Reassociation of annelid giant hemoglobin from the polychaete Perinereis aibuhitensis

Annelid extracellular hemoglobin (Hb) is a supramolecule with molecular mass of ～3,500kDa. The giant Hb consists of 12 subassemblies (globin dodecamers, D) and 18 homodimeric linkers (L) of non-globin chain. The globin dodecamer and linker were isolated from the polychaete Perinereis aibuhittensis Hb separately. Subsequently, these two components were mixed in the presence of 1M urea at a neutral pH to reform a whole molecule of Hb. At first L was refined by reverse phase chromatography in organic solvent. On the other hand, Perinereis Hb was incubated in 4M urea solution at 4°C for 5 min, and applied to two amphoteric ion-exchange resin column to remove L stick to the resin, and to isolate only D. The eluate was condensed and subjected to gel filtration. As a result, an ingredient of molecule mass ～210 kDa, that is D, was provided in high yield. When D and L were mixed in the molar ratio of approximately 1:1 in 50mM phosphate buffer (pH 7.2) in the presence of 1 M urea at room temperature, most of the proteins met to natural Hb size again within about 20 hours. Furthermore, similar experiments were performed in 50 mM Tris-HCl buffer (pH 7.2) containing 1 M urea in the presence of 1 mM CaCl2 or 1mM EDTA. It was observed that the reassociation was affected substantially by the presence of Ca2+. In conclusion, the homodimeric linkers have the key role to form the gigantic Hb

マウス胸腺の脂肪細胞分化過程の速度論による解析

The differential process of adipocytes was investigated in the thymus and spleen of BALB/c mice by the real time PCR method. The transcriptions of PPARγ (PP) ,adiponectin (Ad) and resistin (Re) were considered as indications of differential stages of adipocytes and the transcription ratios, log Ad/PP and log Re/PP , were measured. These logarithmic ratios increased rapidly, at the border between fetal stage and neonatal, from negative value to positive in the thymus and to nearly 0 in the spleen. Subsequently, they were remained virtually constant throughout the experimental period. Kinetics was applied to the differential model of adipocytes in which the quantity of each transcription was assumed to be proportional to the cell number, and the changes of log Ad/PP and log Re/PP with age were interpreted successfullyBALB/cマウスの胸腺および脾臓での脂肪細胞の分化過程を,PPARγ(PP)に対するアディポネクチン(Ad),およびレジスチン(Re)の転写量を指標として,リアルタイムPCR法により検討した。加齢に伴い成熟脂肪細胞が増加してくる胸腺においては,アディポネクチンおよびレジスチンの転写量は,胎生期にはPPARγ の転写量より少なかったが,出生を境に急激に増大し,PPARγ の転写量の数十倍となり,その後ほぼ一定となった。成熟した脂肪細胞が認められない脾臓では,アディポネクチンおよびレジスチンの転写量は,出生を境に増大し,PPARyの転写量とほぼ等しくなった。各遺伝子の転写量は細胞数に比例すると仮定し,脂肪細胞系列の幹細胞を考えることにより,これらの結果を,速度論により説明することができた