Alternative splicing isoform of tau protein kinase I/glycogen synthase kinase 3beta.

Glycogen synthase kinase 3 (GSK3) plays important roles in Wnt and insulin signaling, cell fate determination, and Alzheimer-like tau phosphorylation. We discovered an isoform of tau protein kinase I (TPKI)/GSK3beta with a 13 amino acid insert in the catalytic domain owing to alternative splicing. The alternative transcripts were found in the brains of the mouse, rat and human, with highly conserved sequences.

Generation of different fates from multipotent muscle stem cells.

Although neuronal and mesenchymal stem cells exhibit multipotentiality, this property has not previously been demonstrated for muscle stem cells. We now show that muscle satellite cells of adult mice are able to differentiate into osteoblasts, adipocytes and myotubes. Undifferentiated muscle progenitor cells derived from a single satellite cell co-expressed multiple determination genes including those for MyoD and Runx2, which are specific for myogenic and osteogenic differentiation, respectively.

Protein kinase C-mediated translocation of secretory vesicles to plasma membrane and enhancement of neurotransmitter release from PC12 cells.

In order to elucidate the molecular mechanism of phorbol ester-induced potentiation of neurotransmitter release, changes in the subcellular distribution of secretory vesicles were studied in PC12 cells. Dopamine (DA) and acetylcholine containing vesicles were selectively labelled by expressing green fluorescent protein-conjugated vesicular monoamine transporter and vesicular acetylcholine transporter, respectively. In the resting state, these vesicles were distributed throughout the cytoplasm.

Presence of a novel subset of NKT cells bearing an invariant V(alpha)19.1-J(alpha)26 TCR alpha chain.

CD1d-deficient (CD1d-/-) mouse lymphocytes were analyzed to classify the natural killer T (NKT) cells without reactivity to CD1d. The cells bearing a V(alpha)19.1-J(alpha)26 (AV19-AJ33) invariant TCR alpha chain, originally found in the peripheral blood lymphocytes, were demonstrated to be abundant in the NK1.

Solid-phase synthesis of oligosaccharides and on-resin quantitative monitoring using gated decoupling (13)C NMR.

A general strategy for solid-phase oligosaccharide synthesis capable of nondestructive quantitative monitoring has been developed. The synthesis was carried out on TentaGel using thioglycosides as glycosylating agents and dimethylthiomethylsulfonium triflate as the activator. An acylsulfonamide linker was introduced to cleave the oligosaccharide from the resin.

Direct and indirect inhibition of Th1 development by progesterone and glucocorticoids.

Progesterone may contribute to the maternal suppression of immunity to the fetus by modulating the Th1/Th2 balance. To clarify whether progesterone directly or indirectly affects T cell differentiation, we used two experimental systems with isolated T cells in vitro. In one system, isolated CD4+CD8+thymocytes differentiated into Th1 and Th2 by two pulse stimulations with defined combinations of ionomycin and PMA followed by the treatment with IL-12, IL-4, and IL-2.

Distribution of soluble N-ethylmaleimide fusion protein attachment proteins (SNAPs) in the rat nervous system.

Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) plays an essential role in vesicular transport and the release of neurotransmitters and hormones through associations with NSF and SNAP receptors (SNAREs). Three isoforms (alpha, beta and gamma) of SNAP are expressed in mammals. We have generated isoform-specific antibodies and studied the expression and distribution of these SNAP isoforms in the rat nervous system.

LIRF, a gene induced during hippocampal long-term potentiation as an immediate-early gene, encodes a novel RING finger protein.

We describe here an LTP-induced gene, LIRF, which encodes a novel protein with RING finger and B30.2 domains in its N- and C-terminal portions, respectively. Each domain is encoded by one exon, suggesting that the organization of the gene was generated by exon shuffling.

Induction of CCR7 expression in thymocytes requires both ERK signal and Ca(2+) signal.

CC chemokine receptor 7 (CCR7) expression is crucial for thymocyte trafficking across the corticomedullary junction in the thymus and for lymph node homing of naive T cells. However, the induction mechanism of CCR7 expression is vastly unknown. In isolated CD4+CD8+CCR7-thymocytes, a moderate 20-h pulse stimulation with a combination of the calcium ionophore ionomycin and the protein kinase C activator phorbol myristate acetate induced CCR7 expression and CD8 downregulation.

Random multi-recombinant PCR for the construction of combinatorial protein libraries.

Development of a new methodology to create protein libraries, which enable the exploration of global protein space, is an exciting challenge. In this study we have developed random multi-recombinant PCR (RM-PCR), which permits the shuffling of several DNA fragments without homologous sequences. In order to evaluate this methodology, we applied it to create two different combinatorial DNA libraries.

Regulated expression of an actin-associated protein, synaptopodin, during long-term potentiation.

We report NMDA receptor-dependent expression of synaptopodin mRNA in the dentate granule cells of the hippocampus following induction of long-term potentiation (LTP) in vivo. Synaptopodin did not belong to immediate-early genes, as de novo protein synthesis was required for the induction of synaptopodin gene transcription. An increased level of synaptopodin mRNA was observed at 75 min and 3.

Biochemical activities associated with mouse Mcm2 protein.

Mcm2, a member of the Mcm2-7 protein family essential for the initiation of DNA replication, has several biochemical activities including the ability to inhibit the Mcm4,6,7 helicase. In this study, we characterized the activities associated with Mcm2 and determined the region required for them. It was found that Mcm2 deleted at an amino-terminal portion is able to bind to an Mcm4,6,7 hexameric complex and to inhibit its DNA helicase activity.

A src family tyrosine kinase inhibits neurotransmitter release from neuronal cells.

Tyrosine kinases are expressed in many tissues, particularly in the central nervous system, and regulate various cellular functions. We report here that a src family tyrosine kinase-specific inhibitor, PP2, enhances neurotransmitter release from PC12 cells and primary cultured neurons. PP2 enhances only Ca(2+)-dependent release; it does not affect basal release.

Phosphorylation of Mcm4 at specific sites by cyclin-dependent kinase leads to loss of Mcm4,6,7 helicase activity.

Mcm proteins that play an essential role in eukaryotic DNA replication are phosphorylated in vivo, and cyclin-dependent protein kinase is at least in part responsible for the phosphorylation of Mcm4. Our group reported that the DNA helicase activity of Mcm4,6,7 complex, which may be involved in initiation of DNA replication, is inhibited following phosphorylation by Cdk2/cyclin A in vitro. Here, we further examined the interplay between mouse Mcm4,6,7 complex and cyclin-dependent kinases and determined the sites required for the phosphorylation of Mcm4.

Identification and molecular cloning of a novel brain-specific receptor protein that binds to brain injury-derived neurotrophic peptide. Possible role for neuronal survival.

Brain injury-derived neurotrophic peptide (BINP) is a synthetic 13-mer peptide that supports neuronal survival and protects hippocampal neurons in primary cultures from cell death caused by glutamate. We have developed a monoclonal antibody named mAb 6A22 against the 40-kDa BINP-binding protein, p40BBP. mAb 6A22 inhibits binding between BINP and rat brain synaptosomes and abolishes the protective effect of BINP.

Novel acyl-CoA synthetase in adrenoleukodystrophy target tissues.

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder characterized by demyelination of white matter. The X-ALD gene product adrenoleukodystrophy protein (ALDP) is expressed broadly among various tissues. However, deficiency of functional ALDP exclusively impairs brain, adrenal gland, and testis.