[Molecular organization and function of postsynaptic density (PSD)].
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Actins↗
Biomedical subjects
T Tachibana
Publications and source records attributed to T Tachibana.
[Usefulness of fluorine-18 fluoro-2-deoxyglucose positron emission tomography for diagnosis and therapy assessment of cardiac sarcoidosis].
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Animals↗
Involvement of unique leucine-zipper motif of PSD-Zip45 (Homer 1c/vesl-1L) in group 1 metabotropic glutamate receptor clustering.
Several scaffold proteins for neurotransmitter receptors have been identified as candidates for receptor targeting. However, the molecular mechanism underlying such receptor clustering and targeting to postsynaptic specializations remains unknown. PSD-Zip45 (also named Homer 1c/vesl-1L) consists of the NH(2) terminus containing the enabled/VASP homology 1 domain and the COOH terminus containing the leucine zipper. Here, we demonstrate immunohistochemically that metabotropic glutamate receptor 1alpha (mGluR1alpha) and PSD-Zip45/Homer 1c are colocalized to synapses in the cerebellar molecular layer but not in the hippocampus. In cultured hippocampal neurons, PSD-Zip45/Homer1c and N-methyl-D-aspartate receptors are preferentially colocalized to dendritic spines. Cotransfection of mGluR1alpha or mGluR5 and PSD-Zip45/Homer 1c into COS-7 cells results in mGluR clustering induced by PSD-Zip45/Homer 1c. An in vitro multimerization assay shows that the extreme COOH-terminal leucine zipper is involved in self-multimerization of PSD-Zip45/Homer 1c. A clustering assay of mGluRs in COS-7 cells also reveals a critical role of this leucine-zipper motif of PSD-Zip45/Homer 1c in mGluR clustering. These results suggest that the leucine zipper of subsynaptic scaffold protein is a candidate motif involved in neurotransmitter receptor clustering at the central synapse.
Animals↗
A monoclonal antibody to the COOH-terminal acidic portion of Ran inhibits both the recycling of Ran and nuclear protein import in living cells.
A small GTPase Ran is a key regulator for active nuclear transport. In immunoblotting analysis, a monoclonal antibody against recombinant human Ran, designated ARAN1, was found to recognize an epitope in the COOH-terminal domain of Ran. In a solution binding assay, ARAN1 recognized Ran when complexed with importin beta, transportin, and CAS, but not the Ran-GTP or the Ran-GDP alone, indicating that the COOH-terminal domain of Ran is exposed via its interaction with importin beta-related proteins. In addition, ARAN1 suppressed the binding of RanBP1 to the Ran-importin beta complex. When injected into the nucleus of BHK cells, ARAN1 was rapidly exported to the cytoplasm, indicating that the Ran-importin beta-related protein complex is exported as a complex from the nucleus to the cytoplasm in living cells. Moreover, ARAN1, when injected into the cultured cells induces the accumulation of endogenous Ran in the cytoplasm and prevents the nuclear import of SV-40 T-antigen nuclear localization signal substrates. From these findings, we propose that the binding of RanBP1 to the Ran-importin beta complex is required for the dissociation of the complex in the cytoplasm and that the released Ran is recycled to the nucleus, which is essential for the nuclear protein transport.
Amino Acid Sequence↗
beta-subunit of nuclear pore-targeting complex (importin-beta) can be exported from the nucleus in a Ran-independent manner.
The nuclear export of importin-alpha is mediated by CAS, which is related to importin-beta, whereas the mechanism for the export of importin-beta remains unclear. In this study, we demonstrate that the nuclear export of importin-beta is mediated by the nuclear pore complex-binding domain of this molecule. Insensitivity to leptomycin B indicates that its export is not mediated by a leucine-rich nuclear export signal-specific receptor, CRM1. Furthermore, the nuclear export of importin-beta was not inhibited by co-injection with a GTPase-deficient Ran mutant (G19V). The cell line tsBN2 contains a temperature-sensitive point mutation in the RCC1 gene, which encodes a guanine nucleotide exchange factor of Ran. At the nonpermissive temperature, importin-beta was exported from the nucleus of these cells, even when RanGAP1, a GTPase-activating protein for Ran, was co-injected. These results not only provide support for the view that Ran-dependent GTP hydrolysis is not required for the nuclear export of importin-beta but also indicate that nuclear RanGTP is not essential for its export. As a result, we propose that importin-beta can be recycled from the nucleus alone in a Ran-independent manner.
Animals↗
Up-regulation of nuclear protein import by nuclear localization signal sequences in living cells.
Using an in vivo assay system, nuclear import ability in individual cells was determined by examining the nuclear import rate. It was found that when a small (not excess) amount of SV40 T-NLS peptides was co-injected, the nuclear import rate of SV40 T-NLS-containing substrates apparently increased. This up-regulation was reproduced by the co-injection of peptides containing bipartite type NLS of CBP80, but not mutated non-functional NLS peptides, which suggests that these phenomena are specific for functional NLSs. It was further shown that although, in growth-arrested cells, the nuclear import rate was down-regulated compared to growing cells, the elevation of the functional import rate by co-injected NLS peptides reached the same level as in proliferating cells. This up-regulation was abolished by the addition of a protein kinase inhibitor, staurosporine. These results suggest that although potential nuclear import ability does not vary in each cell, the rate of nuclear import may be controlled by the amount of karyophilic proteins, which need to be carried into the nucleus from the cytoplasm, possibly via an NLS-dependent phosphorylation reaction.
Animals↗
Differential regulation of alpha- and beta-CGRP mRNAs within oculomotor, trochlear, abducens, and trigeminal motoneurons in response to axotomy.
Spinal and cranial motoneurons express alpha- and beta-calcitonin gene-related peptide (CGRP) mRNAs constitutively at variable ratios, and these two mRNAs are differentially regulated following axotomy in spinal, facial, and hypoglossal motoneurons. The purpose of this study was to investigate the change in CGRP mRNA expression following nerve injury in oculomotor, trochlear, abducens, and trigeminal motor nuclei in which beta-CGRP mRNA is predominantly expressed under normal conditions. Using male Sprague-Dawley rats, either the left eyeball and the orbital contents including the bulbar muscles were removed, or the left masseter nerve was ligated and transected. The rats were allowed to survive for 1, 3, 7, 14, 28, 56 days following these procedures. The levels of mRNAs for alpha- and beta-CGRP and growth-associated protein (GAP)-43 were analyzed by in situ hybridization histochemistry using 35S-labeled oligonucleotide probes. Following nerve injury, the expression of alpha-CGRP mRNA rapidly increased on the directly-injured side in all of these nuclei. Thereafter, it gradually decreased and returned to about the control level at postoperative day 56 within oculomotor, trochlear, and abducens motoneurons, but it sustained at a high level within trigeminal motoneurons. The expression of beta-CGRP was quite variable among these nuclei, and significant changes were also seen on the side contralateral to the directly-injured side. These data indicate that the up-regulation of alpha-CGRP mRNA may be a common response of cranial motor neurons following axotomy even if the constitutive expression of beta-CGRP mRNA exceeds that of alpha-CGRP mRNA in these neurons.
Abducens Nerve↗
Existence of activated and memory CD4+ T cells in peripheral blood and their skin infiltration in CD8 deficiency.
CD8 deficiency is a rare primary immunodeficiency caused by the defect of a tyrosine kinase, ZAP-70, which transduces signals from the T cell receptor. We report here a case of CD8 deficiency, having CD4+ T cells with a unique phenotype. The patient's T cells did not respond to anti-CD3 stimulation in vitro, suggesting that they were naive. However, many CD4+ T cells with activated and memory phenotypes, which expressed CD45RO+, HLA-DR+ and CD25+, were present in the peripheral blood, and these cells accumulated in the perivascular area of his infiltrative erythematous skin lesions. The patient's T cells could be activated by a high concentration of phytohaemagglutinin (PHA), indicating the presence of an alternate signalling pathway which bypasses ZAP-70 and activates CD4+ T cells in vivo. The origin and role of activated CD4+ T cells in the pathogenesis involved in the skin lesions are discussed.
CD4-Positive T-Lymphocytes↗
Involvement of CD4+ T cells and macrophages in acquired protection against infection with Sporothrix schenckii in mice.
The cell-mediated immune response against infection with Sporothrix schenckii was investigated in vivo and in vitro. Following primary infection, mice were protected against a secondary subcutaneous inoculation with S. schenckii as determined by the local growth of fungi, and subcutaneously immunized mice were able to survive a lethal intravenous infection. Protection could be transferred to naive congenitally athymic nude mice using lymph node cells taken from immune mice. When immune lymph node cells had CD4+ cells depleted by antibody plus complement, the ability to transfer protection was significantly reduced. Treatment of mice with carrageenan, a macrophage blocker, before and after secondary inoculation abolished protection. An in vitro system, using either immune lymph node cells alone or macrophages alone, failed to kill the organism. However, inhibition of fungal growth was observed when both immune lymph node cells and macrophages were combined. Growth inhibition in vitro was reduced after depletion of CD4+ cells but not CD8+ cells. Addition of anti-interferon (IFN)-gamma monoclonal antibodies (MAb) also reduced growth inhibition. By using reverse transcription-polymerase chain reaction (RT-PCR) analysis, it was shown that immune lymph node cells expressed message for IFN-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-10 after stimulation with heat-killed S. schenckii. These results suggest that acquired immunity against S. schenckii is expressed mainly by macrophages activated by CD4+ T cells.
Adoptive Transfer↗
beta-catenin can be transported into the nucleus in a Ran-unassisted manner.
The nuclear accumulation of beta-catenin plays an important role in the Wingless/Wnt signaling pathway. This study describes an examination of the nuclear import of beta-catenin in living mammalian cells and in vitro semi-intact cells. When injected into the cell cytoplasm, beta-catenin rapidly migrated into the nucleus in a temperature-dependent and wheat germ agglutinin-sensitive manner. In the cell-free import assay, beta-catenin rapidly migrates into the nucleus without the exogenous addition of cytosol, Ran, or ATP/GTP. Cytoplasmic injection of mutant Ran defective in its GTP hydrolysis did not prevent beta-catenin import. Studies using tsBN2, a temperature-sensitive mutant cell line that possesses a point mutation in the RCC1 gene, showed that the import of beta-catenin is insensitive to nuclear Ran-GTP depletion. These results show that beta-catenin possesses the ability to constitutively translocate through the nuclear pores in a manner similar to importin beta in a Ran-unassisted manner. We further showed that beta-catenin also rapidly exits the nucleus in homokaryons, suggesting that the regulation of nuclear levels of beta-catenin involves both nuclear import and export of this molecule.
Animals↗
Molecular shape and ATP binding activity of rat p50, a putative mammalian homologue of RuvB DNA helicase.
Based on partial amino acid sequences of p50 purified from a high-salt buffer extract of a rat liver nuclear matrix fraction, p50 cDNA was cloned and sequenced, and its amino acid sequence was predicted. The sequence contained helicase motifs, and showed homology with RuvB DNA helicase of Thermus thermophilus and an open reading frame for an unknown 50.5 k protein of Saccharomyces cerevisiae. p50 was expressed as a GST-fusion protein and antiserum against the protein was generated. p50 was localized to the nuclear matrix by cell fractionation and immunoblotting. p50 bound to ATP-Sepharose beads. Ultracentrifugation and gel filtration analyses showed that p50 in rat liver and Xenopus egg mitotic extracts exists as large complexes corresponding to 697 k and 447 k, respectively. A 50 k protein reactive with p50 antibodies was detected not only in rat liver nuclei, but also in a Xenopus egg cytoplasm fraction and a S. cerevisiae extract. This suggests that this putative DNA helicase is present in a wide variety of species ranging from yeast to mammals.
ATPases Associated with Diverse Cellular Activitie↗
[A case of left atrial thrombus complicated with coarctation of the aorta].
The case was a sixty one year old female who was diagnosed as having acute arterial embolism of right upper arm and underwent embectomy. The detailed examination which followed revealed left atrial myxoma and coarctation of the aorta. Due to the risk of embolism, an emergency operation was performed to install a right axillofemoral bypass with an 8 mm artificial graft. Left atrial myxoma was then excised in an operation using an artificial heart-lung apparatus. Pathological examinations of excised tumor offered no trace of tumor but only of left atrial thrombus. Postoperative results were satisfactory. The pressure difference between upper and lower arms was improved to 0.8 in pressure index from preoperative 0.5 and it became possible to control the blood pressure of upper arm within the level of 130 to 150 mmHg. Axillofemoral bypass is an effective method for a case like the above where a site of coarctation of the aorta cannot be directly repaired.
Aortic Coarctation↗
[Excess mortality from influenza epidemics in Japan, 1980-1994].
In 1973, WHO proposed that "excess mortality" be used for comparative assessment of the severity of influenza epidemics between countries. In this study, analysis of the data for deaths in Japan between 1980-1994 revealed excess mortality, which could be a useful indicator in Japan. There were six influenza epidemics, and statistically significant excess mortality rates were calculated in both deaths from acute respiratory diseases and all causes. The total number of excess deaths from acute respiratory diseases during influenza seasons for the 15 years was 13,931. Statistically significant excess mortality rates were also shown for other diseases, including ischemic heart disease, cerebro-vascular disease, diabetes, nephritis, chronic liver disease, malignant neoplasm, and chronic respiratory disease. Therefore, "excess mortality" is clearly present in recent years in Japan. In analyses of age-specific excess mortality rates, the epidemic was found to be most fatal to the aged. Further, the value of % excess (= excess rate divided by expected rate x 100) suggested that the number of deaths from all causes would be a more useful indicator than that from acute respiratory diseases. It was confirmed that excess mortality was applicable to regional surveillance of epidemic influenza.
Acute Disease↗
[Chronic dissecting aneurysm of the sinus of valsalva: report of a case].
The subject was a forty five year old female who was admitted to our hospital with chief complaints of cough and thoracic distress. After detailed examination, she was diagnosed as having aneurysm of the noncoronary sinus of Valsalva and aortic regurgitation. The finding during operation showed chronic aortic dissection localized to the noncoronary sinus of Valsalva. The entry of aortic dissection was just above the noncoronary annulus, which was found to be detached and leaned to the left ventricule. An operation for aortic root replacement was performed with Carrel patch method and had a good postoperative course. Only a few reports have been made on cases of aneurysm of the noncoronary sinus of Valsalva caused by chronic regional aortic dissection.
Aortic Dissection↗
Establishment and characterization of cell lines derived from serous adenocarcinoma (JHOS-2) and clear cell adenocarcinoma (JHOC-5, JHOC-6) of human ovary.
The cell lines designated JHOS-2, JHOC-5 and JHOC-6 were established from epithelial ovarian carcinomas. JHOS-2 was established from a serous adenocarcinoma of a 45-year-old Japanese woman, JHOC-5 from a recurrent tumor of a clear cell adenocarcinoma of a 47-year-old Japanese woman and JHOC-6 from a tumor of a clear cell adenocarcinoma of a 43-year-old Japanese woman. These cell lines have grown well and serial passages were successively carried out more than 20 times. The monolayer cultured cells revealed neoplastic and pleomorphic features, and grew in multilayers. Electron micrographs revealed epithelial origins that had desmosomes and tonofilaments.
Adenocarcinoma, Clear Cell↗
Establishment and characterization of human choriocarcinoma cell line derived from a metastatic focus of a testicular mixed germ cell tumor.
A human testicular choriocarcinoma cell line HKRT-II was established by the single-cell cloning method from a mixed cell culture system derived from a retroperitoneal metastatic germ cell tumor composed of a yolk-sac tumor, a choriocarcinoma, and an immature teratoma. Its primary tumor rose from the testis and was comprised of a seminoma, a yolk-sac tumor, a choriocarcinoma and an immature teratoma. The HKRT-II cells were spindle or polygonal in shape and contained multi-nucleated giant cells showing neoplasticity and pleomorphism. The cells proliferated in a stable manner, and the population doubling time was 42 hours. The chromosome numbers showed a wide distribution of aneuploidy, while the mode was in the hypertetraploid range. Double minute chromosomes and homogeneously staining regions were recognized in about 5% to 10% of the metaphase plates, respectively. Heterotransplantation was not difficult. Subcutaneous transplantation of 1 x 10(7) cells into nude mice formed a tumor composed of only a choriocarcinoma. The most noteworthy characteristics of the cell line were that it produced human chorionic gonadotropin (hCG) in an in vitro culture system and in in vivo grafted cells, and that the N-myc gene was amplified about 10 times.
Adult↗
Nuclear transport factor p10/NTF2 functions as a Ran-GDP dissociation inhibitor (Ran-GDI).
The cytosolic nuclear transport factor p10/NTF2 is required for the translocation of karyophilic molecules through nuclear pores [1] [2] [3], and the small GTPase Ran is a key regulator of protein transport between the nucleus and cytoplasm [4] [5]. It has been reported that p10/NTF2 interacts directly and specifically with Ran-GDP but not with Ran-GTP [6]. The precise role(s) of p10/NTF2 in the Ran GTP/GDP cycle are thus far unclear, however. In this study, we show that mammalian p10/NTF2 dramatically inhibits the dissociation of [3H]GDP from Ran and the binding of [35S]GTPgammaS to Ran following the dissociation of non-radioactive GDP by RCC1, the only known mammalian guanine nucleotide exchange factor for Ran (Ran-GEF) [7]. In contrast, the dissociation of [35S]GTP gamma S from Ran, which was also catalyzed by RCC1, was not affected by p10/NTF2. Furthermore, the activities of wild-type p10/NTF2 and the mutant forms M84T and D92G in an assay of nuclear protein import in a digitonin-permeabilized cell-free system correlated with their level of inhibition of the dissociation of nucleotide from Ran-GDP. These results suggest that p10/NTF2 acts as a GDP dissociation inhibitor for Ran (Ran-GDI), thereby coordinating the Ran-dependent reactions that underlie nuclear protein import.
Animals↗
Nuclear localization and export signals of the human aryl hydrocarbon receptor.
The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor that binds DNA in the form of a heterodimer with the Ahr nuclear translocator (hypoxia-inducible factor 1beta). We found in this study that Ahr contains both nuclear localization and export signals in the NH2-terminal region. A fusion protein composed of beta-galactosidase and full-length Ahr translocates from the cytoplasm to the nucleus in a ligand-dependent manner. However, a fusion protein lacking the PAS (Per-Ahr nuclear translocator-Sim homology) domain of the Ahr showed strong nuclear localization activity irrespective of the presence or absence of ligand. A minimum bipartite Ahr nuclear localization signal (NLS) consisting of amino acid residues 13-39 was identified by microinjection of fused proteins with glutathione S-transferase-green fluorescent protein. A NLS having mutations in bipartite basic amino acids lost nuclear translocation activity completely, which may explain the reduced binding activity to the NLS receptor, PTAC58. A 21-amino acid peptide (residues 55-75) containing the Ahr nuclear export signal is sufficient to direct nuclear export of a microinjected complex of glutathione S-transferase-Ahr-green fluorescent protein. These findings strongly suggest that Ahr act as a ligand- and signal-dependent nucleocytoplasmic shuttling protein.
Biological Transport↗