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Y Komiya

Publications and source records attributed to Y Komiya.

At least 55 records · Page 3Linked to original sources
PubMed1991

Subtypes of protein kinase C in isolated nerve growth cones: only type II is associated with the membrane skeleton from growth cones.

The growth cone particle (GCP) fraction was isolated from fetal and neonatal rat brains and the distribution of protein kinase C subtypes in the fraction was examined by using subtype-specific antibodies. The main subtype in the GCP fraction from fetal forebrain was type II, and type III was also present, but not type I. The pattern was not altered from embryonic day 17 to postnatal day 5. The membrane skeleton subfraction from the GCP fraction contained type II, but far less amount of type III. Our results suggest that type II and type III may be closely related to the functions of growth cones but that they appear to be associated with distinct signal transduction processes.

Animals↗
PubMed1991

Maturation and aging of the axonal cytoskeleton: biochemical analysis of transported tubulin.

Changes in solubility and axonal transport of tubulin during maturation and aging have been investigated using sciatic motor fibers of rats at 4, 7, 14, 30, and 80 weeks of age. One to six weeks after injection of L-[35S]methionine into the spinal cord, labeled cytoskeletal proteins in consecutive segments of the sciatic nerve and the ventral roots were fractionated into soluble and insoluble forms by extraction in 1% Triton at low temperature. In 4-week-old rats, the two forms of tubulin were transported coordinately in a single wave with the average rate of 2 mm/day. At 7 weeks of age, two components in tubulin transport were observed to develop, possibly reflecting the maturation of the axonal cytoskeleton. The slower main component (1.5 mm/day) contained most of the insoluble form together with the neurofilament proteins and the faster component (3 mm/day) was enriched in the soluble form. Though significantly different in composition, the two components correspond to slow component a (SCa) and slow component b (SCb) originally defined in the optic system. A progressive decrease in transport rates of both SCa and SCb was observed with rats at 14, 30, and 80 weeks of age. In addition, there was a large decrease in the proportion of insoluble tubulin during the course of transport in animals older than 30 weeks. This loss of the insoluble form seems to be accounted for partly by the proteolytic degradation of the severely retarded SCa proteins. Changes in axonal transport of tubulin may thus reflect age-related changes in dynamics and turnover of the axonal cytoskeleton.

Aging↗
PubMed1991

Tyrosine phosphorylation and immunodetection of vinculin in growth cone particle (GCP) fraction and in GCP-cytoskeletal subfractions.

The growth cone, the motile tip of developing neuronal processes, is considered responsible for the exact guidance of axons and synaptogenesis. High activity of tyrosine kinases in growth cones may contribute to the functions of growth cones. Our previous work revealed that vinculin is one of the endogenous substrates for intrinsic tyrosine kinases in the growth cone particle (GCP) fraction isolated from fetal rat brain. In the present study, we examined tyrosine phosphorylation and immunoblot analysis of vinculin in various fractions from fetal rat brains and adult synaptosomal fraction. Tyrosine phosphorylation of vinculin in the GCP fraction was more prominent than in any other fraction from fetal brain or synaptosomes from adult. Compared to other fractions, however, the enrichment of vinculin in the GCP fraction was not observed. Tyrosine phosphorylation of vinculin in the fraction was inhibited by genistein, a specific tyrosine kinase inhibitor. Although vinculin was also phosphorylated by protein kinase C in the GCP fraction, it incorporated a much smaller amount of 32P than MARCKS protein or GAP-43. The cytoskeletal subfraction from the GCP fraction contained a considerable amount of vinculin and it was one of the major substrates for tyrosine kinases in the GCP cytoskeleton. The membrane skeleton from the GCP fraction contained a low amount of vinculin but showed high kinase activity that phosphorylated vinculin. Taken together, our results suggest that tyrosine phosphorylation of vinculin contributes to the cytoskeletal organization of growth cones.

Animals↗
PubMed1991

Muscarinic acetylcholine receptors are expressed and enriched in growth cone membranes isolated from fetal and neonatal rat forebrain: pharmacological demonstration and characterization.

Nerve growth cones, the motile tips of growing neurites, are closely related to the exact pathway finding, and their roles for synaptogenesis have been proposed to be modified by some neurotransmitters. In the present study, to clarify the expression and the ontogeny of muscarinic acetylcholine receptors in growth cones, growth cone membranes from fetal and neonatal rat forebrain were isolated, and muscarinic receptors in growth cone membrane were pharmacologically characterized, by using the [3H]quinuclidinyl benzilate as a labeled ligand. The specific binding sites for [3H]quinuclidinyl benzilate had already been detected in growth cone membrane on embryonic day (E)17 (Bmax = 557 fmol/mg protein: KD = 19.7 pM) and gradually increased in amount without significant changes in the KD values from E17 to postnatal day (P)5. [3H]Quinuclidinyl benzilate binding sites in growth cone membrane were several times higher than that in the P2-fraction-derived membranes, and in perinuclear membranes. Competitive inhibition studies showed that the proportion of high-affinity sites for pirenzepine (M1-subtype) to total [3H]quinuclidinyl benzilate binding sites in growth cone membrane was significantly lower than that in adult synaptic plasma membranes. In contrast, the proportion of high-affinity sites for AF-DX 116 (M2-subtype) was significantly higher than that in adult synaptic plasma membranes (E17 growth cone membrane: M1, 29.5%; M2, 56.9%; adult synaptic plasma membrane: M1, 63.6%, M2, 5.9%). Electron micrographic examination revealed that there were no significant morphological differences among growth cone particle fractions at the developmental stages which we examined, and that mature synaptic elements did not contaminate the growth cone particle fractions. Biochemical examination by electrophoresis and the phosphorylation study of the growth cone particle fractions showed that the protein composition and the phosphoprotein pattern did not change markedly during these stages. Our results suggest that muscarinic receptors were expressed and more concentrated in growth cone membrane than in other membrane portions from perinatal rat forebrain, and that they may play some role in the axonal guidance in growth cone via receptor subtype-specific signal transduction mechanisms.

Animals↗
PubMed1991

Changes in organization and axonal transport of cytoskeletal proteins during regeneration.

Changes in solubility and transport rate of cytoskeletal proteins during regeneration were studied in the motor fibers of the rat sciatic nerve. Nerves were injured by freezing at the midthigh level either 1-2 weeks before (experiment I) or 1 week after radioactive labeling of the spinal cord with L-[35S]methionine (experiment II). Labeled proteins in 6-mm consecutive segments of the nerve 2 weeks after labeling were analyzed following fractionation into soluble and insoluble populations with 1% Triton at 4 degrees C. When axonal transport of newly synthesized cytoskeleton was examined in the regenerating nerve in experiment I, a new faster component enriched in soluble tubulin and actin was observed that was not present in the control nerve. The rate of the slower main component containing most of the insoluble tubulin and actin together with neurofilament proteins was not affected. A smaller but significant peak of radioactivity enriched in soluble tubulin and actin was also detected ahead of the main peak when the response of the preexisting cytoskeleton was examined in experiment II. It is thus concluded that during regeneration changes in the organization take place in both the newly synthesized and the preexisting axonal cytoskeleton, resulting in a selective acceleration in rate of transport of soluble tubulin and actin.

Actins↗
PubMed1991

Two 68-kDa proteins in slow axonal transport belong to the 70-kDa heat shock protein family and the annexin family.

The major 68-kDa protein found selectively in the faster of the two subcomponents of slow axonal transport [group IV or slow component b (SCb)] in the rat sciatic nerve has been characterized. It was found to contain two distinct classes of proteins, S1 and S2, both of which have isoelectric points of 5.7, but differ in their solubility in the presence of calcium. The S1 protein, which contributes up to 70% of the 68-kDa component, was soluble in the presence or absence of calcium, whereas the S2 protein was bound to the cytoskeleton in a calcium-dependent manner. Further characterization of the two proteins by peptide mapping and immunological methods revealed that the S1 protein belonged to a family of proteins related to the 70-kDa heat shock protein, whereas the S2 protein was identical to 68-kDa calelectrin (annexin VI). Selective occurrence in SCb of these proteins with potential abilities to regulate protein-protein or protein-membrane interactions suggests that they may play important roles in the control of cytoskeletal organization in the axon, because SCb contains mainly cytoskeletal proteins in a more dynamic form compared with the slowest rate component, slow component a, which is enriched in the stably polymerized form of these proteins.

Animals↗
PubMed1990

Vinculin is one of the major endogenous substrates for intrinsic tyrosine kinases in neuronal growth cones isolated from fetal rat brain.

Neuronal growth cones, the motile tips of growing neuronal processes, are responsible for the exact guidance of extending neurites. To elucidate the mechanisms of their biochemical signal transduction in growth cones, the growth-cone-enriched fraction was isolated biochemically from fetal rat brain and the endogenous protein phosphorylation in the fraction was analyzed under the conditions where tyrosine residues were preferentially phosphorylated. One of the major phosphoproteins was a 130-kDa slightly acidic protein which reacted with antiphosphotyrosine antibody. Its phosphoryl residues were alkali-stable. Thus, the 130-kDa protein was concluded to be susceptible to tyrosine phosphorylation. This protein was a component of cytoskeletal proteins thought to be associated indirectly with membranes. All the behavior of the 130-kDa protein was compatible with the properties of vinculin, a component of focal contacts which are responsible for the stable or motile adhesion between cells or between a cell and the substratum. Immunochemical analyses showed that the 130-kDa protein was specifically recognized by anti-vinculin antibody. Therefore, the 130-kDa protein was concluded to be vinculin. Tyrosine phosphorylation of the protein appeared to be relatively more pronounced in the growth-cone-enriched fraction than in adult synaptosomes. The results suggest that tyrosine phosphorylation of vinculin may be regulated developmentally and it may be involved in the functions of growth cones.

Animals↗
PubMed1990

[Three patients with typical sandblaster's silicosis proven by mineralogical analysis].

Three family members who had worked as sandblasters in their own sandblasting factory showed innumerable small nodularities in both lung fields of their chest radiograms. One of those showed conglomerate shadows in the upper lung fields. Those shadows seemed to be consistent with those of silicosis. One of the patients was examined by transbronchial lung biopsy (TBLB) and showed typical silicotic nodules. Mineralogical studies were done on the abrasive particles and the deposited particles in the lung tissue specimen obtained via TBLB and bronchoalveolar lavage fluid sample (BALF) using polarized microscopy, X-ray diffraction and analytical electron microscopy. The particles which had accumulated on the floor of the factory mineralogically consisted of mostly (over 90%) silica quartz containing small amount of chlorite, and the deposited particles in the lung tissue and those in the BALF showed similar composition.

Adult↗
PubMed1989

Axonal transport studied in a single vertebrate neuron: the giant electromotor neuron of the electric catfish, Malapterurus electricus.

Axonal transport was studied using a single vertebrate neuron, the giant electromotor neuron of the electric catfish, Malapterurus electricus. The electric organs of this strongly electric fish are innervated by two neurons whose axons form one electric nerve each. After injection of [35S]methionine into the spinal cord at the level of the two perikarya radioactively labelled material is exported by fast flow as a small wave with a velocity of 5.8 mm/h and a somal release time of 91 min (29 degrees C). Slow flow investigated between 15 and 39 days had a velocity of 1.36 mm/d at 29 degrees C. Analysis of radiolabelled proteins by polyacrylamide gel electrophoresis revealed different patterns of labelling between slow and fast flow. The relative molecular mass of the two major proteins labelled on slow flow correspond to actin and tubulin. Labelled proteins of higher relative molecular mass may correspond to neurofilament proteins. Our results suggest that this vertebrate single-neuron and single-axon system can be used successfully for axonal transport studies.

Animals↗
PubMed1989

Estrogen-binding protein in blood and follicular fluid, and its biochemical properties in human females.

Testosterone-estradiol binding globulin (TeBG) has been known to have specific binding to estradiol (E2) in blood, however an unknown binding protein having higher affinity to E2 than TeBG is thought to exist in blood. Therefore blood serum and follicular fluid were collected in normal females. Ammonium sulfate precipitation showed different maximum bound ratio between E2BP and TeBG. Concanavalin A (Con-A) Sepharose adsorption analysis showed that the Con-A-bound phase demonstrated estradiol-binding protein (E2BP). Isoelectric focusing showed TeBG in pH 4.9 and E2BP in pH 3.9. The substance giving the peak at pH 3.9 has a high affinity to dehydroepiandrosterone sulfate and E2, but the substance with peak at pH 4.9 shows a high affinity to 5-dihydrotestosterone and testosterone. E2BP changed concomitantly with total E2 in blood in the menstrual cycle, but in follicular fluid E2BP was found only in small amounts. It consisted of two components, one protein with low affinity binding and the other with high affinity binding. These results suggest that E2BP exists as a specifically bound fraction to E2, while TeBG, as a nonspecifically bound fraction to E2, is not directly involved in the E2-related biological function.

Adult↗
PubMed1989

Stable and dynamic forms of cytoskeletal proteins in slow axonal transport.

Dynamic organization of the axonal cytoskeleton was investigated by analyzing slow axonal transport of tubulin and other major cytoskeletal proteins in the motor axons of rat sciatic nerve 1-4 weeks after injection of L-35S-methionine into the anterior horn area of L3-L5 lumbar spinal cord. A large proportion (50-65%) of tubulin transported in the axon was found to be insoluble when extracted with 1% Triton at 4 degrees C. This cold-insoluble tubulin was also resistant to other microtubule-destabilizing agents such as Ca2+, colchicine, and nocodazole, suggesting that it corresponded to the stably polymerized tubulin specific to the axon. From the cold-soluble fraction, microtubules containing a distinct set of associated proteins were recovered by the taxol-dependent procedure. Transport pattern of cold-soluble and -insoluble tubulin in this system showed a time-dependent broadening of the tubulin wave resulting in the appearance of a new faster wave enriched in cold-soluble tubulin. The slower and the faster waves of tubulin were defined as group V or slow component a (SCa) and group IV or slow component b (SCb), respectively, with respect to the 2 subcomponents of slow transport originally described in the optic system. However, compositions of groups IV and V in sciatic motor axons differed significantly from those of the optic system. Actin also exhibited a clear dual wave pattern of transport that coincided well with that of tubulin, indicating that both actin and tubulin were the major components of both groups IV and V.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1988

Effects of taxol on slow and fast axonal transport.

Axonal transport of tubulin in the rat sciatic nerve is almost completely inhibited by a single subepineural injection of taxol, without affecting that of neurofilament proteins. Actin and a large number of polypeptides cotransported with actin as minor components are also blocked by taxol, although to a lesser extent. Fast axonal transport is essentially free from the inhibitory effect of this drug. Although previous models have suggested that slow axonal transport involves the bulk movement of cytoskeletal structures, these results suggest that such transport may involve an equilibrium between polymerised and depolymerised forms of the axonal cytoskeleton.

Actins↗
PubMed1987

The axonal transport of dipeptidyl aminopeptidase II, angiotensin-converting enzyme and other peptidases in rat sciatic nerves.

The axonal transport of four peptidases with different subcellular localizations were examined in rat sciatic nerves by the double ligation technique. At 2 days after ligation, rapid retrograde axonal transport of the activity of dipeptidyl aminopeptidase II (a lysosomal enzyme), angiotensin-converting enzyme and leucyl aminopeptidase (membrane-bound enzymes), was found in the segment distal to the proximal ligation. On the other hand, prolyl endopeptidase (a cytosolic enzyme) showed neither anterograde nor retrograde rapid axonal transport. These results indicate that lysosomal and membrane-bound peptidases are transported retrogradely in rat sciatic nerves.

Animals↗
PubMed1987

The recovery of slow axonal transport after a single intraperitoneal injection of beta, beta'-iminodipropionitrile in the rat.

A single intraperitoneal injection of beta, beta'-iminodipropionitrile (IDPN) at a dose of 1.5 g/kg was given to 4-week-old rats. Immediately following, or at 1, 2, 3, 5, 10, and 15 weeks after IDPN injection, [35S]methionine was introduced into the anterior horn area of the lumbar cord. Labeled axonal proteins in the sciatic nerve were analyzed electrophoretically and fluorographically at 5, 10, and 15 weeks post-labeling. Labeled neurofilament proteins halt for a short period just after IDPN injection, then continue migrating distally, though at a slower rate, and finally the transport of affected neurofilament proteins completely recovers by 6 weeks post-labeling.

Animals↗