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2008/09/30, 退職給付ビッグバン研究会2008年度年次総会報告要旨, 科学研究費補助金（特別推進研究） = Grant-in-Aid for Specially Promoted Research

1. 1. Spermine, spermidine and putrescine activated DNA-dependent DNA polymerase from human sera by 47–125% at the concentrations of 0.2, 3 and 30 mM, respectively. 2. 2. The polyamines shifted the optimal MgCl2 concentration for the polymerase activity from 10 mM to more physiological 5 mM. 3. 3. Histamine having amino and imino groups at both ends of the molecule also increased the DNA polymerase activity, while cyclopentylamine and n-butylamine showed no effects on the enzyme activity. 4. 4. The stimulatory effect of polyamines on the DNA polymerase activity was more evident with poly(dC)p(dG) used as a template/primer than with poly(dA)p(dT).

Construction of a bacterial expression vector, pO14001, is described. The vector contains the lac promoteroperator and three sets of ribosome-binding sites (RBSs) tandemly arranged in all possible reading frames. cDNA can be directly cloned downstream from these translational start points in the fixed and proper orientation by using the method of Okayama and Berg [Mol. Cell. Biol. 3 (1982) 280–289]. The open reading frame of any cDNA inserted may be automatically aligned in phase with either of the three ATG start codons, thus enabling its expression with a maximum theoretical probability of unity. Fusion with the lacZ gene (coding for β-galactosidase) has shown that at least two of the three translation initiation sites exhibit high expression capacities and the remaining one can also function at a lower but significant rate. We used the vector to construct a bovine pituitary cDNA library, from which clones coding for prolactin were detected by immunological screening with an efficiency as high as two in three clones. The construction with triple RBSs should also provide a unique experimental model to study the regulation of overlapping translations.

Hepatic glycolytic and gluconeogenic metabolites and enzymes were assayed in obese·hyperglycemic mice (KK), alloxan diabetic mice, and the control mice. Alloxan diabetic mice showed distinctly decreased and increased concentrations of metabolites in fed and starved conditions respectively. There were no differences between KK and the control in these conditions. But in glucose load tests, KK showed the retention of hepatic metabolites in the metabolic pathway of glycolysis at five positions. They were glucose, fructose-6-phosphate, glyceraldehyde-3-phosphate, 2-phosphoglycerate, and oxaloacetic acid. This phenomenon was also observed in alloxan diabetes. In alloxan diabetes, glucokinase, and pyruvate kinase activities were lowered while glucose-6-phosphatase and fructose-1, 6-diphosphatase activities were higher by 75% and 45%, respectively. On the contrary, KK mice showed higher glucokinase and hexokinase activities than the control by 20% as well as higher glucose-6-phosphatase activity (50% increase). Concentrations of corticosterone in the serum were the same levels in KK and the control while insulin-like activity was higher in KK. NAD+/NADH ratio in hepatic cytoplasm suggested the increased reaction in the direction of a-glycerophosphate in KK. High level of cytoplasm ATPIAMP ratio was supposed to be the cause of depression of glycolysis. And this might elevate the citrate concentration resulting in increased fatty acid synthesis.