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Y Komiya

Publications and source records attributed to Y Komiya.

At least 37 records · Page 2Linked to original sources
PubMed1996

Reduced microtubule-nucleation activity of tau after dephosphorylation.

Based on video-enhanced differential interference contrast (DIC) microscopy, we developed a small-scale method which is capable of measuring both the lengths and the number densities of microtubules (MTs) assembled in vitro. With this method, effect of dephosphorylation on the activity of bovine brain tau protein to promote the assembly of tubulin at physiological concentration (15 microM) was quantitatively analyzed. The MT number density was selectively reduced when tau isolated directly in the presence of phosphatase inhibitors (N-tau) was dephosphorylated in vitro (DP-tau), without significant changes in the mean MT length or the binding affinity toward preformed MTs. Tau obtained from brain MTs (MT-tau) also exhibited lower nucleation activity in spite of its high MT-binding affinity. The results indicate that nucleation, elongation and MT-binding are distinct aspects of tau function which are differentially affected by the phosphorylation state of tau.

Animals↗
PubMed1996

Immunohistochemical study of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 human intervertebral discs.

STUDY DESIGN: Immunohistologic staining of human intervertebral discs collected at the time of surgery (100 intervertebral discs from 80 patients) and 10 discs collected from 7 cadavers within 12 hours of death was performed using antimatrix metalloproteinase-3 monoclonal antibody and antitissue inhibitor of metalloproteinase-1 monoclonal antibody. OBJECTIVES: To examine the relationship between matrix destruction and staining for matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 in intervertebral disc degeneration. SUMMARY OF BACKGROUND DATA: Matrix metalloproteinase-3, which decomposes aggregating proteoglycans, has attracted research attention as a substance contributing to matrix destruction in the articular cartilage and intervertebral disc. However, except for a few in vitro studies, the relationship between matrix destruction of the intervertebral disc and matrix metalloproteinase-3 has been little studied. METHODS: Immunohistologic staining was performed to examine the relationship between matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 in the intervertebral disc, and the relationship of these two agents to magnetic resonance imaging, radiographic, and surgical findings. RESULTS: Those cases testing positive for matrix metalloproteinase-3 and negative for tissue inhibitor of metalloproteinase-1 accounted for most of the surgical specimens. The matrix metalloproteinase-3-positive cell ratio was significantly correlated with the magnetic resonance imaging grade of intervertebral disc degeneration, and the matrix metalloproteinase-3-positive cell ratio observed in prolapsed lumbar intervertebral discs was significantly higher than that in nonprolapsed discs. In cervical intervertebral discs, the matrix metalloproteinase-3-positive cell ratio and staining of cartilaginous endplate were correlated with the size of osteophyte formation. CONCLUSIONS: These findings suggested that intervertebral disc degeneration is caused by disturbance in the equilibrium of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1, and that matrix metalloproteinase-3 contributes to degeneration of the cartilaginous endplate.

Adolescent↗
PubMed1996

Signaling pathway downstream of GABAA receptor in the growth cone.

The growth cone is responsible for axonal elongation and pathfinding by responding to various modulators for neurite growth, including neurotransmitters, although the sensor mechanisms are not fully understood. Among neurotransmitters, GABA is most likely to demonstrate activity in vivo because GABA and the GABAA receptor appear even in early stages of CNS development. We investigated the GABAA receptor-mediated signaling pathway in the growth cone using isolated growth cones (IGCs). Both the GABAA binding site and the benzodiazepine modulatory site were enriched in the growth cone membrane. In the intact IGC, GABA induced picrotoxin-sensitive Cl- flux (not influx but efflux) and increased the intracellular Ca2+ concentration in a picrotoxin- and verapamil-sensitive manner. Protein kinase C (PKC)-dependent phosphorylation of two proteins identified as GAP-43 and MARCKS protein was enhanced in the intact IGC stimulated by GABA, resulting in the release of MARCKS protein and GAP-43 from the membrane. Collectively, our results suggest the following scheme: activation of the functional GABAA receptor localized in the growth cone membrane-->Cl- efflux induction through the GABAA-associated Cl- channel-->Ca2+ influx through an L-type voltage-sensitive Ca2+ channel-->Ca(2+)-dependent phosphorylation of GAP-43 and MARCKS protein by PKC.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

Differential axonal transport of soluble and insoluble tau in the rat sciatic nerve.

Axonal transport of microtubule-associated protein tau was studied in the motor fibers of the rat sciatic nerve 1-4 weeks after labeling of the spinal cord with [35S]methionine. As 60-70% of low molecular weight tau in this system was found to be insoluble in 1% Triton-containing buffer, labeled proteins in 6-mm consecutive nerve segments were first separated into Triton-soluble and insoluble fractions. Two-dimensional gel electrophoresis and immunoblotting with anti-tau antibody confirmed the presence of tau among labeled, transported proteins in both fractions. Isoform composition of labeled tau was similar to that of bulk axonal tau, the most acidic species with apparent molecular mass of 66 kDa being the major component. Transport profiles obtained by measuring radioactivities associated with this major isoform showed that soluble and insoluble tau were transported at different rates. Insoluble tau, which contained the majority of tau-associated radioactivity, was transported at 1.7 mm/day in slow component a (SCa), whereas soluble tau was transported faster, at 3 mm/day, corresponding to the rate of slow component b (SCb). Cotransport of insoluble tau with insoluble tubulin in SCa suggests its association with stable microtubules.

Animals↗
PubMed1996

Growth cone collapse and inhibition of neurite growth by Botulinum neurotoxin C1: a t-SNARE is involved in axonal growth.

The growth cone is responsible for axonal growth, where membrane expansion is most likely to occur. Several recent reports have suggested that presynaptic proteins are involved in this process; however, the molecular mechanism details are unclear. We suggest that by cleaving a presynaptic protein syntaxin, which is essential in targeting synaptic vesicles as a target SNAP receptor (t-SNARE), neurotoxin C1 of Clostridium botulinum causes growth cone collapse and inhibits axonal growth. Video-enhanced microscopic studies showed (a) that neurotoxin C1 selectively blocked the activity of the central domain (the vesicle-rich region) at the initial stage, but not the lamellipodia in the growth cone; and (b) that large vacuole formation occurred probably through the fusion of smaller vesicles from the central domain to the most distal segments of the neurite. The total surface area of the accumulated vacuoles could explain the membrane expansion of normal neurite growth. The gradual disappearance of the surface labeling by FITC-WGA on the normal growth cone, suggesting membrane addition, was inhibited by neurotoxin C1. The experiments using the peptides derived from syntaxin, essential for interaction with VAMP or alpha-SNAP, supported the results using neurotoxin C1. Our results demonstrate that syntaxin is involved in axonal growth and indicate that syntaxin may participate directly in the membrane expansion that occurs in the central domain of the growth cone, probably through association with VAMP and SNAPs, in a SNARE-like way.

Animals↗
PubMed1995

Preparation of tau from the peripheral nerve: presence of insoluble low molecular weight tau with high phosphorylation.

Purely axonal tau protein of the peripheral nervous system (PNS) obtained from adult rat ventral roots was composed of both high (HMW) and low molecular weight (LMW) isoforms. While the PNS-specific HMW isoform (110 kDa) was soluble, 60-70% of the LMW isoforms with apparent molecular weights of 67 kDa, 62 kDa and 58 kDa was insoluble. When analyzed by two-dimensional electrophoresis, these axonal LMW isoforms corresponded to the most acidic species among the large number of isoforms found in brain microtubule-associated tau. Immunoreactivities towards phosphorylation-dependent antibody tau-1 and the two anti-phosphopeptide antibodies (PP1 and PP2) indicate that PNS axonal tau is highly phosphorylated at Ser190, Ser193, and Ser387, which are the sites shown to be phosphorylated in fetal brain tau and tau comprising the paired helical filaments of Alzheimer's disease.

Animals↗
PubMed1995

Phosphorylation and transport of neurofilament proteins in the rat spinal ganglion.

Neurofilament proteins (NFs) in rat spinal ganglia were labeled with [32P]orthophosphate injected into ganglia and analyzed by two-dimensional autoradiography and immunoblotting. Three polypeptides of NF were labeled irrespective of the extent of phosphorylation. Most of the labeled NFs were transported from cell bodies to proximal axons within 24 h. A major fraction of low phosphorylated NF-H changed to high phosphorylated form in intraganglionic nerve fibers and peripheral nerves adjacent to spinal ganglia. A small fraction of low phosphorylated NF-H appeared earlier than the high phosphorylated form in adjacent peripheral nerves, suggesting that newly synthesized NF-H in cell bodies migrate a long distance before they are extensively phosphorylated and assembled into the cytoskeleton in proximal axons.

Animals↗
PubMed1995

Two stages in neurite formation distinguished by differences in tubulin metabolism.

Changes in tubulin solubility during neurite formation were studied biochemically using rat dorsal root ganglion neurons in culture. When fractionated with Ca(2+)-containing buffer at low temperature, a considerable proportion of total cellular tubulin was recovered in the insoluble fraction. We designated this cold/Ca(2+)-insoluble tubulin (InsT) and distinguished it from cold/Ca(2+)-soluble tubulin (SoIT). From the relative amount of InsT, neurite formation was found to proceed through two distinct stages. The first 6 days after plating (stage 1) in which the proportion of InsT increased dramatically (from 5 to 60%) coincided with neurite outgrowth. In the following period (stage 2), a constant level of InsT was maintained, whereas neurite maturation took place. Pulse-labeling experiments further revealed that the two stages differed significantly in terms of tubulin metabolism. High rates of synthesis as well as conversion from SoIT to InsT were observed in stage 1, whereas stage 2 was characterized by a decrease in both of these rates and an increase in the rate of degradation. The results show for the first time the coordinated changes in tubulin metabolism that underlie the process of neurite formation.

Animals↗
PubMed1994

Decreased synaptic density in aged brains and its prevention by rearing under enriched environment as revealed by synaptophysin contents.

Changes in synaptic density in various brain regions were assessed among different age groups of rats maintained in ordinary small cages, as determined by synaptophysin assay. The synaptophysin content in hippocampus decreases as early as in the adult stage. The most remarkable decrement occurs in occipital cortex. In other regions, synaptophysin contents decrease in senescence to 60-77% of the respective peak values during young and adult stages. The other rat group reared under enriched environment in a large cage until 30 months of age was examined for synaptic density, and was revealed to maintain the similar levels as in young, or even higher levels in frontal, temporal, entorhinal cortices and hippocampus. These results indicate that the synaptic density in cerebrum decreases in senescence and this decrease can be prevented by rearing under enriched environment.

Aging↗
PubMed1994

Characteristics of gangliosides including O-acetylated species in growth cone membranes at several developmental stages in rat forebrain.

Growth cones, the motile tips of extending neuronal processes, are involved in accurate synaptogenesis. To study the developmental changes in ganglioside composition including O-acetylated gangliosides in growth cones, we analyzed the gangliosides in growth cone membranes (GCM) prepared from rat forebrains at different developmental stages. At several stages, GCM contained significantly larger amounts of gangliosides than the other membrane subfractions. The ganglioside content of GCM increased in amount with development. Moreover, in GCM, the relative amount of GD3 gradually decreased, and that of GD1a dramatically increased. There were significant differences in the composition of ganglioside species between GCM and the perinuclear plasma membrane subfraction (NM); most importantly, GCM had a higher ratio of GD1a to GM3 plus GD3 than NM. There were three different O-acetylated gangliosides in GCM: O-acetyl-GD3, O-acetyl-GT1b, and O-acetyl-GQ1b. The molar ratio of O-acetyl-GD3 decreased in GCM at later stages (5% of the total gangliosides at embryonic day 17, to 1% at postnatal day 5). However, those of the other two O-acetylated gangliosides were almost constant (1-2% of the total). Our results show that there are significant differences in ganglioside content and composition between the membrane subfraction of growth cones and the perinuclear portion. This suggests that several species of gangliosides, including O-acetyl-GD3, play a role in growth cone function.

Acetylation↗
PubMed1994

Axonal transport of actin and actin-binding proteins in the rat sciatic nerve.

Actin is one of the major cytoskeletal proteins carried in slow axonal transport. Since more than 50% of actin in the axon was recovered in the high-speed supernatant, we looked for G-actin-binding proteins in slow axonal transport. Two weeks after injection of L-[35S]methionine into the rat spinal cord (L3-L5), labeled proteins in the sciatic nerve were extracted and those with potential abilities to interact with G-actin were detected by two independent methods: (A) DNAase I affinity chromatography and (B) blot overlay with biotinylated actin. By method (A), a 68 kDa Ca(2+)-dependent binding protein and a 45 kDa Ca(2+)-independent binding protein were detected. The 68 kDa protein was also a major protein binding to actin in method (B). The 68 kDa protein was identified with the Ca(2+)-dependent phospholipid binding protein annexin VI by two-dimensional electrophoresis and Western blotting. As annexin VI is a component of slow axonal transport, it does not seem to be bound to membranous organelles in the axon. Our results suggest that annexin VI may play a role in the control of actin assembly and membrane-microfilament interaction.

Actins↗
PubMed1994

Early effects of beta,beta'-iminodipropionitrile on tubulin solubility and neurofilament phosphorylation in the axon.

To elucidate the role of neurofilaments in microtubule stabilization in the axon, we studied the effects of beta,beta'-iminodipropionitrile (IDPN) on the solubility and transport of tubulin as well as neurofilament phosphorylation in the motor fibers of the rat sciatic nerve. IDPN is known to impair the axonal transport of neurofilaments, causing accumulation of neurofilaments in the proximal axon and segregation of neurofilaments to the peripheral axoplasm throughout the nerve. Administration of IDPN at various intervals after radioactive labeling of the spinal cord with L-[35S]methionine revealed that transport inhibition occurred all along the nerve within 1-2 days. Transport of cold-insoluble tubulin, which accounts for 50% of axonal tubulin, was also affected. A significant increase in the proportion of cold-soluble tubulin was observed, reaching a maximum at 3 days after IDPN treatment and returning to the control level in the following weeks. Preceding this change in tubulin solubility, a transient decrease in the phosphorylation level of the 200-kDa neurofilament protein was detected in the ventral root using phosphorylation-dependent antibodies. These early changes agreed in timing with the onset of segregation and transport inhibition, suggesting that interaction between neurofilaments and microtubules possibly regulated by phosphorylation plays a significant role in microtubule stabilization.

Actin Cytoskeleton↗
PubMed1994

Impairment of cytoskeletal protein transport due to aging or beta,beta'-iminodipropionitrile intoxication in the rat sciatic nerve.

Three major age-related changes in cytoskeletal organization and metabolism in the axon were observed by comparing slow axonal transport in the sciatic nerves of rats aged 7-80 weeks: (a) a progressive decrease in the rate of slow axonal transport, (b) a tight association of cold-insoluble tubulin with the neurofilament (NF) proteins and (c) an accelerated proteolysis of the severely retarded proteins, especially NF proteins. These changes were reproduced to a large extent in the young animal by intoxication with beta,beta'-iminodipropionitrile (IDPN). As IDPN is known to impair the axonal transport of NF proteins and cause segregation of NFs from microtubules, the results indicate that NF-microtubule interaction is one of the major factors regulating the axonal cytoskeleton. The importance of the balance between transport rate and degradation rate in the maintenance of the normal axonal cytoskeleton is stressed especially in the aged animal.

Actins↗
PubMed1993

Effect of an aromatase inhibitor in human follicular fluid on DNA synthesis of granulosa cells, theca cells, and follicles.

An aromatase inhibitor was identified in human follicular fluid. We sought to examine the effects of this inhibitor on granulosa cells, theca cells, a mixture of granulosa and theca cells, and follicles. Granulosa cells, theca cells, the mixture of granulosa and theca cells, and follicles were incubated with [3H]-thymidine and aromatase inhibitor at 37 degrees C for 40 h in Ham F-10 medium. The radioactivities of acid-soluble fractions were counted. Aromatase inhibitor markedly inhibited the DNA synthesis of granulosa cells and follicles, but it did not inhibit the mixture of granulosa and theca cells. This suggested that aromatase inhibitor was a factor in the follicle paracrine system and acted mainly on granulosa cells.

Animals↗
PubMed1992

Actin-binding proteins in the growth cone particles (GCP) from fetal rat brain: a 44 kDa actin-binding protein is enriched in the fetal GCP fraction.

Neuronal growth cones, the motile tips of growing neurites, are thought to play a significant role in nerve growth. To study the role of actin in their motility, we examined actin-binding proteins in growth cone particles (GCP) isolated from fetal rat brain, using a blot-overlay method with biotinylated actin. Among the more than ten species of actin-binding proteins in the GCP, a 44 kDa protein was found specifically in growth cones and was enriched in the cytoskeletal and the membrane skeletal subfractions from the GCP. This protein binds to actin in a Ca(2+)- and Mg(2+)-dependent manner, and ATP enhances its binding to actin. The protein was predominantly present in the fetal GCP, but it is expressed at a much lower level in the neonatal GCP and not detected in adult synaptosomes. The protein also bound to a deoxyribonuclease I column and was eluted by EGTA-containing buffer. The 44 kDa protein appears to be a novel actin-binding protein, since none of the known actin-binding proteins exhibit this combination of properties. Our results suggest that the protein may be involved with the early stages of neurite extension.

Animals↗
PubMed1992

Biochemical characterization of nerve growth cones isolated from both fetal and neonatal rat forebrains: the growth cone particle fraction mainly consists of axonal growth cones in both stages.

Nerve growth cones are responsible for the exact pathway finding, and for the establishment of neurocytoarchitecture. To elucidate the developmental changes of biochemical characteristics of nerve growth cones, growth cone particle (GCP) fractions were isolated biochemically from embryonal day 17 (E17) rat forebrain and from postnatal day 5 (P5). There were no significant differences in protein phosphorylation pattern in a Ca(2+)-dependent manner between E17-GCP fraction and that of P5. As for the membrane lipid composition, molar ratios of cholesterol to total phospholipids were well conserved during these ages. The immunoreactivity to anti-synaptophysin monoclonal antibody as a marker of mature synaptic elements could not be detected either in E17-GCP or P5-GCP fractions. To exclude the possibility of the contamination of dendritic elements, RNA contents and immunoreactivity to anti-high molecular weight microtubule-associated protein 2 (MAP2) monoclonal antibody were examined. RNA contents of the GCP fractions were extremely low compared to those of other subcellular fractions both in E17 and P5. No immunoreactivities to anti-MAP2 antibody were observed in either GCP fraction. Our results suggest that the GCP fractions, isolated from forebrains of E17 to P5 rat, are free from the contamination of the synaptic elements, and that the GCP fractions are mainly composed of axonal growth cones.

Aging↗
PubMed1992

Changes of fast axonal transport by taxol injected subepineurally into the rat sciatic nerve.

In contrast to the complete and long-lasting inhibition of tubulin transport, taxol has no effect on fast axonal transport examined immediately after its sub-epineural application to rat sciatic nerve. However, a significant accumulation of rapidly migrating radioactivity appears at the site proximal to the injection when examined a few weeks after treatment, probably due to mechanical obstruction caused by abnormal aggregation of a huge number of intra-axonal microtubules. It also decreases slightly in amount within a few weeks post-treatment, which may be due to reduction of the number of axons caused by degeneration.

Animals↗