Kazuo Shinozuka

Involvement of the glutamate receptor δ2 subunit in the long-term depression of glutamate responsiveness in cultured rat Purkinje cells

An antisense oligonucleotide against the glutamate receptor delta 2 subunit mRNA, which is selectively expressed only in Purkinje neurons, suppressed the induction of long-term depression (LTD) of glutamate responsiveness in the rat cerebellar culture. LTD of glutamate response is induced by pairing glutamate application and depolarization of a Purkinje cell. Treatment of the culture with the antisense oligonucleotide exerted no appreciable effect on basic physiological and morphological properties of Purkinje cells, except for LTD induction and reduction of delta immunoreactivity which was intense in distal dendrites. Sense and missense oligonucleotides, which were used as controls, did not block LTD induction. These results suggest that the glutamate receptor delta 2 subunit is involved in the cerebellar LTD.

Synthesis of phosphorothioate analogues of oligodeoxyribonucleotides and their antiviral activity against human immunodeficiency virus (HIV)

Nuclease-resistant phosphorothioate analogues of oligodeoxynucleotides (oligos) were synthesized by sulfurization of either internucleoside phosphite linkages, in a repetitive manner during chain extension, or internucleoside hydrogen phosphonate linkages, in a single step following chain assembly. These analogues were tested as antiviral agents against human immunodeficiency virus (HIV). In a cytopathic effect inhibition assay using HIV-uninfected susceptible T cells (tetanus toxoid-specific normal T cells) co-cultured with irradiated chronically HIV-infected cells, phosphorothioate oligomers inhibited the cytopathic effect and replication of several isolates of HIV-1 and HIV-2. Thus phosphorothioate analogues of oligos could inhibit cell-to-cell transmission of the virus as well as the infection by cell-free virus particles and also could inhibit a variety of isolates of human retroviruses.

Cholesterol Biosynthesis Pathway Intermediates and Inhibitors Regulate Glucose-Stimulated Insulin Secretion and Secretory Granule Formation in Pancreatic β-Cells

Cholesterol is reportedly abundant in the endocrine secretory granule (SG) membrane. In this study, we examined the involvement of cholesterol biosynthesis intermediates and inhibitors in insulin secretion and SG formation mechanisms. There are two routes for the supply of cholesterol to the cells: one via de novo biosynthesis and the other via low-density lipoprotein receptor-mediated endocytosis. We found that insulin secretion and content are diminished by β-hydroxy-β-methylglutaryl-coenzyme A inhibitor lovastatin but not by lipoprotein depletion from the culture medium in MIN6 β-cells. Cholesterol biosynthesis intermediates mevalonate, squalene, and geranylgeranyl pyrophosphate enhanced glucose-stimulated insulin secretion, and the former two increased insulin content. The glucose-stimulated insulin secretion-enhancing effect of geranylgeranyl pyrophosphate was also confirmed in perifusion with rat islets. Morphologically, mevalonate and squalene increased the population of SGs without affecting their size. In contrast, lovastatin increased the SG size with reduction of insulin-accumulating dense cores, leading to a decrease in insulin content. Furthermore, insulin was secreted in a constitutive manner, indicating disruption of regulated insulin secretion. Because secretogranin III, a cholesterol-binding SG-residential granin-family protein, coincides with SG localization based on the cholesterol composition, secretogranin III may be associated with insulin-accumulating mechanisms. Although the SG membrane exhibits a high cholesterol composition, we could not find detergent-resistant membrane regions using a lipid raft-residential protein flotillin and a fluorescent cholesterol-Si-pyrene probe as markers on a sucrose-density gradient fractionation. We suggest that the high cholesterol composition of SG membrane with 40-50 mol% is crucial for insulin secretion and SG formation functions.

Facile synthesis of nucleotides containing polyphosphates by Mn(II) and Cd(II) ion-catalyzed pyrophosphate bond formation in aqueous solution

Abstract Mn 2+ and Cd 2+ catalyzed pyrophosphate bond formation from adenosine-5′-phosphorimidazolide and nucleotides or phosphates in neutral aqueous solution, giving nucleotides containing polyphosphates.

Labeling of phosphorothioate antisense oligonucleotides with yttrium-90

Novel yttrium-90 (90Y)-labeled phosphorothioate antisense oligonucleotides were designed as a potential targeted radionuclide therapeutic agent for malignant tumors. A 15-mer phosphorothioate antisense oligonucleotide, which was complementary to the translation start region of the N-myc oncogene mRNA, was conjugated with isothiocyanobenzyl ethylenediamine tetraacetic acid (SCN-Bn-EDTA), via a C-5-substituted deoxyuridine that had replaced a thymine in the oligonucleotide, and was then labeled with 90Y-acetate. Following purification, the radiochemical purity of the 90-Y-Bn-EDTA-phosphorothioate antisense oligonucleotides was estimated by 2.0% agarose gel electrophoresis, and the specific hybridization of 90Y-Bn-EDTA-phosphorothioate antisense oligonucleotide to a phosphorodiester sense oligonucleotide was investigated by 20% polyacrylamide gel electrophoresis in a cell-free system. Radiochemical purity was 98.7 +/- 0.4% at 72 h after labeling and 90.3 +/- 0.9% after 72-h incubation with human normal serum. The 90Y-Bn-EDTA-phosphorothioate antisense oligonucleotide hybridized specifically to a complementary phosphorodiester sense oligonucleotide. In conclusion, phosphorothioate antisense oligonucleotides can be labeled stably with 90Y using SCN-Bn-EDTA without loss of hybridization properties.

SYNTHESIS AND CHARACTERIZATION OF POLYAMINE-BASED BIOMIMETIC CATALYSTS AS ARTIFICIAL RIBONUCLEASE

Several polyamine derivatives (I–V) conjugated with or without an intercalative moiety were prepared as ribonuclease mimics. Although no DNA-cleaving activity was observed for all compounds tested, mimics I,III, and V bearing an intercalative moiety along with the primary amine and/or imidazole moieties exhibited potent RNA-cleaving activity at near physiological pH. The RNA-cleaving reactions of the compounds show characteristic bell-shaped pH dependency, and the optimal pH values for III and V were well correlated to the pK a values of their active sites, primary amine, and imidazole moieties.

Synthesis of a novel 2′-deoxyuridine derivative bearing a cyanomethoxycarbonylmethyl group at C-5 position and its use for versatile post-synthetic functionalization of oligodeoxyribonucleotides

A novel 2′-deoxyuridine derivative bearing a cyanomethoxycarbonylmethyl group at the C-5 position (1) was synthesized, and its use was examined as a convertible nucleoside for the versatile post-synthesis functionalization of oligodeoxyribonucleotides (ODNs). The ODNs containing 1 reacted with primary monoamines such as heptylamine, histamine, and tyramine as well as d- and polyamines under mild conditions, giving the corresponding derivatized ODNs.

Second-sphere ‘communication’ between two cis-bound guanine nucleotides. Factors influencing conformations of dynamic adducts of cis-type platinum anticancer drugs with guanine nucleotides as deduced by circular dichroism spectroscopy

Abstract Adducts of Pt anticancer drugs bound to N7 of guanine are fluxional, regardless of whether the guanine is part of a larger species like DNA or of a small analog like 3′- or 5′-guanosine monophosphate (3′- or 5′-GMP). This dynamic motion problem makes it impossible for NMR methods to reveal the solution conformation of the simple adducts studied here, cis -PtA 2 G 2 ( G =guanine derivative, A=NH 3 , and A 2 =trimethylenediamine ( tn ), or ethylenediamine ( en )). Each adduct has three possible atropisomers (Δ and ΛHT (head-to-tail) and HH (head-to-head) forms). Recently, we demonstrated using NMR methods that the dynamic motion problem could be minimized by the bulky CCC ligands of ( CCC )Pt G 2 complexes ( CCC =chirality-controlling chelate ligand). We identified the atropisomers and determined their conformation. We showed that the chirality of the major HT form correlated with the sign of the CD peaks of the mixture. In the present study, CD spectroscopy was used to evaluate the conformation of the dominant HT atropisomer of dynamic cis -PtA 2 G 2 adducts. All simple cis -PtA 2 G 2 adducts examined had the Δ type CD signal for G =3′-GMP and the Λ type CD signal for 5′-GMP. The CD signal type did not change when the phosphate group was protonated (pH 3), but the intensity decreased. However, the sign changed and the intensity decreased when the guanine ring NH (N1H) was deprotonated (pH ∼10). Interestingly, the 1-methylguanosine 3′-monophosphate (1-Me-3′-GMP) and 1-methylguanosine 5′-monophosphate (1-Me-5′-GMP) adducts had a Λ and Δ CD signal, respectively. 1-Me-3′-GMP was synthesized by phosphorylation of 1-methylguanosine (1-MeGuo) using sodium cyclo -triphosphate hexahydrate. For cis -PtA 2 G 2 adducts of G ’s lacking a 3′ or 5′-phosphate group (e.g. guanosine, guanosine 2′-monophosphate, and 9-ethylguanine), the CD signals were always weak. The inosine 3′-monophosphate adduct, cis -Pt(NH 3 ) 2 (3′-IMP) 2 , has a very strong CD similar to that of the 3′-GMP adduct. These results indicate that the stability of the dominant atropisomer of cis -PtA 2 G 2 complexes depends on the presence and the position of the phosphate group and on the G N1H groups, but not the G amino group. We conclude that intramolecular phosphate–N1H hydrogen bonds formed between two cis G nucleotides favor the ΔHT and ΛHT conformers for the 3′-GMP and 5′-GMP adducts, respectively. The dominance of one chiral form accounts for the characteristic enhanced CD signal.

Divalent Metal Ion-Catalyzed Pyrophosphate Bond Formation in Aqueous Solution. Synthesis of Nucleotides Containing Polyphosphate

Abstract Nucleotides containing polyphosphate were prepared from nucleoside-5′-phosphorimidazolide and nucleotides or phosphoryl compounds by divalent metal ion catalyst such as Mn2, Cd2+ or Mg2+ in aqueous solution.

Synthesis and RNA cleaving activities of polyamine derived novel artificial ribonuclease

Abstract Novel polyamine derivatives (I–IV) bearing imidazole and/or primary amine groups were prepared as artificial ribonuclease. Among the derivatives the compound (III) which has an imidazole and a primary amine groups as catalytic active sites at the ends of certain length of linker arms along with a intercalative RNA binding site exhibited the most potent RNA cleaving activity.