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Biomedical subjects

S Tsuji

Publications and source records attributed to S Tsuji.

At least 19 recordsLinked to original sources
PubMed1999

Cytotoxic T cells against a peptide of Yo protein in patients with paraneoplastic cerebellar degeneration and anti-Yo antibody.

Anti-Yo antibodies are present in the sera and cerebrospinal fluid of some patients with paraneoplastic cerebellar degeneration (PCD), but there is no evidence that the presence of anti-Yo antibodies causes the Purkinje cell loss seen in PCD patients. We examined the level of cytotoxic T lymphocyte (CTL) activity against a nine-amino acid peptide of the Yo protein using the Human Leukocyte Antigen- (HLA-) based approach called reverse immunogenetics. Mononuclear cells (MNCs) were isolated from the peripheral venous blood and fibroblasts were obtained from the skin of three patients with PCD with anti-Yo antibody. After activating the MNCs of the three patients with the peptide, it showed CTL activity against the Yo protein peptide expressed on autologous fibroblasts. Therefore, CTLs may be involved in the loss of Purkinje cells in PCD.

Autoantibodies↗
PubMed1999

Cytosolic calcium changes in a process of platelet adhesion and cohesion on a von Willebrand factor-coated surface under flow conditions.

Recent flow studies indicated that platelets are transiently captured onto and then translocated along the surface through interaction of glycoprotein (GP) Ib with surface-immobilized von Willebrand factor (vWF). During translocation, platelets are assumed to be activated, thereafter becoming firmly adhered and cohered on the surface. In exploring the mechanisms by which platelets become activated during this process, we observed changes in platelet cytosolic calcium concentrations ([Ca(2+)]i) concomitantly with the real-time platelet adhesive and cohesive process on a vWF-coated surface under flow conditions. Reconstituted blood containing platelets loaded with the Ca(2+) indicators Fura Red and Calcium Green-1 was perfused over a vWF-coated glass surface in a flow chamber, and changes in [Ca(2+)]i were evaluated by fluorescence microscopy based on platelet color changes from red (low [Ca(2+)]i) to green (high [Ca(2+)]i) during the platelet adhesive and cohesive process. Under flow conditions with a shear rate of 1,500 s(-1), no change in [Ca(2+)]i was observed during translocation of platelets, but [Ca(2+)]i became elevated apparently after platelets firmly adhered to the surface. Platelets preincubated with anti-GP IIb-IIIa antibody c7E3 showed no firm adhesion and no [Ca(2+)]i elevation. The intracellular Ca(2+) chelator dimethyl BAPTA did not inhibit firm platelet adhesion but completely abolished platelet cohesion. Although both firm adhesion and cohesion of platelets have been thought to require activation of GP IIb-IIIa, our results indicate that [Ca(2+)]i elevation is a downstream phenomenon and not a prerequisite for firm platelet adhesion to a vWF-coated surface. After platelets firmly adhere to the surface, [Ca(2+)]i elevation might occur through the outside-in signaling from GP IIb-IIIa occupied by an adhesive ligand, thereby leading to platelet cohesion on the surface.

Blood Platelets↗
PubMed1999

Real-time analysis of mural thrombus formation in various platelet aggregation disorders: distinct shear-dependent roles of platelet receptors and adhesive proteins under flow.

We evaluated real-time processes of platelet thrombus formation on a collagen surface in a flow chamber with whole blood from patients with various platelet aggregation disorders, such as Bernard-Soulier syndrome (BSS), Glanzmann's thrombasthenia (GTA), type 3 von Willebrand disease (vWD), and congenital afibrinogenemia (Af), who lack platelet glycoprotein (GP) Ib-IX complex, GP IIb-IIIa, von Willebrand factor (vWF), and fibrinogen, respectively. Blood from GTA patients showed impaired thrombus growth but significant initial platelet-surface interaction under all shear conditions tested (50 to 1,500 s(-1)). By contrast, blood from patients with BSS or type 3 vWD showed no platelet-surface interaction under high shear (>/=1, 210 s(-1)) but normal thrombus formation under low shear (</=340 s(-1)). When shear rate was increased stepwise to 1,500 s(-1) during perfusion, the thrombus growth observed in type 3 vWD or BSS under low shear was arrested, whereas that in control blood was sharply accelerated as a function of shear rate. Overall thrombus formation in Af appeared indistinguishable from that of a control under shear rates between 50 and 1,500 s(-1). However, Af thrombi formed under such conditions collapsed immediately when shear rate was further increased to 4,500 s(-1), whereas thrombi of type 3 vWD or BSS formed under low shear were stable even when shear rate was elevated to 9,000 s(-1) during perfusion. These findings suggest that distinct molecular mechanisms underlie the pathologic bleeding in these diseases and point to the distinct roles of two major adhesive proteins, vWF and fibrinogen. In mural thrombus formation under flow conditions, vWF, perhaps mainly through its interaction with GP Ib-IX, acts as an "initiator and promoter," whereas fibrinogen, via its binding to GP IIb-IIIa, acts as a "stabilizer" against heightened shear forces that could lead to peeling off of platelets from the surface.

Blood Coagulation Disorders↗
PubMed1999

Mental stress test is an effective inducer of vasospastic angina pectoris: comparison with cold pressor, hyperventilation and master two-step exercise test.

BACKGROUND: Cold pressor, hyperventilation and exercise stress tests were usually used for inducing an angina attack in patients with vasospastic angina pectoris. We induced vasospastic angina attack using the mental calculation stress test, and compared the results with those using other stress tests. SUBJECTS AND METHODS: Subjects were 29 patients with vasospastic angina pectoris. Their ages were 60.8+/-8.4 years. Coronary vasospasm was induced by an acetylcholine infusion test during coronary angiography. The mental stress test was performed as follows; after memorizing six digits numbers, they repeated these numbers in reverse for 5 min, and performed serial subtraction of 17 from 1000 for 5 min. Blood pressure, heart rate and ECG were recorded every 1-5 min during the mental stress test. The serum concentrations of epinephrine and norepinephrine were measured before and during the mental stress test. We compared these results with those obtained using cold pressor, hyperventilation and the Master two-step exercise stress test. RESULTS: (1) Eight of the 29 patients (28%) showed ischemic ST-T change, which was caused by the mental stress test. (2) The increase in norepinephrine was greater in patients with an ST-T change than without an ST-T change (0.11+/-0.06 vs. 0.04+/-0.04 ng/ml, P<0.01). (3) The incidence of the ST-T change caused by the mental stress test (28%) was similar to the cold pressor test (27%) and greater than that caused by the hyperventilation test (13%). The incidence of ST-T change caused by the Master two-step test was 55%. CONCLUSIONS: The mental stress test is an effective inducer of vasospastic angina attack, and attack may be induced by neurohumoral vasoconstrictive reflex and/or increased left ventricular afterload.

Acetylcholine↗
PubMed1999

No mutation in the entire coding region of the alpha-synuclein gene in pathologically confirmed cases of multiple system atrophy.

To determine whether mutations in the coding region of the alpha-synuclein gene are relevant in cases of multiple system atrophy (MSA), detailed nucleotide sequence analysis of the alpha-synuclein gene was performed using total RNA obtained from autopsied brain specimens of 11 pathologically confirmed cases of MSA. The brain specimens used in this study contained both gray and white matter, which were dissected from the frontal, temporal or occipital lobe. No nucleotide alterations were found in the entire coding region of the alpha-synuclein gene in any of the cases. While mutations in the regulatory or intronic regions of the gene were not ruled out, our results suggest that mutations in the coding region of the alpha-synuclein gene are unlikely to contribute to the pathogenesis of MSA.

Aged↗
PubMed1999

Quantitative analysis of expression of mouse sialyltransferase genes by competitive PCR.

The present paper describes a rapid and systematic method for semi-quantitative analysis of the expression of sialyltransferase genes. So far, fifteen sialyltransferase cDNAs have been cloned from mice. Most of these genes are expressed in developmental stage-dependent and/or tissue-specific manners, and the expression levels of some of them are too low to detect on Northern blot analysis. To resolve how each sialyltransferase contribute to synthesize sialylglycoconjugates, it is necessary to establish the method for quantification of gene expression levels of these fifteen sialyltransferases. Therefore, we developed a competitive PCR-based method for analyzing the quantitative relationship of the gene expression of fifteen sialyltransferases. Using this method, we can investigate the levels of gene expression of sialyltransferases in various cell lines and various tissues of mice, and can accurately determine their expression levels.

Animals↗
PubMed1999

Restricted occurrence of Lewy bodies in the dorsal vagal nucleus in a patient with late-onset parkinsonism.

Lewy body (LB) is consistently found in the substantia nigra in Parkinson's disease. We report a 68-year-old woman with late-onset, dopa-responsive parkinsonism. Her parents were first cousins, but no other affected individuals were present in the family. Autopsy revealed moderate loss of pigmented neurons with gliosis, but neither LBs nor neurofibrillary tangles in the substantia nigra. The locus ceruleus showed neuronal loss with scarce LBs. The most striking change was found in the dorsal vagal nucleus, where marked neuronal loss and fibrillary gliosis with many LBs were evident. Despite the use of ubiquitin and alpha-synuclein immunohistochemistry, no further LBs were identified in other brain regions. These findings suggest that this case was an unusual, anatomically restricted manifestation of LB disease.

Antiparkinson Agents↗
PubMed1999

Regulation of transcription factor C/ATF by the cAMP signal activation in hippocampal neurons, and molecular interaction of C/ATF with signal integrator CBP/p300.

The CCAAT/enhancer binding proteins related activating transcription factor, C/ATF, is a mouse leucine-zipper transcription factor which is structurally homologous to ApCREB2, a suppressor integral to long-term synaptic plasticity in Aplysia. To gain a clue to whether C/ATF is involved in long-term plasticities of brain, we examined if the expression levels of C/ATF are modulated by cAMP, an inducer crucial for memory formation in Aplysia, Drosophila and mice. Our in situ hybridization analysis revealed the expression of C/ATF mRNA in hippocampal neurons. C/ATF protein levels increased after the cAMP signal stimulation in hippocampal neurons, while C/ATF mRNA levels remained constant. The human activating transcription factor 4 (hATF4), another homolog of ApCREB2, interacts with multiple domains of the coactivator CREB-binding protein (CBP), resulting in the potentiation of its ability to activate transcription. As expected, C/ATF was found to interact with three domains of CBP including CREB binding domain or kinase-inducible interaction (KIX) domain, the third cysteine-histidine-rich region (CH3 domain) and the nuclear receptor coactivator p160/SRC-1-interacting domain. Interestingly, C/ATF was further found to interact strongly with CREB binding protein/p300 (CBP/p300) CH1 domain. Mammalian two hybrid assays indicated that the interaction between C/ATF and CBP/p300 can occur in mammalian cells, and that the p300 CH1 domain is critical for the interaction. Thus, C/ATF may be implicated in transcription-dependent phase of hippocampal long-term plasticities through the modulation of its protein level under cAMP signal and the interaction with signal integrator, CBP/p300.

Activating Transcription Factor 4↗
PubMed1999

Administration of prosaposin ameliorates spatial learning disturbance and reduces cavity formation following stab wounds in rat brain.

The effectiveness of prosaposin as a neurotrophic factor was investigated using rats with bilateral stab wounds, injecting 240 ng per day of prosaposin for 3 days. In Morris water maze task, after 3 weeks postoperation, the stab-wounds rats show significant impairment in acquisition compared with the sham-operated rats. In the transfer test the mean number of crossings of the platform place in stab-wounds was significantly lower than that in sham-operated rats (P < 0.01). The stab-wounds rats treated with prosaposin showed significant improvement (P < 0.05). The cavities following stab wounds in the rats treated with prosaposin were significantly smaller than those in the rats treated with (P < 0.05). Our data support that prosaposin is likely to be a new agent for brain injury.

Animals↗
PubMed1999

Induction of cell-cycle arrest and apoptosis by a novel retinobenzoic-acid derivative, TAC-101, in human pancreatic-cancer cells.

In this study, we investigated the effect of a novel retinobenzoic acid, 4-[3,5-bis (trimethylsilyl) benzamido] benzoic acid (TAC-101), on the growth of 4 human pancreatic-cancer cell lines; BxPC-3, MIAPaCa-2, CFPAC-1 and AsPC-1. TAC-101 significantly inhibited the proliferation of BxPC-3 and MIAPaCa-2 cells in a time- and concentration-dependent manner, but not the proliferation of AsPC-1 cells. Furthermore, the anti-proliferative effects of TAC-101 on BxPC-3 and MIAPaCa-2 cells were stronger than those of all-trans retinoic acid. Flow-cytometric analyses indicated that treatment of BxPC-3 with TAC-101 strongly induces cell-cycle arrest at the G1 phase. The cell-cycle arrest induced by TAC-101 was accompanied by reduction of retinoblastoma-gene product (RB) phosphorylation and an increase of 2 cyclin-dependent kinase (CDK) inhibitors, p21(WAF1/Cip1) (p21) and p27Kip1 (p27). TAC-101 also caused a decrease in cyclin A and thymidylate synthase, which are E2F-regulated gene products. No changes were observed in the expression of cyclin D1, cyclin E on CDK2. In addition, Hoechst staining, gel electrophoresis and flow-cytometric analysis indicated that a marked reduction in the number of BxPC-3 cells with TAC-101 was related to the induction of apoptosis. Our results suggest that TAC-101 inhibits the growth of certain pancreatic-cancer cells by means of G1-phase cell-cycle arrest resulting from the reduction of RB phosphorylation and the up-regulation of p21 and p27 as well as the induction of apoptosis. TAC-101 may therefore be a useful agent for new therapeutic strategies focusing on inhibition of pancreatic-cancer-cell proliferation.

Antineoplastic Agents↗
PubMed1999

Molecular cloning and functional expression of two members of mouse NeuAcalpha2,3Galbeta1,3GalNAc GalNAcalpha2,6-sialyltransferase family, ST6GalNAc III and IV.

Two cDNA clones encoding NeuAcalpha2,3Galbeta1,3GalNAc GalNAcalpha2, 6-sialyltransferase have been isolated from mouse brain cDNA libraries. One of the cDNA clones is a homologue of previously reported rat ST6GalNAc III according to the amino acid sequence identity (94.4%) and the substrate specificity of the expressed recombinant enzyme, while the other cDNA clone includes an open reading frame coding for 302 amino acids. The deduced amino acid sequence is not identical to those of other cloned mouse sialyltransferases, although it shows the highest sequence similarity with mouse ST6GalNAc III (43.0%). The expressed soluble recombinant enzyme exhibited activity toward NeuAcalpha2, 3Galbeta1, 3GalNAc, fetuin, and GM1b, while no significant activity was detected toward Galbeta1,3GalNAc or asialofetuin, or the other glycoprotein substrates tested. The sialidase sensitivity of the 14C-sialylated residue of fetuin, which was sialylated by this enzyme with CMP-[14C]NeuAc, was the same as that of ST6GalNAc III. These results indicate that the expressed enzyme is a new type of GalNAcalpha2,6-sialyltransferase, which requires sialic acid residues linked to Galbeta1,3GalNAc residues for its activity; therefore, we designated it mouse ST6GalNAc IV. Although the substrate specificity of this enzyme is similar to that of ST6GalNAc III, ST6GalNAc IV prefers O-glycans to glycolipids. Glycolipids, however, are better substrates for ST6GalNAc III.

Amino Acid Sequence↗
PubMed1999

14-3-3 protein eta chain gene (YWHAH) polymorphism and its genetic association with schizophrenia.

Recent genetic analyses have suggested a linkage between schizophrenia and the chromosomal region 22q12-q13. 14-3-3 protein, abundant in the brain, mediates interactions between diverse molecules of biological activities; its gene was recently mapped to chromosome 22q12.1-q13.1. We therefore investigated allele frequencies of a variable number of tandem repeat (VNTR) in the 5'-noncoding region of the 14-3-3 eta chain gene in controls and schizophrenics. The frequencies of the two-repeat allele were significantly higher (P < 0.05) in the schizophrenics, and particularly in those with onset before age 22 (early-onset schizophrenics, P < 0.02), than in the controls. The odds ratio was significantly increased in the early-onset schizophrenics homozygous for the two-repeat allele (OR = 3.3, 95% CI = 1.1-9.7). The 14-3-3 eta chain gene is a potential susceptibility gene for schizophrenia, and particularly for early-onset schizophrenia.

14-3-3 Proteins↗
PubMed1999

Human MD-1 homologue is a BCG-regulated gene product in monocytes: its identification by differential display.

BCG-CWS is a therapeutically potent immune activator which improves the prognosis of cancer patients. However, the targeting effector cells and molecules for BCG-CWS in the human immune system have not been determined. Here, we found that BCG-CWS activates human monocytes and concomitantly down-regulates expression of a human homologue of chicken MD-1 in the activated monocytes by differential display. According to a previous study, MD-1 forms a complex with the Toll family protein RP-105 on murine B cell lines to facilitate its stable expression. Thus, MD-1 may participate in regulation of innate immune activation on human monocytes. Our results, taken together with these recent findings regarding Toll family proteins, suggest that BCG-CWS acts on monocytes to modulate the human innate immune system via regulation of Toll family proteins.

Amino Acid Sequence↗
PubMed1999

Cytotoxic T lymphocyte-mediated cell death in paraneoplastic sensory neuronopathy with anti-Hu antibody.

Paraneoplastic sensory neuronopathy (PSN) has been shown to harbor characteristic anti-neuronal autoantibody 'anti-Hu' in their sera and cerebrospinal fluid. Creation of animal models exhibiting clinical or pathological features seen in PSN by means of passive transfer of anti-Hu positive IgG has not been achieved. Although, anti-Hu antibody was shown to induce neuronal cell lysis in vitro, this result has not been reproduced so far. Since prominent T cell infiltration are seen in the central nervous system and posterior spinal ganglion of the patients with anti-Hu syndrome, we studied cytotoxic T cell (CTL) activity in peripheral mononuclear cells from a patient with PSN harboring anti-Hu antibody. The activated CD8+ T cells from the patient's venous blood were shown to lyse her own fibroblasts which were incubated with interferon-gamma to induce HLA class I molecules on their surface and the recombinant HuD protein was injected into the cells by microinjector. This is the first report showing the existence of CTL in a patient with PSN.

Autoantibodies↗
PubMed1999

Systematic analysis of N-linked sugar chains from whole tissue employing partial automation.

A partially automated technique for the isolation and characterization of N-linked sugar chains from glycoproteins of crude tissue samples is established. The N-linked sugar chains from the acetone-extracted tissues are made free by a process of hydrazinolysis and subsequently N-acetylated by GlycoPrep 1000 (Oxford Glycosystems). These free sugar chains are further converted to pyridylamino derivatives by GlycoTag (Takara). Characterization of these sugar chains is achieved by a combination of HPLC columns using a highly sensitive fluorescence detector at femtomole levels. Tissue sample can be successfully pyridylaminated and analyzed to give highly reproducible results with consistent yield, requiring fewer purification steps, minimum skills, and less time. Moreover, fixed tissues can also be analyzed employing this technique, giving a similar sugar chain pattern compared to normal tissue samples. Using this method we show that the pattern of N-linked sugar chains present in human sera or in one small region of brain is strikingly similar among the different individuals. However, the absence of a highlighted peak in one of the samples suggests this method can be extrapolated to identify changes, if any, associated with disorders such as inflammation or cancer. Furthermore, this two-dimensional display of sugar chains would discover the function-specific molecules as we see in proteins.

Animals↗
PubMed1999

Rabbit experimental sensory ataxic neuropathy: anti-GD1b antibody-mediated trkC downregulation of dorsal root ganglia neurons.

We previously reported experimental sensory neuropathy in rabbit induced by the immunization of ganglioside GD1b. The major pathological change in diseased rabbits was degeneration of primary sensory neurons with central axons extending to the dorsal column of the spinal cord. The loss of primary sensory neurons that mediate proprioceptive sensation prompted us to investigate the expression of trkC in dorsal root ganglia (DRG) because this type of neuron is thought depend mainly on neurotrophin-3-mediated trkC signaling. Northern blotting analysis revealed markedly reduced expression of trkC in DRG of diseased rabbits in acute phase. This result together with the absence of lymphocytic infiltration in DRG of diseased rabbits at any stage suggests the anti-GD1b antibody-mediated downregulation of trkC expression could be one of the pathogenesis of this experimental sensory ataxic neuropathy.

Animals↗
PubMed1999

Dorsal root ganglia neuron-specific promoter activity of the rabbit beta-galactoside alpha1,2-fucosyltransferase gene.

The rabbit H-blood type alpha1,2-fucosyltransferase (RFT-I), gene and its biosynthetic products, H antigens (Fucalpha1,2Galbeta), are abundantly expressed in a subset of dorsal root ganglia (DRG) neurons. To investigate the regulatory mechanisms for the RFT-I gene expression, we determined the genomic structure and promoter activity of this gene. PCR amplification of the 5' cDNA end analysis revealed two transcriptional start sites, 498 and 82 nucleotides upstream of the translational initiation codon, the latter site yielding a major 3.1-kb transcript specifically expressed in DRG, as revealed by Northern blotting. Promoter analysis of the 5'-flanking region of the RFT-I gene using a luciferase gene reporter system demonstrated strong promoter activity in PC12 cells, which express the rat H-type alpha1,2-fucosyltransferase gene, and Neuro2a mouse neuroblastoma cells. Deletion analysis revealed the 704-base pair minimal promoter region flanking the translational initiation codon, for which two distinct promoter activities were detected and differentially used in PC12 and Neuro2a cells. The minimal promoter region contained a GC-rich domain (GC content 80%), in which a Sp1 binding sequence and a GSG-like nerve growth factor-responsive element were found, but lacked TATA- and CAAT-boxes. Promoter analysis with a primary culture of DRG neurons demonstrated that the minimal promoter region of the RFT-I gene was sufficient for the expression of a reporter gene in DRG neurons. We conclude that the TATA-less GC-rich minimal promoter region of the RFT-I gene controls DRG small neuron-specific expression of the RFT-I gene.

Animals↗