Synthesis and application of a new phosphorylating agent: S-(N-monomethoxytritylaminoethyl)-O-(o-chlorophenyl)phosphorothi oate

A new phosphorylating agent, S-(N-monomethoxytritylaminoethyl)-O-(O-chlorophenyl) phosphorothioate, was prepared and reacted with a 5'-hydroxyl group of an oligonucleotide using 1-mesitylene-sulfonyl-3-nitrotriazole (MSNT) as a condensing agent. After base labile protecting groups were removed, the partially deprotected oligonucleotide was separated on a reversed phase column and converted to the oligonucleotide with an aminoethyl or a phosphoryl group at the 5'-end by treatment with 80% acetic acid or iodine-water, respectively. The syntheses of ppT, pppT, A5'pp5'T and A5'ppp5'T were also performed by treatment of 5'-O-(N-monomethoxytritylaminoethylthiophosphoryl) thymidine with tri-n-octylammonium salt of phosphoric acid, pyrophosphoric acid, pA and ppA, respectively.     

Studies on the structure and stabilizing factor of the CUUCGG hairpin RNA using chemically synthesized oligonucleotides.

A tridecaribonucleotide, r-UGAGCUUCGGCUC, and two analogues r(UGAGC)d(UUCG)r(GCUC) and r-UGAGCUUCIGCUC, which form a hairpin structure with a four-base-paired stem and a UUCG loop, were synthesized by the solid-phase phosphoramidite method. Properties of these three oligomers and d-TGAGCTTCGGCTC, the DNA analogue, were studied by UV, CD and NMR spectroscopy. The melting temperature (Tm) data suggest that the 2'-hydroxy1 groups and the 2-amino group of guanosine in the loop (9G) stabilize the CUUCGG hairpin which is known to have an unusually high Tm. NMR studies show that this 9G takes a syn conformation and the phosphodiester backbone has a turn at 9G-10G which is a junction of the stem and loop.     

Ischemic flow threshold for striatal dopamine release in rats

To determine the level of cerebral blood flow reduction which causes striatal dopamine release, extracellular dopamine and cerebral blood flow was simultaneously determined using in vivo brain dialysis and a hydrogen clearance method, respectively, in the striatum of spontaneously hypertensive rats, before and during experimental cerebral ischemia. The ischemic flow threshold for neurotransmitter dopamine release was found to be 20% of the resting value or 8–10 ml/100g/min of cerebral blood flow, being similar to those for energy and membrane failures.     

Organic anion transport study in mutant rats with autosomal recessive conjugated hyperbilirubinemia

The EHBR is a mutant rat strain with congenital conjugated hyperbilirubinemia bred from a Sprague-Dawley rat. Transport of conjugated bilirubin, indocyanine green, and tetrabromosulfophtalein from liver to bile is severely impaired in these rats. Serum bilirubin amounts to 6.0 +/- 0.05 mg/dl (n = 4) in adult rats, with 97% conjugates. The bile flow is reduced to about 65% of the control group, whereas total bile acid in 10-min bile samples is similar. Liver histology of 10 week-old rats revealed neither intracellular pigmentation nor architectural abnormalities.     

Cell separation system studied by mixed culture of single wild strain with tetrads-forming mutant strain of Micrococcus luteus

Mixed culture study of singly occurring wild strain IFO 3333 of Micrococcus luteus and a tetrads-forming mutant strain MT, in the absence or presence of trypsin, supported our previous assumption that at least two kinds of separation systems were involved in cell separation of M. luteus, the one having a physiological role in cutting off the outermost layer of the cell wall (separation system-Om) and the other in cutting off the inner layer of the "proper" cell wall or the septum (separation system-In). The separation system-Om of IFO 3333 insensitive to trypsin substituted, freely from the cells, for that of MT sensitive to trypsin.     

Raman diagnosis of nucleic acid structure: sugar-puckering and glycosidic conformation in the guanosine moiety.

Observations of Raman spectra of various nucleic acids indicate that the guanine ring breathing frequency is sensitive to the internal rotation angle around the glycosidic bond and to the conformation of the five-membered ring of the ribose residue that is directly connected with the guanine residue in question. It is found that 682 cm-1 for C2'-endo-anti, at 665 cm-1 for C3'-endo-anti, and at 625 cm-1 for C3'-endo-syn. A DNA octamer d(GpGpApApTpTpCpC) shows, in its aqueous solution, a broad Raman band at 680 cm-1 with a tail at 670 cm-1. This fact suggests that the guanosine residues in this oligomer take primarily C2'-endo-anti conformation but an appreciable amount of fluctuation of the ribose ring structure towards C3'-endo is involved.