat

マイクロプレート オ モチイタ オス シバヤギ ケッショウ チュウ 5αジヒドロテストステロン ノ コウソ メンエキ ソクテイ

雄シバヤギ血漿中5α-ジヒドロテストステロン(5α-DHT)に関する酵素免疫測定法(EIA)について検討した。抗血清は抗5α-DHT-11α-Succinate-BSAを,酵素標識ホルモンには5α-DHT-11α-Succinate-peroxidase(5α-DHT-HRP)を用いた。抗血清は100,000倍に希釈・使用が可能であった。また,抗血清にはテストステロンが30%交叉反応するため,Bond Elut CN-Uを用いたカラムクロマトグラフィーによる精製を行い,被検血漿中の5α-DHTとテストステロンを分離・測定した。その結果,酢酸エチル : ベンゼン=2 : 98の展開液を流したところ,1.00-4.25mlの範囲に5α-DHTが溶出された。添加回収試験において添加量0.1-1.0pgの各濃度での回収率は,平均100.45%±2.13となった。再現性試験における頸静脈血および精巣静脈血の測定内変動係数(n=6)は各々6.38%および5.94%となり,測定間変動係数は8.36%ならびに11.53%となった。以上の結果から,雄シバヤギの血漿中5α-DHT濃度を,本法を用いて測定することが可能であることを明らかにした。Enzymeimmunoassay (EIA) for 5α-dihydrotestosterone (5α-DHT) in male Shiba-goat blood plasma was examined. The antiserum used 5α-DHT-11α-succinate-peroxidase (5α-DHT-HRP) for the enzyme labeling hormone in respect of 5α-DHT-11α-succinate-BSA. The use was possible for the antiserum at 100,000 times. The 5α-DHT in plasma was purified and separated from the testosterone by column chromatography using Bond Elut CN-U, since antiserum for 5α-DHT had cross reaction (30%) to the testosterone. The 5α-DHT was washed away by the development liquid of ethyl acetate: benzene=2 : 98 and was collected in the range between 1.00 and 4.25ml. Recovery rates of 5α-DHT each concentration of the addition 0.1～1ng to Shiba-goat plasma were 100.45%±2.13 of the averages. Inter-assay coefficient of variation (C.V.) became respectively 6.38% and 5.94% in jugular and testicular vein blood,while for intra-assay, they became 8.36% and 11.53%. It was possible to analyse 5α-DHT concentration in blood plasma of the male Shiba-goat from the above result using this method

Sardine procalcitonin amino-terminal cleavage peptide has a different action from calcitonin and promotes osteoblastic activity in the scales of goldfish

The nucleotide sequence of a sardine preprocalcitonin precursor has been determined from their ultimobranchial glands in the present study. From our analysis of this sequence, we found that sardine procalcitonin was composed of procalcitonin amino-terminal cleavage peptide (N-proCT) (53 amino acids), CT (32 amino acids), and procalcitonin carboxyl-terminal cleavage peptide (C-proCT) (18 amino acids). As compared with C-proCT, N-proCT has been highly conserved among teleosts, reptiles, and birds, which suggests that N-proCT has some bioactivities. Therefore, both sardine N-proCT and sardine CT were synthesized, and their bioactivities for osteoblasts and osteoclasts were examined using our assay system with goldfish scales that consisted of osteoblasts and osteoclasts. As a result, sardine N-proCT (10− 7 M) activated osteoblastic marker enzyme activity, while sardine CT did not change. On the other hand, sardine CT (10− 9 to 10− 7 M) suppressed osteoclastic marker enzyme activity, although sardine N-proCT did not influence enzyme activity. Furthermore, the mRNA expressions of osteoblastic markers such as type 1 collagen and osteocalcin were also promoted by sardine N-proCT (10− 7 M) treatment; however, sardine CT did not influence their expressions. The osteoblastic effects of N-proCT lack agreement. In the present study, we can evaluate exactly the action for osteoblasts because our scale assay system is very sensitive and it is a co-culture system for osteoblasts and osteoclasts with calcified bone matrix. Both CT and N-proCT seem to influence osteoblasts and osteoclasts and promote bone formation by different actions in teleosts. © 2017 Elsevier Inc.Embargo Period 12 month

Bisphenol A influences the plasma calcium level and inhibits calcitonin secretion in goldfish.

In teleosts, it is well known that plasma calcium levels increase as a result of treatment with estrogen for at least during 2 weeks and that calcitonin secretion is induced by estrogen. The present study examined the influence of bisphenol A on calcium homeostasis in goldfish and compared the above known estrogenic action. In goldfish kept in water containing bisphenol A (10(-6) M), the plasma calcium concentration increased significantly (P<0.001) at 4 days but decreased significantly (P<0.05) at 8 days. By the treatment of bisphenol A, calcitonin secretion was not induced until 4 days. At 8 days, however, plasma calcitonin, as well as calcium, decreased significantly (P<0.05), although vitellogenin was detected in the plasma. Therefore, bisphenol A influences plasma calcium levels, but its action is different from that of estrogen, which indicates that bisphenol A affects the calcium homeostasis and might bring about abnormal conditions in teleosts

Immunoreactive Calcitonin Cells in the Nervous System of Polychaete Perinereis aibuhitensis

Time series variations in protein expression profiles in field Chattonella marina cells were investigated during a HAB occurred in the inner part of Ariake Sea, Japan (5–14 September, 2012). This study aimed to gather information on the molecular mechanisms underlying the physiological responses in field C. marina population during HAB. Proteomic analysis showed that the abundance of ~37% protein spots (40 out of 108 detected from 2–DE gel images) significantly varied with the sampling date. Significant decreases in the abundances of proteins involved in photosystem II (LHCP 4), electron transfer chain (Cyt c553), Calvin cycle (GAPDH), and chloroplast antioxidant system (2–Cys Prx) were observed as the bloom progressed, suggesting the efficiencies of those photosynthetic pathways declined during the bloom. In addition, the abundances of the above proteins showed significant positive correlations with the F_v/F_m ratio and growth rate of C. marina and with DIN concentrations (except LHCP 4). Our findings suggested that declined expressions of those photosynthesis–related proteins presented some molecular foundation of the decreases in F_v/F_m ratio and growth rate of C. marina during the bloom, and also provided insight into mechanistic links between the external/internal factors and physiological responses of C. marina that may ultimately dictate the ecology of the bloom