Enzymatic synthesis of modified DNA by PCR.

We synthesized various 5'-triphosphates of C5-substituted 2'-deoxyuridine derivatives bearing methylene linker at C5-alpha position. We examined whether the C5-substituted 2'-deoxyuridine 5'-triphosphates (dUTP) can work as a substrate for the modified DNA synthesis by PCR. We found that only KOD dash DNA polymerase, a thermostable DNA polymerase from extremely thermophilic archaeum, accepted the modified substrates in place of TTP for PCR forming the corresponding modified DNAs. On the other hand, no other DNA polymerase could accept these TTP analogues.

Inhibition of ATP-sensitive K+ channel by a non-sulfonylurea compound KAD-1229 in a pancreatic beta-cell line, MIN 6 cell.

We studied the mechanism of action of KAD-1229, a non-sulfonylurea compound shown to stimulate insulin secretion, in a glucose responsive insulinoma cell line, MIN 6 cells. In microsomal fraction of MIN 6 cells, KAD-1229 displaced binding of [3H]glibenclamide in a concentration-dependent manner. The dissociation constant and the maximum binding capacity were 0.61 nM and 8.70 pmol/mg.protein, respectively. In inside out configuration of patch-clamp technique, KAD-1229 attenuated the opening of ATP-sensitive K+ channels. The effect of KAD-1229 was detected at 10(-8) M, and 10(-5) M KAD-1229 almost completely blocked the activity of ATP-sensitive K+ channel. When membrane potential was monitored by a perforated mode of patch clamp, KAD-1229 induced depolarization of plasma membrane, which was followed by a burst of action potentials. These action potentials were blocked by cobalt. In a fura-2-loaded single MIN 6 cell, KAD evoked an elevation of intracellular free Ca2+ concentration, [Ca2+]i. The KAD-1229-mediated elevation of [Ca2+]i was attenuated by either removal of extracellular Ca2+ or an addition of nifedipine. Finally, KAD-1229 augmented insulin secretion in MIN 6 cells in a concentration-dependent manner. KAD-1229 also enhanced the effect of glucose and nifedipine inhibited the action of KAD-1229 on insulin secretion. These results indicate that KAD-1229 stimulates insulin secretion by stimulating Ca2+ influx and that, despite the lack of sulfonylurea structure, KAD-1229 binds to sulfonylurea receptors and inhibits the activity of ATP-sensitive K+ channel in MIN 6 cells.

[Mucosubstance and lectin histochemistry of DMH induced colonic tumor in rats].

Histo-pathological appearances of DMH-induced colonic tumor in Wistar rats were sequentially observed upto the 35th week after the drug administration. In our series, 28 tumors were successfully induced in the colonic mucosa of 19 out of 64 rats treated with DMH, and they were histologically diagnosed as adenocarcinoma. However, there were differences in the histo-pathological findings of the carcinoma between the distal colon and the proximal colon in rats. That is, the slowly growing type of well differentiated adenocarcinoma was likely to originate from the proper mucosa in the distal colon, while in the proximal colon the rapidly growing type of tumor did from atypical glands in the lymphoid follicles. Therefore, it was suggested that histogenesis and growing processes of the carcinoma were differed in the distal colon from that in the proximal colon in rats. Second, epithelial mucosubstances and lectin-binding properties of these lesions were examined histochemically. There were differences in the lectin-binding patterns of UEA-I and PNA between carcinomas and/or atypical glands in the lymphoid follicle and normal background mucosa in rat colon. In the UEA-I staining, almost all tumors were positively stained except for one case, and in the well differentiated type a positive staining was discernible at the cell apex and secretory product in tumors, while in the undifferentiated type, it was seen at the cytoplasma and secretory product. From these histopathological and histochemical studies it may be concluded that these lectins is likely to be useful as tumor markers for the large bowel, and also effective for a diagnosis of minute carcinoma in the large bowel.