ABSTRACT
In this report we describe the synthesis of oligonucleotides containing sulfide-linked dinucleoside units, namely rT(2'OH)sdT, rT(2'OMe)sdT, dTsrU(2'OMe) and dT(2'OMe)srU(2'OMe). We also describe the interactions of such oligomers with complementary DNA and RNA targets, and provide the structural basis for their remarkable RNA binding selectivity. In all cases, the Tm values of the S/P-chimera duplexes were lower than those of the corresponding unmodified duplexes. We attribute this to steric interactions between the 5'sulfur and the atoms of the nearby base/sugar residues. The 2'-substituents (i.e., 2'OH or 2'OMe) vicinal to the alkylsulfide internucleoside linkage significantly perturb the structure and stability of the duplexes formed with DNA, and more so than with RNA. The introduction of three rT(2'OH)sdTp (or rT(2'OMe)sdTp) units into an oligodeoxynucleotide sequence was sufficient to abolish binding to complementary DNA but not RNA. The same three substitutions with dTsrU(2'OMe)p and dT(2'OMe)srU(2'OMe)p did not abolish binding to DNA but the resulting complexes had poor thermal stability. The RNA-binding 'selectivity' exhibited by these oligomers is attributed to the tendency of the 2'-substituted (branched) furanoses to adopt the C3'-endo pucker, a conformation that is inconsistent with the B-form structure of helical DNA. The preference of these sugars to exist often exclusively in the C3'-endo form is attributed to stereoelectronic effects, namely gauche and anomeric effects. Our findings support the hypothesis that nucleoside analogues puckered exclusively in the C3'-endo form may result in them being especially good binders of targeted mRNA [S.H. Kawai (1991), Ph.D. Thesis, McGill University; Kawasaki et al. (1993) J. Med. Chem. 36, 831-841].
Subject(s)
Deoxyribonucleosides/chemistry , Oligonucleotides/chemistry , Oligonucleotides/metabolism , RNA/metabolism , Sulfides/chemistry , Base Sequence , DNA, Complementary/metabolism , DNA, Single-Stranded/metabolism , Drug Stability , Hot Temperature , Methylation , Models, Molecular , Molecular Sequence Data , Molecular Structure , Nucleic Acid Conformation , Nucleosides/chemistry , RNA, Messenger/metabolism , Structure-Activity RelationshipABSTRACT
The liver is a major site of production of insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGF-BPs). GH decisively influences IGF-I production. To study the role of GH and glucagon in the regulation of IGF-I and IGF-BP production, we examined IGF-I and IGF-BPs secreted by primary rat hepatocytes cultured in a serum-free medium. Glucagon (1 x 10(-8) M) stimulated IGF-I secretion and IGF-BP secretion. Bovine GH (bGH, 300 ng/ml) stimulated IGF-I secretion but suppressed IGF-BP secretion. Combining bGH and glucagon significantly augmented IGF-I secretion above the level seen with each individual agent. The inhibitory effect of bGH on IGF-BP secretion was reversed by glucagon. The major species of IGF-BPs secreted by hepatocytes were found, on Western ligand blotting, to be 24K and 30-34K. All species of secreted IGF-BPs appeared to be comparably affected by glucagon, bGH, and their combination. Northern analysis of IGF-I mRNA revealed three transcripts of 0.7-1.1 kilobases (kb), 1.8 kb, and 7.0 kb. Glucagon stimulated IGF-I mRNA levels 1.8- to 2.0-fold, whereas bGH stimulated IGF-I mRNA levels 2.0- to 2.5-fold. When hepatocytes were incubated with glucagon and bGH for 6 h, IGF-I mRNA levels were augmented 10-fold. Glucagon, in the presence of 50 ng/ml bGH, had a dose-dependent effect on IGF-I mRNA accumulation from a 6-fold level of stimulation at 50 ng/ml of glucagon to a 9-fold level of stimulation at 1000 ng/ml glucagon to a 9-fold level of stimulation at 1000 ng/ml glucagon. This study has demonstrated that glucagon, as well as GH, has significant effects on the production of both IGF-I and IGF-BPs. Of particular interest was the marked augmentation of hepatic IGF-I messenger RNA levels and the reversal of the low levels of IGF-BP production seen on adding glucagon to bGH.
Subject(s)
Carrier Proteins/biosynthesis , Glucagon/pharmacology , Growth Hormone/pharmacology , Insulin-Like Growth Factor I/biosynthesis , Liver/metabolism , Animals , Carrier Proteins/genetics , Cells, Cultured , Dose-Response Relationship, Drug , Gene Expression Regulation/drug effects , Glucagon/administration & dosage , Growth Hormone/administration & dosage , Insulin-Like Growth Factor Binding Proteins , Insulin-Like Growth Factor I/genetics , Kinetics , Liver/drug effects , Male , Molecular Weight , Nucleic Acid Hybridization , RNA, Messenger/analysis , Rats , Rats, Inbred StrainsABSTRACT
This study was planned to investigate whether part of the energy produced by cardiac contraction to propel blood toward the systemic circulation can be used to increase the coronary flow in systole. Ten devices (I-X) designed to divert laterally part of a flow column (without substantially increasing resistance to flow) were tested in a mock circulation and in nine anesthetized dogs. An increase of 11.69 +/- 1.97% (mean +/- SEM, P less than 0.005) in "coronary flow" and of 8.98 +/- 0.56% (mean +/- SEM, P less than 0.001) in coronary sinus flow were obtained with device I mounted on a stylet and placed above the "aortic valve" in the mock circulation and the dogs, respectively, without increasing the flow resistance. The results in;dicate the possibility of diverting part of the aortic flow toward the coronary arteries without increasing mean aortic pressure.