Maiko Higuchi

Synaptic adhesion molecule OBCAM; synaptogenesis and dynamic internalization.

Opioid-binding cell adhesion molecule (OBCAM) is the member of the IgLON family, a subgroup of the immunoglobulin superfamily. In the present study, the functions and dynamics of OBCAM were investigated in hippocampal neurons in vitro. Western blotting revealed that OBCAM expression was low at early stages of culture but it was increased as culture development. Double labeling immunofluorescence microscopy showed that OBCAM immunoreactivity was localized mainly at postsynaptic spines labeled with phalloidin and anti-PSD-95. The inhibition of OBCAM function with the specific antibody resulted in a significant decrease in the number of synapses on dendrites compared with control mouse IgG. The suppression of OBCAM expression using the antisense oligodeoxynucleotide also impaired the formation of synapses compared with control universal ones. The overexpression of OBCAM mRNA using a plasmid vector augmented the formation of synapses. Moreover, the internalization of OBCAM was promoted with increased neuronal activity by 4-aminopyridine. This internalization was reduced with the treatment of filipin, a sterol agent, indicating that this process is a raft-dependent pathway. These results indicate that OBCAM is a synaptic cell adhesion molecule concerning synaptogenesis and its surface localization is dynamically regulated in response to neuronal activity.

Selective Photo-Cross-Linking of 2′-O-Psoralen-Conjugated Oligonucleotide with Rnas Having Point Mutations

It has been reported that point mutations in genes are responsible for various cancers and the selective regulation of the gene expression is an important issue to develop a new type of anticancer drugs. In this report, we present a new type of antisense molecule that photo-cross-links to an oligoribonucleotide having a point mutation site in a sequence specific manner. 2′-O-psoralen-conjugated adenosine was synthesized in four steps from adenosine and introduced in the middle of an oligodeoxyribonucleotide (2′-Ps-oligo). Compared with 5′-O-psoralen-conjugated oligodeoxyribonucleotide (5′-Ps-oligo), which has a psoralen at the 5′-end, 2′-Ps-oligo more selectively photo-cross-linked to a pyrimidine base of the site of alteration from purine to pyrimidine in the oligoribonucleotide.

Homogeneous fluorescence assays for RNA diagnosis by pyrene-conjugated 2'-O-methyloligoribonucleotides.

We developed a bispyrene-conjugated 2 ′-O-methyloligoribonucleotide as an RNA-specific RNA-probe. The probe hybridized with the complementary RNA, greatly enhancing fluorescence and discriminating RNA from DNA. The assay was carried out in homogeneous aqueous media without removing the unbound probe from the detection solution. This homogeneous fluorescence assay also discriminated mismatch sequences in the target RNA. These pyrene probes could possess high potential to detect RNA in biological specimens simply.