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Biomedical subjects

G Sakaguchi

Publications and source records attributed to G Sakaguchi.

At least 19 recordsLinked to original sources
PubMed1998

Interaction of Doc2 with tctex-1, a light chain of cytoplasmic dynein. Implication in dynein-dependent vesicle transport.

Doc2 has one Munc13-interacting domain at the N-terminal region and two C2-like domains interacting with Ca2+ and phospholipid at the C-terminal region. Doc2 consists of two isoforms, Doc2alpha and -beta. Doc2alpha is specifically expressed in neuronal cells and implicated in Ca2+-dependent neurotransmitter release, whereas Doc2beta is ubiquitously expressed and its function is unknown. We show here that both Doc2alpha and -beta interact with rat tctex-1, a light chain of cytoplasmic dynein, in both cell-free and intact cell systems. Overexpression of the N-terminal fragment of Doc2 containing the tctex-1-interacting domain induces changes in the intracellular localization of cation-independent mannose 6-phosphate receptor and its ligand, cathepsin D, which are transported from trans-Golgi network to late endosomes. Overexpression of the C-terminal fragment containing two C2-like domains shows the similar effect, but to a lesser extent, whereas overexpression of full-length Doc2 or the C-terminal fragment of rabphilin3 containing two C2-like domains does not show this effect. Because dynein is a minus-end-directed microtubule-based motor protein, these results suggest that Doc2, especially Doc2beta, plays a role in dynein-dependent intracellular vesicle transport.

Amino Acid Sequence↗
PubMed1998

Role of the Doc2 alpha-Munc13-1 interaction in the neurotransmitter release process.

Doc2alpha and Munc13-1 proteins are highly concentrated on synaptic vesicles and the presynaptic plasma membrane, respectively, and have been implicated in Ca2+-dependent neurotransmitter release. Doc2alpha interacts with Munc13-1 through the N-terminal region of Doc2alpha (the Mid domain; amino acid residues 13-37). Here we examine whether the interaction between Doc2alpha and Munc13-1 is required for Ca2+-dependent neurotransmitter release from intact neuron. A synthetic Mid peptide (the Mid peptide), but not a control mutated Mid peptide or a scrambled Mid peptide, inhibited the interaction between Doc2alpha and Munc13-1 in vitro. Introduction of the Mid peptide into presynaptic neurons of cholinergic synapses, formed between rat superior cervical ganglion neurons, reversibly inhibited synaptic transmission evoked by action potentials. In contrast, the control peptides did not inhibit synaptic transmission. This inhibitory effect depended on the presynaptic activity and was affected by extracellular Ca2+ concentrations. The onset of the Mid peptide effect was shortened when the neuron was stimulated at a higher frequency, and the inhibition was more potent at 1 mM Ca2+ than at 5.1 mM Ca2+. These results suggest that the Doc2alpha-Munc13-1 interaction plays a role in a step before the final fusion step of synaptic vesicles with the presynaptic plasma membrane in the evoked neurotransmitter release process.

Animals↗
PubMed1998

A novel brain-specific isoform of beta spectrin: isolation and its interaction with Munc13.

Munc13 is a component of the neurotransmitter release machinery which is specifically expressed in brain. Munc13 interacts with Doc2 and syntaxin which are also implicated in the neurotransmitter release process. Here we isolated another Munc13-interacting molecule from a rat brain cDNA library by use of the yeast two-hybrid system, identified it to be a novel type of beta spectrin, and named it beta SpIII sigma 1. beta SpIII sigma 1 was specifically expressed in brain, where it was enriched in the synaptic vesicle and plasma membrane fractions. Because spectrin has been shown to interact with the actin cytoskeleton which is involved in the exocytotic process, the present results suggest that the Munc13-beta SpIII sigma 1 interactions play a role in neurotransmitter release.

Amino Acid Sequence↗
PubMed1998

[A case report of aortoesophageal fistula due to thoracoabdominal aortic aneurysm].

Aortoesophageal fistulas due to thoracic aneurysms are usually fatal, with few reported survivors. A 57-year-old man with aortoesophageal fistula due to thoracoabdominal aortic aneurysm underwent the graft replacement of thoracoabdominal aorta. In the postoperative course, prosthetic graft infection had occurred in the result of residual esophageal fistula. On the 32nd postoperative day (POD), a subtotal esophagectomy was performed and the esophagus was reconstructed by gastrointestinal interposition technique via a retrosternal route. Following the second operative procedure, inflammatory reactions had been improved with systemic administration of antibiotics and continuous irrigation of the infected cavity. On 77th POD, he was discharged.

Aortic Aneurysm, Abdominal↗
PubMed1998

Aortic valve replacement after previous coronary artery bypass grafting in a patient with antiphospholipid syndrome.

We report a 55-year-old female patient with antiphospholipid syndrome secondary to systemic lupus erythematosus. The patient had undergone coronary artery bypass grafting for myocardial infarction due to left main trunk stenosis at the age of 52. Subsequently, she developed aortic insufficiency and underwent aortic valve replacement without any hemodynamic or hemostatic problems. Both coronary and valve disease should be considered in patients with antiphospholipid syndrome secondary to systemic lupus erythematosus.

Antiphospholipid Syndrome↗
PubMed1998

[A case report of gastric perforation after coronary artery bypass grafting with right gastroepiploic artery].

A 71-year-old man, who had received coronary angioplasty to right coronary artery 1 year before, was admitted because of unstable angina. An urgent CABG was performed using the left internal thoracic artery and the right gastroepiploic artery. Coronary anastomosis was performed under ventricular fibrillation due to porcelain aorta. Seven days after surgery, abdominal pain was suddenly experienced. A chest X-P showed subphrenic free air. So an emergent laparotomy was performed, and a 2 x 2 cm gastric perforation was found on the anterior wall of the greater gastric curvature. Partial gastrectomy was performed. However, he unfortunately died on the 58th postoperative day for multiple organ failure. Pathological examination of the excised gastric wall revealed ischemic change, not ulcer. This gastric perforation was possibly caused by ischemia after harvesting the right gastroepiploic artery.

Aged↗
PubMed1997

HUB1, a novel Krüppel type zinc finger protein, represses the human T cell leukemia virus type I long terminal repeat-mediated expression.

We have shown that human T-cell leukemia virus type I (HTLV-I) gene expression is negatively regulated by the U5 repressive element (U5RE) of its long terminal repeat (LTR). To isolate factors binding to U5RE, we screened a cDNA expression library by south-western blotting with a U5RE probe. Screening 2 x10(6) clones gave a positive clone with a 3.8 kb insert encoding a novel 671 residue polypeptide, named HTLV-I U5RE binding protein 1 (HUB1), with five zinc finger domains and a Krüppel-associated box like domain; HUB1 may be related to a repressor belonging to the Krüppel type zinc finger protein. A 4.0 kb mRNA for HUB1 is ubiquitously expressed among all human tissues tested. HUB1 recognizes the TCCACCCC sequence as a core motif and exerts a strong repressive effect on HTLV-I LTR-mediated expression. A new repressive domain, named HUB1 repressive (HUR) domain, was identified, rather than the Krüppel-associated box like domain. The N-terminal region upstream of HUR domain seemed to be also indispensable to the repression. Thus, we propose that HUB1 is a new type repressor and plays an important role in the HTLV-I U5-mediated repression.

Amino Acid Sequence↗
PubMed1997

Physical and functional interactions of Doc2 and Munc13 in Ca2+-dependent exocytotic machinery.

Doc2 has two C2 domains that interact with Ca2+ and phospholipid. Munc13 has two C2 domains and one C1 domain that interacts with phorbol ester or diacylglycerol (DAG) and phospholipid. Both Doc2 and Munc13 are implicated in Ca2+-dependent neurotransmitter release, but their modes of action still remain unclear. We show here that Doc2 interacts with Munc13 both in a cell-free system and in intact PC12 cells during the high K+-induced Ca2+-dependent exocytosis. The Doc2-Munc13 interactions are stimulated by phorbol ester through the C1 domain of Munc13. Overexpression of the Doc2-interacting domain of Munc13 reduces the Ca2+-dependent exocytosis from PC12 cells, and co-expression with Doc2 suppresses this reduction. These results, together with the earlier findings that secretagogues produce DAG and elevate cytoplasmic Ca2+, suggest that the DAG-induced Doc2-Munc13 interactions play an important role in Ca2+-dependent exocytotic machinery.

Animals↗
PubMed1997

Molecular cloning of mouse Doc2alpha and distribution of its mRNA in adult mouse brain.

We have previously isolated from a human brain cDNA library, a new protein having two C2-like domains which interact with Ca2+ and phospholipid, and named Doc2alpha. Doc2alpha is abundantly expressed in brain, where it is highly concentrated on the synaptic vesicle fraction, and is implicated in Ca2(+)-dependent exocytosis. We have isolated here a mouse Doc2alpha cDNA and determined the localization of its mRNA in adult mouse brain. The amino acid sequence of the mouse Doc2alpha cDNA is 92% identical with that of the human counterpart. Northern blot analysis and in situ hybridization on adult mouse brain sections have revealed that Doc2alpha is predominantly expressed in mouse brain, where it is expressed in neuronal cells, but not in non-neuronal cells. Doc2alpha is highly expressed in the olfactory bulb, cerebral cortex, hippocampus, amygdaloid complex, and ventromedial hypothalamus nucleus, but not in the cerebellum, caudate-putamen, or ventral thalamus. These results indicate that Doc2alpha is expressed heterogeneously in mouse brain, where it is predominantly expressed in neuronal cells, and suggest that Doc2alpha plays a specific role in the area where it is expressed.

Amino Acid Sequence↗
PubMed1996

The recombination mediated by double-strand breaks in extrachromosomal DNA substrate carrying mouse immunoglobulin switch regions S mu and S gamma 2b.

Recombination in mouse cells was analyzed using extrachromosomal DNA substrates carrying the mouse immunoglobulin switch regions S mu and S gamma 2b. Recombination was detected at a frequency of 10(-2)-10(-3) in mouse fibroblasts and in pre-B cell lines, but at a low frequency in a scid fibroblast cell line. Restriction enzyme digestion profile revealed that most recombination occurred between the CMV promoter region, which neighbors the S mu upstream region, and the S gamma 2b region. However, frequency of direct recombination between the CMV promoter region and the S gamma 2b region was low as measured by the substrate-lacking S mu region. Nucleotide sequence analysis showed that recombination occurred between several homologous base-pairs, and extranucleotides were frequently found at the recombination junctions. These results indicate that recombination took the form of the recombination mediated by double-strand breaks. Double-strand breaks likely occurred in the S mu and/or S gamma 2b region, and the ends joined.

Animals↗
PubMed1996

Sp1 family proteins recognize the U5 repressive element of the long terminal repeat of human T cell leukemia virus type I through binding to the CACCC core motif.

We have identified several nuclear proteins binding to the U5 repressive element (U5RE) at the U5 region of the human T cell leukemia virus type I (HTLV-I) long terminal repeat (LTR). In gel mobility shift assays with the U5RE DNA probe, Jurkat T cell nuclear proteins generated five different complexes, named U5RE binding protein complexes (U5RP)-A1, -A2, -A3, -B, and -C. Only U5RP-C was affected by pretreatment with an excess of poly(dI-dC) and was immunodepressed by anti-Ku/p80 antibodies, suggesting that U5RP-C is a nonspecific complex involving Ku antigen. UV cross-linking showed at least six nuclear proteins involved in the other complexes, including U5RP-A1, -A2, -A3, and -B. The sequence of the binding core element of these specific complexes, determined by competition assays and gel mobility shift assays using a series of the U5RE mutants, is CACCC which is identical to that for the Sp1 transcription factor. LTR with a mutant U5RE, which has no ability to bind with the nuclear proteins, showed stronger promoter activity than LTR with the wild U5RE, suggesting that the specific interaction of these U5RE-binding proteins might result in the U5-mediated repression. U5RP-A1 was supershifted by anti-Sp1 antibodies and U5RP-A2 and -B were supershifted by anti-Sp3 antibodies, suggesting that Sp1 or Sp3 is involved in U5RP-A1 or U5RP-A2 and -B, respectively. Although the other nuclear proteins remain to be characterized, these findings suggest that U5RE-binding proteins in U5RP-A1, -A2, -A3, and -B are involved in HTLV-I gene repression.

Base Sequence↗
PubMed1996

Doc2 enhances Ca2+-dependent exocytosis from PC12 cells.

We previously isolated a new protein having two C2-like domains which interacted with Ca2+ and phospholipid and named Doc2 (Double C2). Because Doc2 was abundantly expressed in brain where it was highly concentrated on the synaptic vesicle fraction, we have examined here whether Doc2 is involved in Ca2+-dependent exocytosis from cultured PC12 cells. For this purpose, we took advantage of the growth hormone (GH) co-expression assay system of PC12 cells in which GH is stored in dense core vesicles and released in response to high K+ in an extracellular Ca2+-dependent manner. Northern and Western blot analyses indicated that Doc2 is present in PC12 cells. Overexpression of hemagglutinin-tagged Doc2 stimulated the Ca2+-dependent, high K+-induced release of co-expressed GH without affecting the basal release. In the PC12 cells transfected with a plasmid with the coding sequence of Doc2 in the antisense orientation, the high K+-induced release of co-expressed GH was inversely inhibited. The Doc2 mutant expressing an N-terminal fragment or a C-terminal fragment containing two C2-like domains inhibited the high K+-induced release of co-expressed GH. These results indicate that Doc2 enhances Ca2+-dependent exocytosis of dense core vesicles from PC12 cells.

Amino Acid Sequence↗
PubMed1995

Molecular cloning of an isoform of Doc2 having two C2-like domains.

We previously isolated a novel protein having two C2-like domains known to interact with Ca2+ and phospholipid, and named Doc2 (Double C2). Doc2 is predominantly expressed in brain and is implicated in Ca(2+)-dependent neurotransmitter release. We have isolated here an isoform of Doc2 and named the original one Doc2 alpha and the new one Doc2 beta. Doc2 beta alsp has two C2-like domains and is 61% identical to Doc2 alpha at the amino acid level. In contrast to Doc2 alpha, the Doc2 beta mRNA is expressed ubiquitously. These results indicate that there are at least two isoforms of Doc2, and suggest that Doc2 beta is involved in Ca(2+)-dependent intracellular vesicle trafficking in various types of cells.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Autoantigen Ku protein is involved in DNA binding proteins which recognize the U5 repressive element of human T-cell leukemia virus type I long terminal repeat.

We have identified and analyzed a 27-nucleotide sequence (U5 repressive element, designated as U5RE) at the U5 region of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) which is required for HTLV-I basal transcriptional repression. The basal promoter strength of constructs that contained deletions in the U5 region of the LTR was analyzed by chloramphenicol acetyltransferase (CAT) assays following transfection of HeLa cells or Jurkat T-cells in the presence or absence of viral transactivator tax protein. We consistently observed a 2- to 5-fold increase in basal promoter activity when sequences between +277 to +306 were deleted. In vivo competition experiments suggested that the U5 DNA fragment from +269 to +295 contains a functional repressive element (U5RE). Using gel mobility shift assays, we have purified a highly enriched fraction that could specifically bind U5RE. This DNA affinity column fraction contained three major detectable proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining: 110-, 80- and 70-kDa proteins. The 110-kDa protein appeared to be a novel DNA-binding protein whose characteristics are still obscure, while the 70- and 80-kDa proteins were shown to be related to the human autoantigen Ku, the Ku (p70/p80) complex, as demonstrated by amino acid sequencing and immunological analyses. As Ku is known to be involved in transcriptional regulation, the specific interaction of Ku with U5RE raises intriguing possibilities for its function in HTLV-I basal transcriptional repression.

Amino Acid Sequence↗
PubMed1994

Multiplication of Clostridium botulinum in dead honey-bees and bee pupae, a likely source of heavy contamination of honey.

Multiplication of Clostridium botulinum in honey-bees was examined to explain the heavy contamination of honey which may occur with this pathogen. When dead bees were inoculated with C. botulinum spores at levels of 10(2)-10(3) and incubated aerobically for 10 days, the organisms increased to 10(4)-10(5). When botulinum spores were inoculated together with Bacillus alvei, the growth of most strains was significantly enhanced (10(5)-10(7)). Similar results were obtained in bee pupae, but not in bee larvae. The heavy contamination of honey with botulinum spores that we have sometimes encountered may have been caused by contamination from dead bees in which C. botulinum had proliferated.

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↗
PubMed1992

Detection of Clostridium botulinum in natural sweetening.

Various sugar products were examined for contamination with C. botulinum spores. Type A, B and C spores were detected in three of 56 samples of sugar for apiculture, which may attest the significance of bee-feed as a source of contamination of honey. The heavy contamination of honey with C. botulinum spores sometimes encountered, however, can not be explained unless some other factors, e.g., that allowing germination and multiplication of the spores somewhere during honey production, are found. Type A spores were detected in some samples of raw sugar and molasses and also in two of 41 samples of brown sugar lump, but not in refined sugar or other various samples taken at a sugar factory or in sugar cane left on the field in Okinawa. The fact that some natural sweetenings are contaminated with C. botulinum spores, even in low concentrations, may be food-hygienically important.

Animals↗