Hyaluronic acid (HA), a natural anionic mucopolysaccharide, can be deposited onto the cationic surface of DNA/polyethyleneimine (PEI) complexes to recharge the surface potential and reduce nonspecific interactions with proteins. HA can also be used as a ligand to target specific cell receptors. Furthermore, HA-coating enhanced the transcriptional activity of the plasmid/PEI complexes, probably by loosening the tight binding between DNA and PEI, which facilitated the approach of transcription factors. Amphoteric HA derivative having spermine side chains (Spn-HA) with a structure similar to HMG protein showed higher transcription-enhancing activity than HA. Plasmid/PEI/Spn-HA ternary complex exhibited 29-fold higher transgene expression efficiency than naked plasmid/PEI complexes in CHO cells.

A novel sulfotransferase gene (designated GP3ST) was identified on human chromosome 2q37.3 based on its similarity to the cerebroside 3-sulfotransferase (CST) cDNA (Honke, K., Tsuda, M., Hirahara, Y., Ishii, A., Makita, A., and Wada, Y. (1997) J. Biol. Chem. 272, 4864 -4868). A full-length cDNA was obtained by reverse transcription-polymerase chain reaction and 5-and 3-rapid amplification of cDNA ends analyses of human colon mRNA. The isolated cDNA clone predicts that the protein is a type II transmembrane protein composed of 398 amino acid residues. The amino acid sequence indicates 33% identity to the human CST sequence. A recombinant protein that is expressed in COS-1 cells showed no CST activity, but did show sulfotransferase activities toward oligosaccharides containing nonreducing ␤-galactosides such as N-acetyllactosamine, lactose, lacto-N-tetraose (Lc4), lacto-N-neotetraose (nLc4), and Gal␤1-3GalNAc␣-benzyl (O-glycan core 1 oligosaccharide). To characterize the cloned sulfotransferase, a sulfotransferase assay method was developed that uses pyridylaminated (PA) Lc4 and nLc4 as enzyme substrates. The enzyme product using PA-Lc4 as an acceptor was identified as HSO 3 -3Gal␤1-3GlcNAc␤1-3Gal␤1-4Glc-PA by two-dimensional 1 H NMR. Kinetics studies suggested that GP3ST is able to act on both type 1 (Gal␤1-3Glc-NAc-R) and type 2 (Gal␤1-4GlcNAc-R) chains with a similar efficiency. In situ hybridization demonstrated that the GP3ST gene is expressed in epithelial cells lining the lower to middle layer of the crypts in colonic mucosa, hepatocytes surrounding the central vein of the liver, extravillous cytotrophoblasts in the basal plate and septum of the placenta, renal tubules of the kidney, and neuronal cells of the cerebral cortex. The results of this study indicate the existence of a novel ␤-Gal-3-sulfotransferase gene family.Sulfated glycoconjugates, whose sulfate groups are biologically relevant, occur in a wide range of biological compounds (reviewed in Ref. 1), including glycoproteins, proteoglycans, glycolipids, and polysaccharides. The enzymes responsible for the sulfation of these compounds, sulfotransferases, utilize in common the sulfate donor 5Ј-phosphoadenosine 3Ј-phosphosulfate (PAPS). 1We recently reported on the purification of the glycolipid 3-sulfotransferase (cerebroside sulfotransferase (CST); galactosylceramide sulfotransferase, EC 2.8.2.11) to homogeneity from human renal cancer cells (2) and the cloning of the human CST cDNA on the basis of the amino acid sequence of the purified enzyme (3). The deduced amino acid sequence shows no overall homology to other sulfotransferases, except for the PAPS-binding motifs, suggesting that CST has a different evolutionary origin (3).Carbohydrate structures with 3Ј-sulfo-␤-Gal linkages have been found in both N-glycans (4, 5) and O-glycans (6 -13) of glycoproteins, and the ␤-Gal-3Ј-sulfotransferase activities responsible for the synthesis of these glycoproteins have been demonstrated (4, 13-16). The ␤-Gal-3Ј-sulfotransferase synthesizing O-glycans has bee...

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Abstract. NIP-142 is a novel benzopyran compound that was shown to prolong the atrial effective refractory period and terminate experimental atrial fibrillation in the dog. In the present study, we examined the effects of NIP-142 on isolated guinea pig myocardium and on the Gprotein-coupled inwardly rectifying potassium channel current (acetylcholine-activated potassium current; I KACh ) expressed in Xenopus oocytes. NIP-142 (10 and 100 µM) concentration-dependently prolonged the refractory period and action potential duration in the atrium but not in the ventricle. E-4031 and 4-aminopyridine prolonged action potential duration in both left atrium and right ventricle. Prolongation by NIP-142 of the atrial action potential duration was observed at stimulation frequencies between 0.5 and 5 Hz. In contrast, the prolongation by E-4031 was not observed at higher frequencies. Tertiapin, a blocker of I KACh , prolonged action potential duration in the atrium but not in the ventricle. NIP-142 completely reversed the carbachol-induced shortening of atrial action potential duration. NIP-142 (1 to 100 µM), as well as tertiapin (0.1 to 100 nM), concentration-dependently blocked I KACh expressed in Xenopus oocytes; the blockade by NIP-142 was not affected by membrane voltage. In conclusion, NIP-142 was shown to prolong atrial refractory period and action potential duration through blockade of I KACh which may possiblly explain its previously described antiarrhythic activity. NIP-142 has pharmacological properties that are different from classical class III antiarrhythmic agents such as atria specificity and lack of reverse frequency dependence, and thus appears promising for the treatment of supraventricular arrhythmia.

Abstract. Mechanisms for the atria-specific action potential-prolonging action of NIP-142 ((3R*,4S*)-4-cyclopropylamino-3,4-dihydro-2,2-dimethyl-6-(4-methoxyphenylacetylamino)-7-nitro-2H-1-benzopyran-3-ol), a benzopyran compound that terminates experimental atrial arrhythmia, was examined. In isolated guinea-pig atrial tissue, NIP-142 reversed the shortening of action potential duration induced by either carbachol or adenosine. These effects were mimicked by tertiapin, but not by E-4031. NIP-142 concentration-dependently blocked the human G protein-coupled inwardly rectifying potassium channel current (GIRK1 / 4 channel current) expressed in HEK-293 cells with an EC 50 value of 0.64 µM. At higher concentrations, NIP-142 blocked the human ether a go-go related gene (HERG) channel current with an EC 50 value of 44 µM. In isolated guinea-pig papillary muscles, NIP-142 had no effect on the negative inotropic effect of carbachol under β-adrenergic stimulation, indicating lack of effect on the muscarinic receptor and Gi protein. These results suggest that NIP-142 directly inhibits the acetylcholine-activated potassium current.