Yasuteru Iijima

Identification of 2′-Phosphodiesterase, Which Plays a Role in the 2-5A System Regulated by Interferon

The 2-5A system is one of the major pathways for antiviral and antitumor functions that can be induced by interferons (IFNs). The 2-5A system is modulated by 5′-triphosphorylated, 2′,5′-phosphodiester-linked oligoadenylates (2-5A), which are synthesized by 2′,5′-oligoadenylate synthetases (2′,5′-OASs), inactivated by 5′-phosphatase and completely degraded by 2′-phosphodiesterase (2′-PDE). Generated 2-5A activates 2-5A-dependent endoribonuclease, RNase L, which induces RNA degradation in cells and finally apoptosis. Although 2′,5′-OASs and RNase L have been molecularly cloned and studied well, the identification of 2′-PDE has remained elusive. Here, we describe the first identification of 2′-PDE, the third key enzyme of the 2-5A system. We found a putative 2′-PDE band on SDS-PAGE by successive six-step chromatographies from ammonium sulfate precipitates of bovine liver and identified a partial amino acid sequence of the human 2′-PDE by mass spectrometry. Based on the full-length sequence of the human 2′-PDE obtained by in silico expressed sequence tag assembly, the gene was cloned by reverse transcription-PCR. The recombinant human 2′-PDE expressed in mammalian cells certainly cleaved the 2′,5′-phosphodiester bond of 2-5A trimer and 2-5A analogs. Because no sequences with high homology to this human 2′-PDE were found, the human 2′-PDE was considered to be a unique enzyme without isoform. Suppression of 2′-PDE by a small interfering RNA and a 2′-PDE inhibitor resulted in significant reduction of viral replication, whereas overexpression of 2′-PDE protected cells from IFN-induced antiproliferative activity. These observations identify 2′-PDE as a key regulator of the 2-5A system and as a potential novel target for antiviral and antitumor treatments.

Purification of α-l-fucosidase from the liver of a marine gastropod, Turbo cornutus, and its action on blood group substances

Abstract Using p -nitrophenyl α- l -fucoside as substrate, α- l -fucosidase was purified 120-fold from the liver of a marine gastropod, Turbo cornutus , by the combination of ammonium sulfate fractionation, pH 4 treatment, heating at pH 3.0, SE-cellulose column chromatography, and Sephadex G-200 column chromatography. The purified enzyme was practically free from other glycosidases and had a pH optimum of 4.0. The Michaelis constant toward p -nitrophenyl α- l -fucoside was 5.7 × 10 −4 m. The purified enzyme released fucose from hog gastric blood group substance, and rapidly destroyed type H specificity of human blood group substance.

Biological properties of griseolic acid, a cyclic AMP phosphodiesterase inhibitor with an adenine group

Griseolic acid inhibited cAMP phosphodiesterase (PDE) at low concentrations, the I 50 being of the order of 0.01–0.1 μM. Administration of griseolic acid to rats increased the cAMP level in liver and plasma several‐fold. It increased glycogen degradation in mouse liver and stimulated lipolysis in isolated rat fat cells. Griseolic acid did not block the adenosine‐elicited accumulation of cAMP in guinea pig brain slices. It had no effect on cAMP‐dependent protein kinase from rat liver nor on the adenyl cyclase from rat brain.

Effects of dietary fatty acids on hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity in hamsters on a high-glucose diet

Abstract Hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity in hamsters given a fat-free high-glucose diet for 21 days was approximately 20 times higher than that in chow-fed hamsters. The increase in enzyme activity by dietary glucose was affected by saturated or unsaturated fatty acids or cholesterol added to the high-glucose diet. Ethyllinoleate or ethyloleate, added to the diet at a concentration of 5%, suppressed the increase in the enzyme activity. In contrast, addition of ethylpalmitate to the diet further stimulated the increase in the enzyme activity. Addition of 2% cholesterol to the high-glucose diet moderately suppressed, and addition of both cholesterol and ethyllinoleate completely prevented, the increase in the enzyme activity. The enzyme activity closely correlated with the incidence of formation of cholesterol gallstones but not with the liver cholesterol level. Marked increase in the enzyme activity was observed by feeding the high-glucose diet to starved hamsters for even a short period. On the third day after feeding was resumed, the enzyme activity was increased 500-fold compared to that during starvation. This increase in the enzyme activity was also reduced by dietary unsaturated fatty acid esters and stimulated by a dietary saturated fatty acid ester.

Nahocols and isonahocols, endothelin antagonists from the brown alga, sargassum autumnale

Novel endothelin antagonists, nahocols A, A1, B, C, D1 and D2, and isonahocols D1 and D2, were isolated from the brown alga, Sargassum autumnale. Their structures were determined through detailed analysis of NMR spectra and chemical reactions. Nahocols have an aryl prenyl ether structure, which are speculated to be biogenetic precursors of the ubiquitous prenyl hydroquinones or prenyl benzoquinones in the plant and animal kingdoms.

Inhibition of 15-hydroxyprostaglandin dehydrogenase by antiallergic agents

Abstract Some antiallergic agents having protective action against anaphylaxis were found to inhibit NAD + -linked 15-hydroxyprostaglandin dehydrogenase partially purified from guinea pig lung. The concentrations required to inhibit the enzyme by 50% were 3.0 μM for baicalein, 9.8 μM for xanoxic acid and 350 μM for disodium cromoglycate. The inhibitory effects of these compounds on 15-hydroxyprostaglandin dehydrogenase were greater than on cAMP phosphodiesterase activity in the same tissue. Inhibitory effects of flavonoids other than baicalein were also investigated.

Effect of an anti-ulcer drug, plaunotol, and its metabolites on NAD+ dependent 15-hydroxyprostaglandin dehydrogenase from gastric mucosa

15-Hydroxyprostaglandin dehydrogenase was partially purified from hog gastric mucosa by about 1000-fold with a 13.5% yield. Its molecular weight was estimated to be 32,000 daltons by gel filtration. The enzyme was inhibited by some metabolites of plaunotol [(2E, 6Z, 10E)-7-hydroxymethyl-3,11,15-trimethyl-2,6,10,14-hexadecatetrae n-1- ol], a new anti-ulcer drug. The inhibition patterns for substrates, prostaglandin E1 and NAD+ were both uncompetitive with Ki values of 7.8 and 19.7 microM, respectively.

Studies on griseolic acid derivatives. X: Synthesis and phosphodiesterase inhibitory activity of 2-substituted derivatives of griseolic acid

Abstract Griseolic acid derivatives which were modified at the 2-and/or 6-positions were first synthesized from griseolic acid by a ring opening—reclosure reaction of the adenine ring. Among these derivatives, the 2-amino-6-deamino-6-hydroxyl (guanine) derivative showed 3.3 and 45 times stronger inhibitory activity against cAMP and cGMP PDE, respectively, than those of griseolic acid. Structure-activity relationships among these derivatives are also discussed.

Properties of base-substituted and carboxyl-esterified analogues of griseolic acid, a potent cAMP phosphodiesterase inhibitor

Griseolic acid (GA) is a potent cyclic AMP (cAMP) phosphodiesterase (PDE) inhibitor that has an adenine base and two carboxyl groups in its molecule (Nakagawa F, Okazaki T, Naito A, Iijima Y and Yamazaki M, J Antibiot 38: 823-829, 1985). GA analogues were synthesized in which the adenine group was substituted with guanine (6-deamino-2-amino-6-hydroxygriseolic acid, G-GA) or hypoxanthine (6-deamino-6-hydroxygriseolic acid, H-GA). Their inhibitory activities to cyclic GMP (cGMP) PDE and cAMP PDE were compared with GA. For cGMP PDE from rod outer segments of bovine retina, the IC50 values of GA, G-GA and H-GA were 18, 0.040 and 0.12 microM, respectively, with 0.25 microM cGMP as substrate. For type IV PDE isozyme from mouse 3T3 fibroblast cells, the IC50 values of GA, G-GA and H-GA were 0.021, 15 and 11 microM, respectively, with 0.25 microM cAMP as substrate. Thus, GA and G-GA were found to be base-selective inhibitors of type IV PDE of 3T3 cells and type V PDE of bovine retinas, respectively. Esters of carboxylic acids of GA were synthesized in order to increase permeability into cells, and their efficacy was tested by measuring the accumulation of cAMP in 3T3 cells. The dipivaloyloxymethyl ester of GA was found to increase cAMP levels at 0.1 microM, while GA and 3-isobutyl-1-methylxanthine were active only above 100 microM, and the dimethyl ester of GA was inactive. The dipivaloyloxymethyl ester of GA seems to exert its activity after conversion to GA in the cell, since the pivaloyloxymethyl ester was easily hydrolysed by the enzyme action and the dipivaloyloxymethyl ester of GA itself was much less potent an inhibitor of PDE. The dipivaloyloxymethyl ester of GA inhibited thrombin-induced aggregation of platelets and stimulated lipolysis of adipocytes at low concentrations.