Tomohisa Moriguchi

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.

Alternate stranded triplex formation of chimeric DNA composed of tandem α- and β-anomeric strands

A chimeric oligoDNA composed of a natural β-anomeric oligonucleotide portion and an unnatural α-anomeric oligonucleotide portion forms an alternate stranded triplex possessing enhanced thermal stability compared to the triplexes composed of the parental oligomers.

Development of the excimer probe responsible for DNA target bearing the silylated pyrenes at base moiety.

For the development of the excimer probe responsible for DNA target, the deoxyuridine phosphoramidite derivative bearing the silylated pyrene attached at the C-5 position was prepared and incorporated into oligonucleotides. The modified oligonucleotides showed the excimer emission in the absence of the target DNA, on the other hands, the excimer emission was quenched in the presence of the target DNA. For the utilization of the fluorescence behavior, the novel molecular beacon probe containing the silylated pyrene-modified nucleoside at the stem region was designed and the fluorescence property of the probe found to show the responsibility for DNA target.

Synthesis and Duplex-Forming Property of Oligonucleotides Bearing a Novel Polyamine-Modified Intercalator at the Terminal or the Internal Position

Novel oligonucleotides bearing a polyamine-intercalator conjugate modified at the terminal or the internal position were reported. These modified oligonucleotides showed duplex-stabilization effect, and the thermodynamic analysis and the salt concentration dependency of the duplex stability revealed that the polyamine moiety also acted as the duplex stabilizer by neutralization of the phosphate negative charge.

Synthesis and Spectroscopic Properties of a Novel Bifunctional Pyrene Derivative and its Incorporation into OligoDNA

A new pyrene derivative bearing a modifiable silyl function (electron-donating group) and cyano function (electron-withdrawing group) was synthesized as a labelling agent for biological substances. The modified pyrene exhibited a marked bathochromic shift in both absorption and fluorescence spectra. The fluorescence quantum yield of the modified pyrene was also significantly increased compared with that of the unmodified pyrene. The modified pyrene was successfully incorporated into oligoDNA.

Synthesis and Properties of Fluorescent Biological Molecules Labeled with Novel Silylated Perylene Derivative

We have synthesized novel perylene derivatives bearing a silyl function with modifiable functional groups as a feasible material for visualizing biological substances. The fluorescent perylene derivative was coupled to C-5 position of pyrimidine nucleotide and the nucleotide was subsequently incorporated to the middle position of oligoDNA using an automated DNA synthesizer. Also, the derivative was coupled with a cholesterol analog with different length of linking alkyl function. The resulting modified fluorescent oligoDNA exhibited a small change in its fluorescent signal upon hybridization with the complementary oligoDNA. While the cholesterol analogs bearing the silylated perylene exhibited marked fluorescent signal in living cells. The signal is stronger in the analog having longer linker function compared to the analog having shorter linker function. In each case, however, the signal was much prominent compared to that of dehydroergosterol (DHE), a fluorescent cholesterol analog widely used in biological study.

Synthesis and Properties of Molecular Beacon DNA Probe Bearing Novel Silylated Pyrene Derivative

A novel stem-loop structured fluorescent oligoDNA probe (molecular beacon probe) bearing a silylated pyrene derivative at C-5 position of deoxyuridine has been synthesized. The fluorescently labeled modified nucleoside has been incorporated into two consecutive positions in the stem segment of the DNA using an automated DNA synthesizer. The resulting modified DNA exhibited an excimer fluorescent signal upon binding to the fully matched complementary DNA strand. The excimer emission was, however, effectively quenched while it stays alone or it hybridizes to a single base mismatched complementary target.

Novel method of the synthesis and hybridization properties of an oligonucleotide containing non-ionic diisopropylsilyl internucleotide linkage

Efficient synthesis of a dithymidine dinucleotide analog bearing a diisopropylsilyl linkage instead of a phosphodiester linkage is described with respect to its incorporation into oligonucleotides. The diisopropylsilyl linkage was introduced into the oligonucleotide by preparation of the phosphoramidite derivative of a dithymidine dimer unit. The diisopropylsilyl-modified oligonucleotide exhibited hybridization behavior with both single strand and duplex DNA. The thermal stability of both the duplex and triplex showed a relative instability compared to the corresponding natural phosphodiester DNA, because of the steric hindrance of the isopropyl group on the silicon atom.