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Biomedical subjects

Y Kamata

Publications and source records attributed to Y Kamata.

At least 73 records · Page 4Linked to original sources
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

Vesicle-associated membrane protein 2 is essential for cAMP-regulated exocytosis in rat parotid acinar cells. The inhibition of cAMP-dependent amylase release by botulinum neurotoxin B.

Amylase exocytosis of the parotid gland is mediated by intracellular cAMP. To investigate whether cAMP-dependent secretion has a mechanism similar to that of regulated neuroexocytosis, we examined the expression of synaptosome-associated proteins. In rat parotid acinar cells, we found 25 (p25) and 18 kDa (p18) proteins reacted with antibodies against Rab3A and vesicle-associated membrane protein 2 (VAMP-2), respectively. On the other hand, syntaxin 1 and SNAP-25, which interact with VAMP-2 at synapses, were undetectable. Rab3A-like p25 and VAMP-2-like p18 were also expressed in other exocrine acinar cells. The latter was localized at secretory granule membranes, and the former was detected in secretory granule and cytosolic fractions. The antibody against VAMP-2 used in this study did not react with cellubrevin, and p18 was cleaved with botulinum neurotoxin B. Thus, we identified p18 as VAMP-2. Botulinum neurotoxin B inhibited the cAMP-induced amylase release from streptolysin O-permeabilized acinar cells. Therefore, VAMP-2 is required for cAMP-regulated amylase release in rat parotid acinar cells. This is the first report that VAMP-2 is involved in regulated exocytosis that is independent of Ca2+.

Amylases↗
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

Cleavage of vesicle-associated membrane protein (VAMP)-2 and cellubrevin on GLUT4-containing vesicles inhibits the translocation of GLUT4 in 3T3-L1 adipocytes.

We have identified VAMP isoforms, VAMP-2 and cellubrevin, on GLUT4-containing vesicle membranes isolated from 3T3-Ll adipocytes. These proteins translocate from a low density microsomal fraction to the plasma membrane upon insulin stimulation in a fashion similar to GLUT4. VAMP-1 was not detected in this low density microsomal fraction nor on purified GLUT4-containing vesicles. In streptolysin-O permeabilized 3T3-L1 adipocytes, both VAMP-2 and cellubrevin were cleaved with botulinum neurotoxin isoform B, BoNTx/B. In addition, BoNTx/B partially inhibited insulin-stimulated GLUT4 translocation and glucose transport activity. We conclude that the synaptobrevin isoforms are important components of the insulin-dependent translocation of GLUT4 to the cell surface in adipocytes.

3T3 Cells↗
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

Bcl-2 expression is correlated with a low apoptotic index and associated with progesterone receptor immunoreactivity in endometrial carcinomas.

A total of 103 endometrial carcinomas (endometrioid type), as well as 15 samples of normal (atrophic or proliferative phase) and 26 of hyperplastic endometrium, were immunohistochemically investigated for expression of Bcl-2, and oestrogen and progesterone receptors (ER and PR), and the results compared with findings for apoptosis and cell proliferation. Carcinoma cases were subdivided into tubular and solid components on the basis of tumour growth patterns. Immunopositivity for Bcl-2, ER, and PR in tubular components was significantly higher than in the solid category, being negatively associated with histological grading. Immunoreactivity scores revealed that Bcl-2 in the tubular group was positively correlated with PR but not ER, while its expression in normal and hyperplastic endometrium was closely linked with both. Apoptotic and mitotic indices (AI and MI) were both significantly lower in tubular than in solid areas. In the tubular areas, AI, values were significantly lower in the subgroup with a high level of Bcl-2 expression than in either low-level or negative groups. These results indicate that Bcl-2 expression may play a central role in the inhibition of apoptosis in endometrial carcinoma, in particular those cases with tubular components, possibly being associated with PR rather than ER. Changes in the propensity for apoptosis may be related to alterations of tumour growth pattern and of features of differentiation.

Apoptosis↗
PubMed1996

Bcl-2 expression and allelic loss of the p53 gene in gastric carcinomas.

In order to clarify the association between bcl-2 protein (Bcl-2) expression and genetic alteration, we investigated p53 and DCC (deleted in the colon carcinoma gene locus) gene abnormalities in Bcl-2-positive and -negative gastric carcinomas using a polymerase chain reaction/loss of heterozygosity (LOH) assay. Bcl-2 immunoreactivity was found in 25 of 178 (14%) gastric carcinoma cases. With these 25 positive cases, the proportion 18/87 (20.6%) of the total in early stages demonstrating invasion of the mucosa and/or submucosa was significantly greater (P = 0.013) than the 7/91 (7.7%) found for advanced tumors exhibiting invasion into or through the muscularis propria. However, there was no statistically significant variation between the proportions for differentiated (17/98 cases, 17.3%) and undifferentiated (8/80 cases, 10%) lesions. Sixteen Bcl-2-positive cases (9 cases were not studied because of insufficient specimen material to allow extraction of DNA) and 31 cases randomly selected from a Bcl-2-negative group were analyzed for the presence of p53-LOH or DCC-LOH and for p53 by immuno-histochemistry. The minority of the Bcl-2-positive group had p53-LOH and were immunopositive for p53 (P = 0.033, P = 0.028 respectively), while no association was found in the Bcl-2-negative category. In contrast, there was no correlation at all between Bcl-2 expression and DCC-LOH although the number of informative cases analyzed was too small to allow definite conclusions. These results indicate that Bcl-2 may be predominantly expressed at an early stage in gastric carcinomas, possibly in negative association with p53 gene abnormalities.

Alleles↗
PubMed1996

The genome sequence of the virulent Kabete 'O' strain of rinderpest virus: comparison with the derived vaccine.

We have compared the complete genome sequences of the vaccine strain of rinderpest virus and the virulent strain from which it was derived. Only 87 bases differed between the two genomes (0.55%). Possibly significant differences in amino acid sequence were found in the N, P, F, H and L proteins. A number of differences were also found in the leader region (3' end of the genome), whilst the trailer region appears to be more conserved. In addition, the length of the genome was found in both cases to be 15882, an exact multiple of six, fulfilling predictions made earlier based on work with Sendai and measles viruses.

Base Sequence↗
PubMed1995

Inhibition of purified (Na+,K+)-ATPase activity from porcine cerebral cortex by NO generating drugs.

We tested the effects of several nitric oxide (NO) generating compounds on the activity of sodium-potassium adenosine 5'-triphosphatase [(Na+,K+)-ATPase] purified from porcine cerebral cortex. Sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine (SIN-1) and (d1)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (NOR 3) inhibited the (Na+,K+)-ATPase activity dose dependently. Superoxide dismutase, a NO scavenger, and sulfhydryl (SH) compounds, reduced-form glutathione (rGSH) and dithiothreitol (DTT), prevented the inhibitory action of SNAP, SIN-1 and NOR 3 but not of SNP, when applied simultaneously with NO generating compounds, and this enzyme inhibition could be reactivated by the incubation with these SH compounds but not with SOD. The inhibitory action by SNP was magnified by simultaneous application of DTT. These results suggest that NO generating compounds, SNAP, SIN-1 and NOR 3 but not SNP, may release NO or NO-derived products and may inhibit (Na+,K+)-ATPase activity by interacting with a SH group at the active site of the enzyme.

Animals↗
PubMed1995

Immunological characterization of the neurotoxin produced by Clostridium botulinum type A associated with infant botulism in Japan.

The neurotoxin associated with type A infant botulism in Japan shows different antigenic properties from those produced by authentic strains. The monoclonal antibodies recognizing the light chain reacted to both neurotoxins, whereas half the antibodies recognizing the heavy chain reacted specifically to the respective neurotoxin. Each neurotoxin showed its own manner of binding to brain synaptosomes. These results indicate that the distinguishable characteristics are ascribable to the heavy chain but not to the light chain. In both neurotoxins, an epitope recognized by the monoclonal antibody that reacts to the light chain and neutralizes the toxin was found to be very close to the amino-terminal half (H-1 fragment) of the heavy chain. This may support the hypothesis that the H-1 fragment functions in the transport of the light chain in the target cell.

Amino Acid Sequence↗
PubMed1995

Purification and characterization of phosphoribulokinase from the cyanobacterium Synechococcus PCC7942.

Phosphoribulokinase (PRK) was purified to electrophoretic homogeneity from Synechococcus PCC7942 with high specific activity. Molecular masses of the native enzyme and its subunit were 178 and 42 kDa, respectively. Cys-17 and Cys-38 were conserved in the cyanobacterial PRK, but 18 amino acid residues between them were missing among the 40 residues found in higher plant PRKs.

Amino Acid Sequence↗
PubMed1994

The light chain of botulinum neurotoxin forms channels in a lipid membrane.

The ability of botulinum neurotoxin and its isolated subunits, the heavy and light chains, to bind to a lipid membrane and to form channels in the membrane was examined. At pH 4.0, the neurotoxin caused aggregation of calcein-containing liposomes, providing evidence of binding of the neurotoxin to the surface of the outer lipid membrane. Aggregation was followed by the release of calcein, as a result of the formation of channels. The heavy chain evoked the same responses as those of the neurotoxin. The light chain did not cause aggregation of the liposomes but did evoke the release of calcein. The channel-forming ability of the light chain appeared to be higher than that of the neurotoxin or the heavy chain. This novel property of the light chain may help us to understand the mechanism of action of botulinum neurotoxin.

Botulinum Toxins↗
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

Involvement of phospholipids in the intoxication mechanism of botulinum neurotoxin.

Phospholipids were examined for their potential to interact with botulinum neurotoxin by an in vivo toxin-inactivation assay and a direct binding assay on a thin layer plate. Type E neurotoxin was inactivated by negatively charged phospholipids, phosphatidylserine (PS) and phosphatidylinositol (PI). The toxicity of the neurotoxin was not affected by phosphatidylcholine (PC) without an electric charge or phosphatidylethanolamine (PE) with a positive electric charge. The neurotoxin bound directly to PS and PI but not to PC or PE. These results suggest that the negatively charged phospholipids in the cell membranes are involved in the intoxication mechanism of botulinum neurotoxin. The phospholipids PS and PI were tested for their potential to interact within three domains [L, H-1, and H-2] which compose the neurotoxin. All three domains bound to PS; whereas, PI specifically accepted the binding of the H-1 domain relative to the penetration of the neurotoxin into the lipid membrane. In this paper, we discuss the interaction between the neurotoxin and the lipid membrane in the intoxication mechanism.

Animals↗
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↗
PubMed1993

Solubilization and characterization of the acceptor for Clostridium botulinum type B neurotoxin from rat brain synaptic membranes.

The acceptor for Clostridium botulinum type B neurotoxin was solubilized from rat brain synaptic membrane with nonionic detergent, nonanoyl-N-methylglucamide (MEGA-9). The solubilized acceptor was assayed for the binding activity by precipitating the acceptor with acetone in the presence of phosphatidylcholine. 125Ilabeled neurotoxin specifically bound to the lipid vesicles having incorporated the acceptor together with gangliosides. The lipid vesicles having incorporated either the acceptor or gangliosides alone showed extremely low binding activity. The treatment of the solubilized acceptor with lysyl endopeptidase and glycopeptidase F but not with sialidase resulted in decreased toxin binding, indicating that the putative acceptor is a glycoprotein accompanying an N-linked carbohydrate moiety. The observations suggest also that a protein acceptor/ganglioside complex may be required to form the functional toxin receptor.

Animals↗
PubMed1993

Purification and characterization of the ganglioside-binding fragment of Clostridium botulinum type E neurotoxin.

A way of fragmentation of Clostridium botulinum neurotoxin was carried out to elucidate the structure-function relationship of neurotoxin. The hitherto only plausible fragment was isolated from the trypsin-treated heavy chain of botulinum type E neurotoxin. In the presence of 4 M urea, one protein peak emerged from QAE-Sephadex column loaded with the heavy chain mildly treated with trypsin by elution with 0.1 M sodium chloride. Although many protein bands were detected in SDS-PAGE of the treated heavy chain, the eluted protein migrated in a single band to the position of 41,000 Da. The recovery of the 41,000-Da fragment was 28.6%, but with a 2 M urea-containing buffer as eluant, the recovery was less than 12%. The 41,000-Da fragment bound to gangliosides GD1a, GT1b, and GQ1b, to which neurotoxin and the heavy chain bound. The 41,000-Da fragment partially interfered with the binding of 125I-labeled neurotoxin to mouse brain synaptosomes. We have proposed a three-fragment structure (L.H-1.H-2) for botulinum type E neurotoxin. The characters of the 41,000-Da fragment described in this paper seem to substantiated our proposal that type E neurotoxin consists of three fragments, L.H-1.H-2, and that the ganglioside-binding fragment is H-2.

Animals↗