Contribution of occlusal activity to synaptogenesis in masticatory muscles.

Synaptogenesis in the neuromuscular junction involves a nicotinic acetylcholine receptor (nAChR) switch and elimination. The microphthalmic mouse (mi/mi) with a mutation in the mitf gene cannot perform occlusal activity, because its teeth do not erupt. The present study attempted to elucidate the contribution of occlusal activity to synaptogenesis in masticatory muscles. In the masseter of the mi/mi, the nAChR elimination initiated, but did not progress normally, after 3 weeks of age, when the occlusal activity emerged in the +/+ mouse, whereas the nAChR switch progressed normally during the entire period of synaptogenesis. The mRNA expression patterns of nAChR subunits in the temporalis and digastric of the mi/mi differed from those in its masseter. These findings suggest that, in the masseter, occlusal activity is essential for the completion of nAChR elimination, but not for the nAChR switch, and that the contribution of occlusal activity to synaptogenesis varies among the masticatory muscles.

Mononuclear platinum(II) complex with 2-phenylpyridine ligands showing high cytotoxicity against mouse sarcoma 180 cells acquiring high cisplatin resistance.

Mouse sarcoma 180 cell with a 25-fold higher cisplatin (CDDP) resistance, termed S-180cisR, is newly established. S-180cisR cells grow quite slowly in the presence of CDDP with high concentration. This may show that S-180cisR cells modulate the cell cycle to acquire CDDP resistance. P-Glycoprotein is selectively expressed on the surface of S-180cisR, which is not on CDDP-sensitive S-180 parent cells. In an experiment using an inhibitor (verapamil) of P-glycoprotein, cytotoxicity of CDDP against S-180cisR is significantly increased (viz., IC(50) value is decreased) and accumulation of CDDP in S-180cisR cells is also increased. These results indicate that enhanced pumping-out of CDDP by P-glycoprotein should be one of the reasons for the CDDP resistance of S-180cisR. A platinum(II) complex with a cyclometalated 2-phenylpyridine ligand and a nonchelated one (complex 5) is synthesized, and its structure is determined by X-ray structural analysis. Complex 5 has a cyctotoxicity against S-180cisR higher than that of CDDP and its derivatives with 2- or 3-substituted pyridine ligands (complexes 2-4, 6, 7). Complex 5 is incorporated in S-180cisR to an enormously greater extent than CDDP; that is, the ratio of accumulated platinum amount after 3 h is 61.9. In S-180 parent cells, on the other hand, the ratio remains 8.1. This high accumulation of complex 5 into S-180cisR must account for the higher activity of complex 5 against S-180cisR compared to CDDP.

Platinum (II) complex with cyclometalating 2-phenylpyridine ligand showing high cytotoxicity against cisplatin-resistant cell.

A platinum (II) mononuclear complex with two kinds of 2-phenylpyridine which coordinate as cyclometalated and non-chelated ligands shows high cytotoxicity against cisplatin-resistant mouse sarcoma 180 cell in comparison with its related complexes.

Preparation of functional liposomes with peptide ligands and their binding to cell membranes.

Two novel lipopeptides, which have the peptide ligands [alpha-melanocyte stimulating hormone (alpha-MSH)] sequence and repeated [Gly-Arg-Gly-Asp-Se (GRGDS) sequence], are designed, synthesized by the solid-phase method, and introduced into liposome membranes by the freeze-thaw method. These liposomes bearing the peptide ligands on their surface are expected to bind to cell membranes. We have confirmed that the lipopeptides are introduced into liposome membranes almost quantitatively, while such a high degree of incorporation has not been accomplished in conventional methods. In this respect, the present method is superior to prepare surface-modified liposomes that are applicable to drug carriers and so on. We have also confirmed by using immunoelectron microscopy that the peptide ligands are actually located in an aqueous phase. It has been shown by flow cytometry that the liposome bearing alpha-MSH peptide ligand binds to B16 cells and the liposome bearing the repeated GRGDS sequence binds to NIH3T3 cells.

Lipid analog with 2-nitrophenol trigger designed for liposome fusion at physiological pH.

A novel lipid analog with two long alkyl (C16) chains, an aspartate skeleton, a connecting alkyl (C8) chain, and 2-nitrophenol trigger group is synthesized by an efficient synthetic route, which can induce liposome fusion at physiological pH.

Selective synthesis and kinetic measurement of 1:1 and 2:2 cyclic compounds containing 1,4,7,10-tetraazacyclododecane and azobenzene units

1:1 cyclic compounds 8a-c (51-55%) and 2:2 cyclic compounds 9a-c (20-49%) containing 1,4,7,10-tetraazacyclododecane (cyclen) and azobenzene units were selectively synthesized under UV irradiation (330 nm < lambda < 380 nm) and in the dark. Synthesis depended on the wavelength of irradiation light and the length of methylene chains of the linker between the cyclen and azobenzene units. A study of NMR and UV-vis spectra indicated that properties of 8a-c and 9a-c are closely related to their structural flexibility. Rate constants (k) and thermodynamic parameters (DeltaG(), DeltaH(), and DeltaS()) of 8a-c and 9a-c were studied in nonpolar media (benzene) and polar media (methanol). The cis to trans isomerization rates in the dark for these cyclic compounds increase with ring size or structural flexibility (8a < 8c < 8b < 9a < 9b < 9c). In principle, DeltaS() dominates DeltaG() in cyclic compounds.

Synthesis of a novel lipopeptide with alpha-melanocyte-stimulating hormone peptide ligand and its effect on liposome stability.

Introduction of liposomes into target cells is important for drug delivery systems. For this purpose, the surface of the liposome is equipped with ligand peptides, which may bind to specific receptors on the cell membrane. An artificial novel lipopeptide (MSH-C4A2) containing the alpha-melanocyte-stimulating hormone (alpha-MSH) sequence and two long alkyl chains was designed and synthesized, and the liposome, composed of egg phosphatidylcholine (EPC) and MSH-C4A2, was prepared. The stability of the liposome was estimated by measuring calcein leakage from the liposome inner phase. The stability of the liposome decreased upon addition of MSH-A4C2, which seemed to be attributable to the amphiphilic property of the peptide moiety (alpha-MSH) of MSH-A2C4. The stability was, however, recovered fairly well upon addition of cholesterol (Ch) or phosphatidylglycerol (PG). It was concluded therefore that the ternary system, MSH-C4A2/Ch/EPC or MSH-C4A2/PG/EPC, is suitable for preparing the functional liposome.