Detection of vitellogenin incorporation into zebrafish oocytes by FITC fluorescence

<p>Abstract</p> <p>Background</p> <p>Large volumes of lymph can be collected from the eye-sacs of bubble-eye goldfish. We attempted to induce vitellogenin (Vtg) in the eye-sac lymph of bubble-eye goldfish and develop a method for visualizing Vtg incorporation by zebrafish oocytes using FITC-labeling.</p> <p>Methods</p> <p>Estrogen efficiently induced Vtg in the eye-sac lymph of goldfish. After FITC-labeled Vtg was prepared, it was injected into mature female zebrafish.</p> <p>Results</p> <p>Incorporation of FITC-labeled Vtg by zebrafish oocytes was detected in <it>in vivo </it>and <it>in vitro </it>experiments. The embryos obtained from zebrafish females injected with FITC-labeled Vtg emitted FITC fluorescence from the yolk sac and developed normally.</p> <p>Conclusion</p> <p>This method for achieving Vtg incorporation by zebrafish oocytes could be useful in experiments related to the development and endocrinology of zebrafish oocytes.</p

Study on the usefulness of Meg1Crb10 transgenic mouse as aType2 diabetes mellitus model animal. -Analysis of blood plasma component and expression of genes related to onset of diabetes -

Meg1/Grb10遺伝子導入マウス（Meg1マウス）はインスリンのシグナル伝達阻害による高インスリン血症を呈することから2型糖尿病モデルと考えられている。Meg1マウスは肥満を伴なわずに高血糖を発症するが､脂肪･カロリーの過剰摂取によっても糖尿病の発症が著しく増加する｡本研究はMeg1マウスの2型糖尿病モデルとしての有用性を検討するために､Meg1マウスと対照マウスを高脂肪･高カロリー飼料（HFD）及び対照飼料（NFD）で飼育した時の血漿アディポネクチン量とBMI値を比較するとともに糖尿病関連遺伝子の発現量について他の糖尿病モデルマウスと比較検討した。血漿アディポネクチン量はMeg1マウスのHFDが最も高く､対照マウスのNFDが最も低い値を示した。一方､BMI値は対照マウスのHFDが最も高い値を示し､血漿アディポネクチン量とBMI値は逆相関が認められ､ヒト2型糖尿病と類似することが認められた。また､Grb10､Glut4遺伝子の発現量はMeg1マウスと他の糖尿病モデルマウスでは異なる値を示し､Meg1マウスでのGrb10遺伝子の発現量は高く､Glut4遺伝子の発現量は低かった。以上のことから､Meg1マウスには他の糖尿病モデルと異なる発症機構の存在が示唆され､Meg1マウスは2型糖尿病モデルとしての有用性が考えられた

Analysis of plasma components related to onset of diabetes in Meg1/Grb10 transgenic mice

2型糖尿病は肥満から糖尿病を発症すると考えられており､Meg1マウスは通常飼料では肥満を伴わないが､高カロリー飼料を摂取することによりグルコース代謝の異常によって糖尿病を発症する｡Meg1/Grb10遺伝子導入マウス(Meg1マウス)はインスリンのシグナル伝達阻害によるインスリン抵抗性を呈することから2型糖尿病モデルと考えられている｡本研究は､糖尿病発症機序の解明を目的として､Meg1/Grb10遺伝子導入マウス(Meg1マウス､TG+)とコントロールのマウス(Meg1マウスの親系統であるC57BL/6N;TG-)の雄を用いて高カロリー･高脂肪飼料を給餌した場合のグルコース代謝におけるシグナル伝達阻害の影響について血漿成分の面から検討した｡その結果､血漿中のIGF､BUN値はいずれもTG+がTG-より有意に低く､一方､中性脂肪量はTG+ がTG-より有意に高かった｡Meglマウスで認められたインスリン値の増加とIGF-1値の減少はインスリン伝達経路においてGrb10がインスリン受容体への結合を阻害していることの証明といえる｡Meg1マウスにおける高インスリン血症はインスリン抵抗性の結果と考えられる

Effect of High-Fat and High-Calorie Diets on the Onset of Diabetes in Meg1/Grb10 Transgenic Mice

Meg1/Grb10遺伝子導入マウスはインスリンのシグナル伝達阻害による高インスリン血症を呈することから､II型糖尿病モデルと考えられている｡そこで､本モデルマウスを用いてII型糖尿病発症に及ぼす飼料の影響について検討した結果､ヒトII型糖尿病モデルとして有用性を確認することが出来た

Root tip-dependent, active riboflavin secretion by Hyoscyamus albus hairy roots under iron deficiency

Hyoscyamus albus hairy roots with/without an exogenous gene (11 clones) were established by inoculation of Agrobacterium rhizogenes. All clones cultured under iron deficient condition secreted riboflavin from root tips into the culture medium and the productivity depended on the number and size of root tips among the clones, although the addition of sucrose was essential for riboflavin production. A decline of pH was observed before riboflavin production and root development using either a root tip or propagated roots: propagated roots were employed for further work due to their lesser variation. Additions of proton-pump inhibitors, N,N’-dicyclohexylcarbodiimide (DCCD) at 100 and 10 μM and erythrosine B at 100 μM, suppressed the pH decline at 100 and 10 μM accompanied by inhibition of riboflavin secretion and root growth; at 10 μM of erythrosine B, pH decline occurred with a moderate delay, but both growth and riboflavin efflux were inhibited. Neither inhibition of the pH decline nor riboflavin production was observed at 1 μM. To examine the necessity of acidification and riboflavin secretion by the roots themselves, artificial pH reduction of culture medium with organic acids and the addition of exogenous riboflavin with/without pH reduction were performed. When hairy roots were cultured in iron-deficient medium acidified with citric acid (pH 4.0) or malic acid (pH 3.7), pH increased rapidly to around 5 overnight, following which riboflavin production and root growth occurred. Addition of riboflavin did not affect riboflavin secretion by the roots, but acidification with citric acid (pH 4.0) helped achiever greater riboflavin production and earlier pH elevation. These results indicate that riboflavin efflux does not directly connected to active pH reduction, and more significantly active riboflavin secretion occurs by internal requirement in H. albus hairy roots under iron deficiency

Quantitative in vivo fluorescence cross-correlation analyses highlight the importance of competitive effects in the regulation of protein-protein interactions.

Computer-assisted simulation is a promising approach for clarifying complicated signaling networks. However, this approach is currently limited by a deficiency of kinetic parameters determined in living cells. To overcome this problem, we applied fluorescence cross-correlation spectrometry (FCCS) to measure dissociation constant (Kd) values of signaling molecule complexes in living cells (in vivo Kd). Among the pairs of fluorescent molecules tested, that of monomerized enhanced green fluorescent protein (mEGFP) and HaloTag-tetramethylrhodamine was most suitable for the measurement of in vivo Kd by FCCS. Using this pair, we determined 22 in vivo Kd values of signaling molecule complexes comprising the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway. With these parameters, we developed a kinetic simulation model of the EGFR-Ras-ERK MAP kinase pathway and uncovered a potential role played by stoichiometry in Shc binding to EGFR during the peak activations of Ras, MEK, and ERK. Intriguingly, most of the in vivo Kd values determined in this study were higher than the in vitro Kd values reported previously, suggesting the significance of competitive bindings inside cells. These in vivo Kd values will provide a sound basis for the quantitative understanding of signal transduction