Rika Kato

Neurotransmitter Release from Synaptotagmin-Deficient Clonal Variants of PC 12 Cells

Synaptotagmin (p65) is an abundant synaptic vesicle protein of neurons and contains regions similar to the regulatory domain of protein kinase C. These domains are thought to be involved in calcium-dependent interaction with membrane phospholipids during exocytosis. To assess the functional role of synaptotagmin, synaptotagmin-deficient clonal variants of PC12 cells were isolated. All of the variant cells released catecholamine and adenosine triphosphate in response to elevated intracellular concentrations of calcium, which suggests that synaptotagmin is not essential for secretion of catecholamine and adenosine triphosphate from PC12 cells.

Localization and Developmental Changes of τ Protein Kinase I/Glycogen Synthase Kinase‐3β in Rat Brain

Abstract: τ protein kinase I (TPKI) purified from bovine brain extract has been shown to phosphorylate τ and to form paired helical filament (PHF) epitopes and was found recently to be identical to glycogen synthase kinase‐3β (GSK‐3β). Before elucidating a role of TPKI/GSK‐3β in PHF formation, it is necessary to investigate the normal function of the enzyme. To study the distribution and developmental changes of the enzyme, specific polyclonal antibodies were prepared against TPKI and GSK‐3α. Immunoblot analysis demonstrated that TPKI was nearly specifically localized in the brain of adult rats. The level of TPKI in the rat brain was high at gestational day 18, peaked on postnatal day 8, and then decreased rapidly to a low level, which was sustained up to 2 years. Immunohistochemistry indicated primarily neuronal localization of TPKI. Growing axons were stained most intensely in the developing cerebellum, but the immunoreactivity became restricted to the gray matter in the mature tissue. Parallel fibers had a high level of TPKI and also stained intensely for τ. These findings indicate that τ is one of the physiological substrates of TPKI and suggest that the enzyme plays an important role in the growth of axons during development of the brain.

τ Protein Kinase II Is Involved in the Regulation of the Normal Phosphorylation State of τ Protein

Abstract: To study the phosphorylation state of τ in vivo, we have prepared antisera by immunizing rabbits with synthetic phosphopeptides containing phosphoamino acids at specific sites that are potential targets for τ protein kinase II. Immunoblot experiments using these antisera demonstrated that τ in microtubule‐associated proteins is phosphorylated at Ser144 and at Ser315. Almost all τ variants separated on two‐dimensional gel electrophoresis were phosphorylated at Ser144 and nearly one‐half of them at Ser315. Phosphorylation at Ser144 and at Thr147 of τ isolated from heat‐stable brain extracts was shown to be developmentally regulated, with the highest level of phosphorylation found at postnatal week 1. In vitro phosphorylation of τ by τ protein kinase I, a kinase responsible for abnormal phosphorylation of τ found in paired helical filaments of patients with Alzheimers disease, was enhanced by prior phosphorylation of τ by τ protein kinase II. Thus, we suggest that τ protein kinase II is indirectly involved, at least in part, in the regulation of the phosphorylation state of τ in neuronal cells.

Bicyclic turned dipeptide (BTD) as a β-turn mimetic; its design, synthesis and incorporation into bioactive peptides

Abstract After reviewing the development of β-turn mimetics briefly, design, synthesis and incorporation into bioactive peptides of BTD will be mentioned. The biological activity of the BTD containing peptides and synthesis of some BTD derivatives will also be described.

Properties of ω conotoxin MVIIC receptors associated with α1A calcium channel subunits in rat brain

Solubilized 125I‐ω conotoxin MVIIC receptors from rat cerebellum were immunoprecipitated by antibodies directed against the calcium channel α1A subunit. Anti‐αIA antibodies recognized a 240‐220, 180 and 160 kDa proteins in immunoblots of cerebellar membranes. Disuccinimidyl suberate cross‐linked 125I‐ω conotoxin MVIIC to an α2δ‐like 200‐180 kDa subunit, which migrated at 150‐140 kDa after disulfide reduction. These observations are consistent with a heteromeric structure in which high affinity ω conotoxin MVIIC binding sites formed by α1A subunits are located in close proximity to peripheral α2 subunits.

Improved protective groups for phosphate of o-phosphoserine useful for the solid-phase peptide synthesis

Abstract 2-Methylphenyl and 2,6-dimethylphenyl groups on phosphate moiety of O-phosphoserine were more stable in HF treatment than phenyl group and could be effectively removed by hydrogenolysis. They were shown to be protective groups for phosphoserine practically applicable in the Boc/Bzl mode of solid-phase peptide synthesis.

Amphiphilic helix is essential for the activity of brain injury-derived neurotrophic peptide (BINP)

To study the structure‐activity relationships of brain injury‐derived neurotrophic peptide (BINP), 12 analogs were synthesized by replacing each amino acid residue with Gly. BINP showed CD spectra typical of an α‐helical conformation in TFE solution which mimics the membrane environment. In the α‐helical conformation, BINP showed an amphiphilic profile. Neurotrophic activities of BINP and its analogs were estimated from the effects on supporting septal cholinergic neurons and on rescuing hippocampal neurons from injury caused by glutamate. Both assays showed that the residues on the hydrophobic side of the amphiphilic helix were essential for the neurotrophic activity.

1021 Regional expression of an o-acetylated ganglioside epitope in subsets of neuroepithelial cells of chicken embryonic forebrain

Shinobu C. Fujita, Rika Kato, Hideyoshi Higashi Monoclonal antibody(MAb) 85A9 stains a certain subset of neurons and undifferentiated neuroepithelial cells of restricted regions of the chicken embryonic brain. Immunochemical results had indicated a glycolipid nature of the antigen. In this work MAb 85A9-immunoreactive bands were identified on TLC of extracts of embryonic brain. The stained bands were similar but not identical to those of MAbs 8A2 or 493D4 with reported specificity to certain 0-acetylated gangliosides. The 85A9 immunoreactivity was lost by treatment with sialidase or sodium methylate, a deacetylating reagent. The histochemical immunoreactivity was destroyed by sialidase but not by periodate treatments. Thus the antigen was considered to be 0-acetylated gangioside(s). A whole mount immunostaining procedure that does not use detergents was developed to visualize the glycolipid antigen in chicken embryos up to embryonic day 5, and the diencephalic staining patterns deduced from the sections were confirmed.

1123 Antigen 802C11, expressed in a subset of cranial ganglia, is a membrane glycoprotein

1121 DIFFERENTIAL EXPRESSION OF TAG-l/F3 AND Ll AxCAM SUBGROUP MEMBERS IN THE DEVELOPING AND ADULT RAT NERVOUS SYSTEM. ATSUSHI TAMADA’j2, YOSHIHIRO YOSHIHARArs, KENSAKU MORItp2, lDeDt. of Neuroscience. Osaka Bioscience Institute, Osaka 565, *Lab. Neuronal Recovnition Molecules. Frontier Research Proeram, RIKEN. Saitama 351-01, 3DeDt. of Biochemistrv. Osaka Medical College. Osaka 569. laDan. BIG-l/BIG-2/TAG-1 /F3 and Ll /NrCAM/neurofascin constitute two axon-associated cell adhesion molecule (AxCAM) subgroups of the immunoglobulin superfamily. We previously reported that TAGl/F3 subgroup mRNAs are differentially expressed in the rat nervous system. To elucidate the role of the two AxCAM subgroups, we compared expression of the seven different AxCAM mRNAs by in situ hybridization. We also examined immunohistochemical localization of BIG-l and BIG-2 proteins using specific antisera. Ll subgroup mRNAs were expressed in distinct regions of the developing and adult nervous system. Ll was strongly expressed in the thalamus and hypothalamic paraventricular and supraoptic nuclei; NrCAM in the inferior olive, the median eminence (ME) and the floor plate; and neurofascin in the motor nuclei and the white matter, suggesting distinct roles of each AxCAM member. BIG-l and BIG-2 proteins were localized in subsets of axonal processes. In the ME, BIG-l and BIG-2 were expressed in the tanycyte processes and axonal terminal region of the external layer, respectively, suggesting the possible interaction of the two molecules.