Tatsumi Hirata

The A5 antigen, a candidate for the neuronal recognition molecule, has homologies to complement components and coagulation factors

The A5 antigen is a neuronal cell surface protein of Xenopus presumed to be involved in the neuronal recognition between the optic nerve fibers and the visual centers. Analyses of cDNA clones revealed that the A5 antigen is a class I membrane protein containing two different internal repeats in the extracellular segment. The first repeat bears homology to domain III of complement components C1r and C1s, and the second repeat is homologous to the C1 and C2 domains of coagulation factors V and VIII. The mRNA for the A5 antigen was present in retinal ganglion cells and visual center neurons. Nonneuronal cells in the peripheral and central nervous systems did not express the mRNA for the A5 antigen.

ROLES OF A NEURONAL CELL-SURFACE MOLECULE, NEUROPILIN, IN NERVE FIBER FASCICULATION AND GUIDANCE

Abstract. Neuropilin is a cell-surface glycoprotein that was first identified in Xenopus tadpole nervous tissues and then in chicken and mouse. The primary structure of neuropilin is highly conserved among these vertebrate species. The extracellular part of the molecule is composed of three domains referred to as a1/a2, b1/b2, and c, each of which is expected to be involved in molecular and/or cellular interactions. Neuropilin can mediate cell adhesion by heterophilic molecular interaction. In all vertebrate species examined, the neuropilin protein is restricted to axons of particular neuron classes, and at stages when axon growth is active. The gain and loss of function of neuropilin in developing mouse embryos causes defasciculation and incorrect sprouting of nerve fibers. These findings suggest that neuropilin serves in a variety of neuronal cell interactions by binding to a variety of molecules, and that it plays essential roles in nerve fiber fasciculation and guidance.

Growth-Associated Expression of a Membrane Protein, Neuropilin, in Xenopus Optic NerveFibers

Neuropilin (previously referred to as A5) is a neuronal cell surface protein which is widely distributed in such vertebrate species as Xenopus, chicken and mouse. In these vertebrate species, neuropilin is expressed in particular neuron circuits at particular developmental stages when axonal growth is active. To test whether the expression patterns of neuropilin is associated with axonal growth, we examined expression patterns of neuropilin in developing and regenerating Xenopus optic nerves. In embryos, a neuropilin-specific monoclonal antibody A5 (MAbA5) bound strongly to the optic nerves, and in situ hybridization signals for neuropilin mRNA were prominent in the retinal ganglion cells (RGCs), indicating that developing optic nerve fibers express neuropilin. The binding of MAbA5 to the optic nerve fibers was maximal at stages 41-43, then decreased in the subsequent tadpole life. In tadpoles after stage 50, the binding of MAbA5 to the optic nerves was extremely weak or almost nil, although RGCs expressed considerable amounts of neuropilin mRNA. When the tadpole optic nerves were crushed and prompted to regenerate, however, neuropilin protein reappeared in the optic nerve fibers. The binding of MAbA5 to the regenerating optic nerve fibers was detectable as early as the 5th day and maximal at the 2nd or 3rd weeks after the optic nerve crush, and declined thereafter. These findings suggest that the expression of neuropilin in the optic nerve fibers is regulated in an axonal growth-associated manner.

Catch-relaxing peptide isolated from Mytilus pedal ganglia

A peptide that relaxes catch tension of the anterior byssus retractor muscle of Mytilus edulis was purified from pedal ganglion extracts of the mussel. Its primary structure was determined to be H-Ala-Met-Pro-Met-Leu-Arg-Leu-NH2. This peptide was found to have not only catch-relaxing action on the byssus retractor muscle but also modulatory actions on contractions in various molluscan muscles.

CHARACTERIZATION OF C-KIT-POSITIVE NEURONS IN THE DORSAL ROOT GANGLION OF MOUSE

Previously, we showed that c-kit receptor tyrosine kinase is expressed by a subpopulation of dorsal root ganglion (DRG) neurons, and that the ligand for the c-kit receptor, stem cell factor (SCF), induces the neurite outgrowth and supports the survival of these neurons in culture [16]. However, it is unknown which class of DRG neurons express c-kit receptor and which factor regulates differentiation and survival of c-kit-positive neurons. In the present study, we attempted to characterize c-kit positive neurons in the mouse DRG. The c-kit-positive neurons were small or medium in size, and 44% of these neurons contained substance P. Central fibers of the c-kit-positive neurons terminated in laminae I and II of the gray matter of the spinal cord. These results suggest that c-kit-positive neurons in the DRG belong to a functional subpopulation. The c-kit receptor protein was presented on the membrane of processes and growth cones in neurons. When DRG cells of embryonic day 15.5 or 17.5 were cultured, the survival of c-kit-positive neurons was supported by SCF, nerve growth factor (NGF) or leukemia inhibitory factor. SCF and NGF synergistically supported the survival of c-kit-positive neurons at submaximal concentrations. c-kit-positive DRG neurons from neonatal mice survived without addition of any factor in culture, suggesting that the requirement for trophic support in c-kit-positive neurons changes during development.

The membrane protein A5, a putative neuronal recognition molecule, promotes neurite outgrowth

The A5 is an unique membrane protein that is expressed in the neurons of the visual system and general somatic sensory system of Xenopus laevis. We cultured retinal explants or trigeminal ganglion neurons on the monolayers of A5-expressing line cells obtained by transfection with the cDNA, and found that the A5-expressing transfectants promote neurite outgrowth for these A5-expressing neurons. The neurite outgrowth-promoting effect was inhibited by anti-A5 antibodies. While, the neurite outgrowth-promotion by the A5 transfectants was not observed for the vestibulocochlear ganglion neurons which lack the A5. These results indicate that the membrane protein A5 is a potent substrate for neurite extension of the A5-expressing neurons but not for the A5-negative ones, suggesting its involvement in specific neuronal interaction.

Effects of the catch-relaxing peptide on molluscan muscles

Abstract 1. Effects of the catch-relaxing peptide (CARP) on contractile responses were examined in the anterior byssus retractor muscle (ABRM) of Mytilus edulis and in various other molluscan muscles. 2. In the ABRM, phasic contraction in response to repetitive electrical pulses of stimulation was potentiated by low concentrations of CARP and inhibited by high concentrations. The inhibitory effect reached maximum in 2–10 min after applying the peptide. Contractions of the muscle in response to acetylcholine (ACh) and 100 mM K + artificial seawater (ASW) were also potentiated by low concentrations of CARP and inhibited by high concentrations. Contractions in response to FMRFamide and caffeine were simply inhibited by CARP. 3. In the radula protractor and retractor muscles of Rapana thomasiana and Fusinus perplexus , contractions in response to electrical pulse, ACh and glutamate were inhibited by CARP. 4. In the herats of Mytilus edulis , Merelrix lusoria , Tapes japonica and Rapana thomasiana , their activities were inhibited by CARP. The ACh-antagonist benzoquinonium did not affect the inhibitory effect of the peptide. 5. The results of the present study suggest that CARP has modulatory effects on contractions in many molluscan muscles and that its principal modulatory effect is one of inhibition.

Localization and developmental expression of a novel protein kinase C δ gene

The expression and localization of a novel protein kinase C delta (nPKC delta) mRNA were investigated using Northern blotting and in situ hybridization in the developmental process of mouse brain. In adult mice, nPKC delta was abundantly expressed in the thalamus, moderately in the pons and the cerebellum, but faintly in the cerebral cortex and the spinal cord. By in situ hybridization, the signals were observed specifically at the sensory and motor relay nuclei of the thalamus, the dorsal cochlear nuclei of the pons, and the molecular layer of the cerebellum. When developmental changes in the expression of nPKC delta gene were analyzed by in situ hybridization, it was not detectable in embryonic and neonatal brains, very weakly expressed in the thalamus in the first week, and highly expressed at two weeks of age. These results suggest that the gene expression of nPKC delta is strictly controlled by both the cell type and the developmental process.

Pharmacology of relaxing response of mytilus smooth muscle to the catch-relaxing peptide

Abstract 1. Pharmacological properties of relaxing action of the catch-relaxing peptide (CARP) on the anterior byssus retractor muscle of Mytilus were examined and compared with those of the actions of other relaxing stimuli. 2. Butaclamol (10 −5 M), a dopamine blocker, showed little effect on relaxing responses to CARP, serotonin and repetitive electrical pulses of stimulation. 3. Mersalyl (5 × 10 −4 M), a serotonin blocker, depressed but did not abolish relaxing response to CARP. Pre-treatment of the muscle with KCl-EGTA solution also depressed but did not abolish the response to CARP. 4. Propranolol (10 −3 M) depressed or abolished relaxing responses to serotonin, dopamine and repetitive electrical stimulation, whereas it enhanced the response to CARP. 5. Phenoxybenzamine (10 −4 M) enhanced relaxing responses to CARP, serotonin and repetitive electrical stimulation, but it showed little effect on the response to dopamine. 6. Simple exposure of the muscle to La 3+ (5 mM) showed little effect on relaxing response to CARP. 7. When the muscle was continuously immersed (for 30 min or more) in CARP (5 × 10 −8 M), relaxing response to repetitive electrical stimulation was depressed or abolished. 8. It is suggested that CARP relaxes catch tension of the ABRM by acting on postsynaptic sites in the muscle.

Effects of Mytilus inhibitory peptides on mechanical responses of various molluscan muscles

Abstract 1. Effects of the Mytilus inhibitory peptides (MIPs), Ala 2 -MIP and Ser 2 -MIP, on mechanical responses of various molluscan muscles were examined. 2. In the ABRM of Mytilus edulis , all contractions in response to repetitive electrical pulses of stimulation, ACh, FMRFamide and caffeine were inhibited by MIPs. Contraction by 400 mM K + ASW was invariably not affected, but that by 100 mM K + ASW was slightly inhibited in some muscles. 3. In the pedal retractor muscle of Mytilus edulis , contractions in response to repetitive electrical pulses of stimulation and ACh were also inhibited by MIPs. 4. In the heart of Meretrix lusoria , its activity was inhibited by MIPs. Ala 2 -MIP was found to be more potent than Ser 2 -MIP. The ACh-antagonist benzoquinonium did not change the inhibitory effects of the peptides. In the heart of Tapes japonica , both of the peptides showed weak inhibitory effect at 100 nM. At 10 μM, however, Ala 2 -MIP showed inhibitory effect followed by excitatory effect, and Ser 2 -MIP showed excitatory effect and then brought about a transient cystolic arrest. 5. Twitch and tetanic contractions of the penis retractor muscle of Achatina fulica were inhibited by MIPs. Ala 2 -MIP was found to be more potent than Ser 2 -MIP. 6. MIPs seem to have an inhibitory effect on many molluscan muscles. There may exist a family of MIP-related peptides in molluscs.