Biomedical subjects

T Nishiki

Publications and source records attributed to T Nishiki.

At least 19 recordsLinked to original sources

PubMed2001

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. Each antibody specifically recognized alpha-, beta- or gamma-SNAP in an isoform-specific manner in immunoblots of brain homogenate. Alpha- and gamma-SNAP were ubiquitously expressed in various tissues, whereas beta-SNAP was expressed only in brain. After subcellular fractionation of brain homogenates, all three isoforms were recovered in both soluble and particulate fractions. Immunohistochemistry revealed that alpha- and beta-SNAP were generally differentially distributed both in synaptic and non-synaptic regions, including brain white matter. The presynaptic location of both alpha- and beta-SNAP was confirmed by immunoelectron microscopy. At the neuromuscular junction, immunoreactive alpha-SNAP was identified in synaptic vesicles, while in the cerebellum, beta-SNAP was present in the presynaptic membranes of basket neuron and mossy fiber terminals. From these results we suggest that both alpha- and beta-SNAP may play an important role in neurotransmitter release as well as in constitutive vesicular transport.

Animals↗

PubMed2000

Anaerobic oxidation of dissolved hydrogen sulfide in continuous culture of the phototrophic bacterium Prosthecochloris aestuarii.

The anaerobic oxidation of dissolved H2S to elemental sulfur was studied at 23 degrees C and pH 6.5+/-0.3 in continuous culture of the phototrophic green sulfur bacterium Prosthecochloris aestuarii. The number of cells formed in the cultures was proportional to the amount of H2S oxidized, and the growth yield was independent of light intensity. The specific growth rate was significantly dependent on the dissolved H2S concentration and light intensity. The kinetic data were analyzed with a rate expression as a function of each rate-limiting factor. Under illumination by white fluorescent lamps, the specific oxidation rate of P. aestuarii reached a maximum of 2.02 x 10(-14) mol-H2S.h(-1).cell(-1) when the dissolved H2S concentration was 2.1 mM at 5000 lx. Simultaneous use of near infrared LED (light-emitting diode) and white fluorescent lamps provided a 35% increase in the maximum specific H2S oxidation rate.

Journal Article↗

PubMed1998

Ganglioside GT1b as a complementary receptor component for Clostridium botulinum neurotoxins.

Clostridium botulinum type B neurotoxin (BoNT/B) recognizes a complex of synaptotagmin II and ganglioside GT1b or GD1a as the high-affinity toxin binding site. Recombinant deletion mutants of synaptotagmin II allowed us to demonstrate that the N-terminal domain including the transmembrane region retains BoNT/B binding activity while the C-terminal domain is not involved in constituting the BoNT/B receptor. BoNT/B binding to reconstituted lipid vesicles containing synaptotagmin II and gangliosides showed that GT1b and GD1a confer the difference in the maximum binding capacity but not in the dissociation constant. The direct binding of GT1b to the deletion mutants revealed that the transmembrane region is required to bind GT1b, suggesting that synaptotagmin II binds to the ceramide portion of gangliosides within the plasma membrane. A monoclonal antibody against GT1b effectively inhibited not only BoNT/B binding to the reconstituted lipid vesicles and brain synaptosomes but also type A BoNT (BoNT/A) binding to brain synaptosomes. In addition, the monoclonal antibody antagonized the action of both BoNT/A and BoNT/B on synaptic transmission of rat superior cervical ganglion neurons. These results suggest that GT1b functions as a component of the receptor complex.

Animals↗

PubMed1998

Characterization of Clostridium botulinum type B neurotoxin associated with infant botulism in japan.

The neurotoxin of strain 111 (111/NT) associated with type B infant botulism showed antigenic and biological properties different from that (Okra/NT) produced by a food-borne botulism-related strain, Okra. The specific toxicity of 111/NT was found to be about 10 times lower than that of Okra/NT. The monoclonal antibodies recognizing the light chain cross-reacted with both neurotoxins, whereas most of the antibodies recognizing the carboxyl-terminal half of the heavy chain of Okra/NT did not react to 111/NT. Binding experiments with rat brain synaptosomes revealed that 125I-labeled 111/NT bound to a single binding site with a dissociation constant (Kd) of 2.5 nM; the value was rather lower than that (0.42 nM) of 125I-Okra/NT for the high-affinity binding site. In the lipid vesicles reconstituted with ganglioside GT1b, 125I-Okra/NT interacted with the amino-terminal domain of synaptotagmin 1 (Stg1N) or synaptotagmin 2 (Stg2N), fused with the maltose-binding protein, in the same manner as the respective full-length synaptotagmins, and the Kd values accorded with those of the low- and high-affinity binding sites in synaptosomes. However, 125I-111/NT only exhibited a low capacity for binding to the lipid vesicles containing Stg2N, but not Stg1N, in the presence of ganglioside GT1b. Moreover, synaptobrevin-2, an intracellular target protein, was digested to the same extent by the light chains of both neurotoxins in a concentration-dependent manner. These findings indicate that the 111/NT molecule possesses the receptor-recognition site structurally different from Okra/NT, probably causing a decreased specific toxicity.

Animals↗

PubMed1998

[Exocytosis].

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Adenosine Triphosphate↗

PubMed1997

Comparison of exocytotic mechanisms between acetylcholine- and catecholamine-containing vesicles in rat pheochromocytoma cells.

The molecular mechanisms of exocytosis from two types of secretory organelles, synaptic-like microvesicles and secretory vesicles, were compared by measuring acetylcholine (ACh) and catecholamine (CA) release from a newly isolated PC12 subclone, PC12-C3 which contains a high level of Ach. Digitonin-permeabilized PC12-C3 cells released both transmitters with similar Ca(2+)-dependency. Ca(2+)-evoked Ach and CA release from permeabilized cells were increased in the presence of MgATP, suggesting the existence of a MgATP-dependent priming step prior to the Ca(2+)-triggered fusion step in both ACh release and CA release. The non-hydrolyzable analogue of GTP guanosine 5'-(gamma-thio)triphosphate (GTP gamma S), produced both ACh and CA release from permeabilized cells in the absence of Ca2+. Pretreatment with a phorbol ester which activates protein kinase C, potentiated depolarization-induced ACh and CA release from unpermeabilized cells. These results indicated that exocytosis from two distinct vesicle populations are mediated by the same basic molecular mechanisms.

Acetylcholine↗

PubMed1997

Localization of hepatocyte growth factor and tubulointerstitial lesions in IgA nephropathy.

To investigate the relationship between localization of hepatocyte growth factor (HGF) and tubulointerstitial lesions (TILs) in the cortical area of renal biopsy specimens, a clinicopathological study was performed in 35 patients with IgA nephropathy. HGF was detected by an enzyme-antibody method and TILs were assessed semiquantitatively by light microscopy. HGF was observed mainly on epithelial cells in the tubules, but not in the glomeruli. Fourteen patients had biopsies that were positive for HGF. There was a correlation between HGF positivity and histological damage, the TIL grade, and several clinical parameters determined at biopsy. Thus, HGF is related to TILs in IgA nephropathy, and may be a factor in the exacerbation of this disease.

Adolescent↗

PubMed1997

A case of nephrotic syndrome with rapid spontaneous remission in an elderly patient.

In July 1994, a 70-year-old woman was diagnosed as having nephrotic syndrome with proteinuria of 8 to 10 g/day and a serum albumin level of 1.8 g/dl. She was hospitalized in August 1994 for investigation. The urinary findings then normalized, with urinary protein and occult blood both negative and total urinary protein excretion at 0 g/day. A renal biopsy was performed, and spontaneous remission of minimal change nephrotic syndrome was diagnosed. This is an interesting case involving rapid remission of minimal change nephrotic syndrome in an elderly patient.

Aged↗

PubMed1996

Dissociation of SNAP-25 and VAMP-2 by MgATP in permeabilized adrenal chromaffin cells.

In digitonin-permeabilized adrenal chromaffin cells, Ca(2+)-induced catecholamine release can be resolved into at least two sequential steps: a MgATP-dependent priming step and a MgATP-independent Ca(2+)-triggered step. Botulinum neurotoxins types A and E cleaved SNAP-25, and blocked MgATP-independent Ca(2+)-induced catecholamine release from the permeabilized chromaffin cells. When the permeabilized cells were primed by pretreatment with MgATP, the amount of SNAP-25 associated with VAMP-2 decreased, and the fraction of SNAP-25 proteolyzed by the neurotoxins increased. These results suggest that dissociation of SNAP-25 and VAMP-2 occurs during the MgATP-dependent priming step, and SNAP-25 plays some important roles in the subsequent MgATP-independent step.

Adenosine Triphosphate↗

PubMed1996

Phosphorylation of 25-kDa synaptosome-associated protein. Possible involvement in protein kinase C-mediated regulation of neurotransmitter release.

Protein kinase C-mediated phosphorylation of a 25-kDa synaptosome-associated protein (SNAP-25) was examined in living PC12 cells. Phorbol 12-myristate 13-acetate treatment enhanced high potassium-induced [3H]-norepinephrine release, and a 28-kDa protein recognized by an anti-SNAP-25 antibody was phosphorylated on Ser residues. The molecular size of the phosphorylated band decreased slightly following treatment with Clostridium botulinum type A neurotoxin, whereas the band disappeared after treatment with botulinum type E neurotoxin, indicating that the 28-kDa protein was SNAP-25. A phosphorylation is likely to occur at Ser187, as this is the only Ser residue located between the cleavage sites of botulinum type A and E neurotoxins. SNAP-25 of PC12 cells was phosphorylated by purified protein kinase C in vitro, and the amount of syntaxin co-immunoprecipitated with SNAP-25 was decreased by phosphorylation. These results suggest that the phosphorylation of SNAP-25 may be involved in protein kinase C-mediated regulation of catecholamine release from PC12 cells.

Animals↗

PubMed1996

Binding of botulinum type B neurotoxin to Chinese hamster ovary cells transfected with rat synaptotagmin II cDNA.

We have previously identified synaptotagmin, a synaptic vesicle membrane protein from rat brain, as a binding protein for Clostridium botulinum type B neurotoxin. In this report, rat synaptotagmin II was expressed by transfection in Chinese hamster ovary cells and interaction with the neurotoxin was studied. In stable transfectants, the NH(2)-terminal region of synaptotagmin was exposed to the extracellular medium. Synaptotagmin-expressing cells were shown to possess an extremely low binding activity for the radiodinated toxin. However, toxin-binding was markedly increased to cells which had been treated with gangliosides G T1b or G D1a. In synapses, the intravesicular NH(2)-terminus of synaptotagmin becomes exposed at the cell surface after following exocytosis. These findings suggest that the NH(2)-terminal domain of synaptotagmin II forms the binding site for type B neurotoxin by associating with specific gangliosides in presynaptic plasma membranes.

Animals↗

PubMed1996

Interaction of SNARE complexes with P/Q-type calcium channels in rat cerebellar synaptosomes.

P- and Q-type calcium channels, which trigger rapid neurotransmitter release at many mammalian synapses, are blocked by omega-conotoxin MVIIC. 125I-omega-Conotoxin MVIIC binding to rat cerebellar synaptosomes was not displaced by omega-conotoxins GVIA or MVIIA (Ki > 1 microM), which are selective for N-type calcium channels. Solubilized 125I-omega-conotoxin MVIIC receptors were specifically recognized by antibodies directed against alpha1A calcium channel subunits, proteins known to constitute a pore with P/Q-like channel properties. Antibodies against syntaxin 1, SNAP 25, and VAMP 2 (synaptobrevin) each immunoprecipitated a similar fraction (20-40%) of omega-conotoxin MVIIC receptors. Immunoprecipitation was not additive, suggesting that heterotrimeric (SNARE) complexes containing these three proteins interact with P/Q-type calcium channels. Immobilized monoclonal anti-syntaxin antibodies retained alpha1A calcium channel subunits of 220, 180 and 160 kDa monitored by immunoblotting with site directed antibodies. Synaptotagmin was detected in channel-associated complexes, but not synaptophysin, Rab 3A nor rat cysteine string protein. Trimeric SNARE complexes are implicated in calcium-dependent exocytosis, a process thought to be regulated by synaptotagmin. Our results indicate that these proteins interact with P/Q-type calcium channels, which may optimize their location within domains of calcium influx.

Animals↗

PubMed1996

The high-affinity binding of Clostridium botulinum type B neurotoxin to synaptotagmin II associated with gangliosides GT1b/GD1a.

125I-labeled botulinum type B neurotoxin was shown to bind specifically to recombinant rat synaptotagmins I and II. Binding required reconstitution of the recombinant proteins with gangliosides GT1b/GD1a. Scatchard plot analyses revealed a single class of binding site with dissociation constants of 0.23 and 2.3 nM for synaptotagmin II and synaptotagmin I, respectively, values very similar to those of the high- (0.4 nM) and low-affinity (4.1 nM) binding sites in synaptosomes. The high-affinity binding of neurotoxin to synaptosomes was specifically inhibited by a monoclonal antibody recognizing with the amino-terminal region of synaptotagmin II. These results suggest that this region of synaptotagmin II participates in the formation of the high-affinity toxin binding site by associating with specific gangliosides.

Amino Acid Sequence↗

PubMed1996

A new model of transient cerebral ischemia in neonatal rats.

A new model of transient cerebral ischemia in 10-day- old rats is described. Under microscopic guidance, the right external and internal carotid arteries were electrically coagulated. A solid 0.47 mm diameter nylon thread was inserted into the right common carotid artery toward the ascending aorta up to 10-12 mm from the upper edge of the sternomastoid muscle (preischemic rats). A 60-min cerebral ischemia was induced by clamping the left external and internal carotid arteries (ischemic rats), followed by 3-h recirculation. 31P magnetic resonance (MRS) spectroscopic studies revealed that severe intracellular acidosis occurred and ATP disappeared completely for a least the last 20 min of ischemia. Cerebral blood flow (CBF), measured by the hydrogen clearance technique, decreased to approximately 11% of the preischemic level in the frontal cortex soon after the induction of ischemia. On resuscitation, ATP recovered completely and the preischemic intracellular pH level was restored within 180 min. CBF has recovered to approximately 30% of the preischemic level at 5 min after resuscitation. The CBF recovery was not compete even at 180 min after resuscitation. With this model, the effects of pure ischemia without hypoxia on the neonatal brain and the process of recovery from transient ischemia can be studied.

Adenosine Triphosphate↗

PubMed1994

Identification of protein receptor for Clostridium botulinum type B neurotoxin in rat brain synaptosomes.

The protein receptor for Clostridium botulinum type B neurotoxin was purified 340-fold from rat synaptosomes by successive chromatography on DEAE-Sepharose, phenyl-Toyopearl, and heparin-Toyopearl columns. 125I-Labeled neurotoxin bound to lipid vesicles containing the protein receptor and ganglioside GT1b or GD1a. The reconstituted receptor showed the same affinities as the native receptor on synaptosomes. Chemical cross-linking of 125I-toxin to the receptor in the presence of gangliosides resulted in formation of a cross-linked product of 161 kDa under reducing conditions. Cross-linking was specific, as it was inhibited by the presence of excess unlabeled toxin. A monoclonal antibody against the purified 58-kDa receptor protein and a monoclonal antibody against the heavy chain (103 kDa) of the neurotoxin reacted with the cross-linked product of 161 kDa in immunoblotting experiments. We determined partial amino acid sequences of the 58-kDa protein, which were identical to synaptotagmin, a synaptic vesicle membrane protein. In addition, the monoclonal antibody against the 58-kDa receptor protein recognized recombinant rat synaptotagmin. These results suggest that synaptotagmin in association with ganglioside GT1b or GD1a may be a natural receptor for C. botulinum type B neurotoxin at the nerve terminals.

Amino Acid Sequence↗

PubMed1994

Patient with unusual alternating hemiplegia.

A patient with unusual alternating hemiplegia in childhood is reported. The frequency of hemiplegic episodes was lower than that in other reported patients. Interictal positron emission tomography revealed decreased cerebral blood flow in the left hemisphere. After cerebral angiography, postanesthetic irritability occurred which evolved into coma with hemiplegic episodes. We believe that cerebral angiography in a child with alternating hemiplegia may cause hemiplegic episodes and coma. The episodes disappeared almost completely for 6 years after treatment with flunarizine, thus indicating the benefit of the medication for the treatment of alternating hemiplegia.

Basal Ganglia↗

PubMed1994

Morphological effects, rate of incorporation, and the enzymatic action of botulinum ADP-ribosyltransferase, known as C3 exoenzyme, on human neuroblastoma GOTO cells.

The susceptibility of various lines of cultured cells to botulinum ADP-ribosyltransferase, known as C3 exoenzyme, was examined. Human neuroblastoma GOTO cells were most sensitive. The C3 exoenzyme caused a change in cell shape that involved extension of neurites. The exoenzyme evoked the outgrowth of neurites from chick ganglion as effectively as nerve growth factor, suggesting that C3 exoenzyme possesses neurotropic activity. Experiments with 125I-labeled enzyme revealed that C3 exoenzyme was rapidly incorporated into cells but the number of incorporated enzyme molecules was small. Once C3 exoenzyme had been incorporated, ADP-ribosylation of the substrate (Rho protein) in GOTO cells occurred immediately and rapidly reached a maximum level. However, some of Rho proteins remained unmodified even after induction of the change in morphology. These findings suggest that ADP-ribosylation by C3 exoenzyme is directly associated with the differentiation of GOTO cells but that other events may also participate in this process.

ADP Ribose Transferases↗

PubMed1994

Efficacy of thyrotropin-releasing hormone in the treatment of spinal muscular atrophy.

Children with spinal muscular atrophy were treated by the administration of thyrotropin-releasing hormone. In three infants with spinal muscular atrophy type I, thyrotropin-releasing hormone showed little efficacy, but in children with types II and III, there was improvement in motor function and electromyographic findings after the thyrotropin-releasing hormone therapy. Thyrotropin-releasing hormone has a neurotrophic effect on the spinal anterior motor neurons of spinal muscular atrophy patients and thus may be warranted for the management of spinal muscular atrophy.

Age of Onset↗