Biomedical subjects

S Tsuji

Publications and source records attributed to S Tsuji.

At least 235 records · Page 13Linked to original sources

PubMed1999

Role of Bcl-2 family proteins (Bax, Bcl-2 and Bcl-X) on cellular susceptibility to radiation in pancreatic cancer cells.

The aim of this study was to examine Bax, Bcl-2 and Bcl-XL proteins in human pancreatic cancer cell lines and to clarify the mechanism of radiation resistance. PANC-1 and AsPC-1 pancreatic cell lines were used, both having mutated p53. Radioresistant PANC-1/Rad cells and AsPC-1/Rad cells were obtained by repeated 5 Gy irradiation of PANC-1 cells and AsPC-1 cells, respectively. Radiation was found to inhibit the growth of PANC-1 cells and AsPC-1 cells. After exposure to radiation, detached cells were subjected to FITC-TUNEL staining to calcualte the ratio of apoptosis. TUNEL positive ratios increased dose-dependently in both cell lines. Western blotting showed that the basal level of the Bax/Bcl-2 ratio reflected the radiosensitivity of these cell lines, and Bax expression was obviously upregulated after irradiation in the presence of mutated p53, but Bcl-2 expression remained almost constant. Both PANC-1/Rad and AsPC-1/Rad cells had greater Bcl-XL expression than the parental cells, and the basal level of the Bax/Bcl-2 ratio was no longer predictive of radiosensitivity. Upregulated expression of Bax protein after irradiation was not related to induction of apoptosis in these cells, suggesting that overexpression of Bcl-XL and functional reconstruction of Bcl-2 family proteins are important factors in acquired radioresistance.

Blotting, Western↗

PubMed1999

Novel chloride channel gene mutations in two unrelated Japanese families with Becker's autosomal recessive generalized myotonia.

We investigated the skeletal muscle voltage-gated chloride channel gene (CLCN1) in two unrelated Japanese patients with Becker's myotonia congenita. The non-myotonic parents of each patient were consanguineous. The proband of each family shares generalized myotonia, transient weakness after rest, and leg muscle hypertrophy. However, the disease severity related to the degree of myotonia differed, even in view of the response to long train nerve stimulation tests. CLCN1 gene analysis revealed a novel Ala659Val missense mutation identified to be homozygous in the more severe patient, while a novel Gln445Stop nonsense mutation was present in the other patient. Both mutations were absent in 90 Japanese normal controls. This is the first report of Japanese cases of Becker's myotonia congenita with CLCN1 gene mutations.

Adult↗

PubMed1999

Antibody-independent classical complement pathway activation and homologous C3 deposition in xeroderma pigmentosum cell lines.

Of human malignantly transformed cell lines, xeroderma pigmentosum (XP) cell lines were found to be highly susceptible to homologous complement (C): cells were opsonized by C3 fragments on incubation with diluted normal human serum. C3 fragment deposition on XP cells was Ca2+-dependent and occurred on live cells but not UV-irradiated apoptotic cells. (Ca2+ is required for activation of the classical C pathway via C1q and the lactin pathway via mannose binding lectin (MBL), and the surface of apoptotic cells usually activates the alternative C pathway.) In this study we tested which of the pathways participates in XP cell C3 deposition. In seven cell lines that allowed C3 deposition (i), Clq was shown to be essential but MBL played no role in C activation, (ii) Cls but not MASP bound XP cells for activation, (iii) no antibodies recognizing XP cells were required for homologous C3 deposition, and (iv) the alternative pathway barely participated in C3 deposition. Furthermore, the levels of C-regulatory proteins for host cell protection against C, decay-accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), were found to be relatively low in almost all XP cell lines compared with normal cells. These results indicate that XP cells activate the classical C pathway in an antibody-independent manner through the expression of a molecule which directly attracts C1q in a C-activating form, and that relatively low levels of DAF and MCP on XP cells facilitate effective C3 deposition. The possible relationship between the pathogenesis of XP and our findings is discussed.

Antibodies↗

PubMed1999

Epidermal tissue-derived T-cell growth factor, colony stimulating factor and nerve growth factor in chickens.

The purpose of this study was to determine the mechanism of the local cytokine-mediated immune response in the skin of chickens. The incorporation of 3H-thymidine into spleen T lymphocytes from 9-to 10-week-old chickens was augmented by the addition of epidermal tissue culture supernatant (ESN) from 11-day-old embryos. The colony formation of neonatal chicken bone marrow cells in the methylcellulose medium was also significantly increased by addition of ESN. When axonal outgrowth in matrigel was investigated, the embryonal sympathetic ganglion was found to grow axons outwards towards the epidermal tissue specimens. The above results suggest that chicken epidermal cells (probably keratinocytes) produce T-cell growth factor (corresponding to IL-1), colony-stimulating factor for macrophages (M-CSF) and granulocytes (G-CSF), and nerve growth factor (NGF).

Animals↗

PubMed1999

Isolation of 10 differentially expressed cDNAs in differentiated Neuro2a cells induced through controlled expression of the GD3 synthase gene.

Recently, we showed that transfection of GD3 synthase cDNA into Neuro2a cells, a mouse neuroblastoma cell line, causes cell differentiation with neurite sprouting. In a search for the genes involved in this ganglioside-induced Neuro2a differentiation, we used a tetracycline-regulated GD3 synthase cDNA expression system combined with differential display PCRs to identify mRNAs that were differentially expressed at four representative time points during the process. We report here the identification of 10 mRNAs that are expressed highly at the Neuro2a differentiated stage. These cDNAs were named GDAP1-GDAP10 for (ganglioside-induced differentiation-associated protein) cDNAs. It is interesting that in retinoic acid-induced neural differentiated mouse embryonic carcinoma P19 cells, GDAP mRNA expression levels were also up-regulated (except that of GDAP3), ranging from three to >10 times compared with nondifferentiated P19 cells. All the GDAP genes (except that of GDAP3) were developmentally regulated. The GDAP1, 2, 6, 8, and 10 mRNAs were expressed highly in the adult mouse brain, whereas all the other GDAP mRNAs were expressed in most tissues. Our results suggested that these GDAP genes might be involved in the signal transduction pathway that is triggered through the expression of a single sialyltransferase gene to induce neurite-like differentiation of Neuro2a cells.

Aging↗

PubMed1999

Involvement of cyclooxygenase-2 in proliferation and morphogenesis induced by transforming growth factor alpha in gastric epithelial cells.

Transforming growth factor alpha is one of the most potent growth factors for gastrointestinal epithelium. In this study, we examined the roles of cyclooxygenase-2 on proliferation and morphogenesis of RGM1 rat gastric epithelial cells after stimulation with transforming growth factor alpha in vitro, RGM1 cells increased expression of cyclooxygenase-2 messenger RNA 20-60 min after stimulation with transforming growth factor alpha. Transforming growth factor alpha stimulated [3H]thymidine incorporation and tubulogenesis of RGM1 cells in collagen matrix, both of which were significantly suppressed by treatment with a cyclooxygenase-2 specific inhibitor, NS-398 or cyclooxygenase-2 antisense oligonucleotide. Both of the treatment lowered prostanoid production by enzyme immunoassay. The transforming growth factor alpha-induced expression of cyclooxygenase-2 is followed by cell proliferation and development of tubular morphology of RGM1 gastric epithelial cells. Treatment with cyclooxygenase-2 inhibitor and cyclooxygenase-2 antisense oligonucleotide suppressed these responses induced by transforming growth factor alpha suggesting the involvement of cyclooxygenase-2 in proliferation and morphogenesis in gastric mucosal epithelium.

6-Ketoprostaglandin F1 alpha↗

PubMed1999

Endoscope disinfection using acidic electrolytic water.

BACKGROUND AND STUDY AIMS: The aim of the present study was to evaluate a new endoscope disinfector (WM-1) that uses acidic electrolytic water (AEW). MATERIALS AND METHODS: AEW was produced by electrolysis of a 0.05% NaCl-water mixture, with a redox potential greater than 1000 mV and a pH lower than 2.7. In the first study, an endoscope artificially contaminated with 15 species of bacteria and four strains of viruses was treated using the WM-1. In the second study, endoscopic contamination after clinical use was examined by culture for Helicobacter pylori and other bacteria, and by polymerase chain reaction for the H. pylori urease gene and hepatitis C virus. The extent of contamination was then examined after exposing the WM-1 to AEW. The safety of AEW was examined using both in vivo and in vitro studies. RESULTS: All of the bacteria and viruses were destroyed or inactivated after the instrument had been exposed to AEW. Clinical contamination was detected from the instrument in 19 of 30 endoscopic procedures, whereas no bacteria or viruses were detected after five minutes' exposure to AEW. AEW was found to be nonirritant, nontoxic to cells, and nonmutagenic. CONCLUSION: The WM-1 successfully and safely disinfected the endoscopes. With running costs of yen 24 per day ($0.21 per day), the WM-1 provides an effective and inexpensive alternative to conventional disinfection equipment.

Acids↗

PubMed1999

Paroxysmal kinesigenic choreoathetosis locus maps to chromosome 16p11.2-q12.1.

Paroxysmal kinesigenic choreoathetosis (PKC), the most frequently described type of paroxysmal dyskinesia, is characterized by recurrent, brief attacks of involuntary movements induced by sudden voluntary movements. Some patients with PKC have a history of infantile afebrile convulsions with a favorable outcome. To localize the PKC locus, we performed genomewide linkage analysis on eight Japanese families with autosomal dominant PKC. Two-point linkage analysis provided a maximum LOD score of 10.27 (recombination fraction [theta] =.00; penetrance [p] =.7) at marker D16S3081, and a maximum multipoint LOD score for a subset of markers was calculated to be 11.51 (p = 0.8) at D16S3080. Haplotype analysis defined the disease locus within a region of approximately 12.4 cM between D16S3093 and D16S416. P1-derived artificial chromosome clones containing loci D16S3093 and D16S416 were mapped, by use of FISH, to 16p11.2 and 16q12.1, respectively. Thus, in the eight families studied, the chromosomal localization of the PKC critical region (PKCR) is 16p11.2-q12.1. The PKCR overlaps with a region responsible for "infantile convulsions and paroxysmal choreoathetosis" (MIM 602066), a recently recognized clinical entity with benign infantile convulsions and nonkinesigenic paroxysmal dyskinesias.

Adolescent↗

PubMed1999

Cloning and expression of a human gene encoding an N-acetylgalactosamine-alpha2,6-sialyltransferase (ST6GalNAc I): a candidate for synthesis of cancer-associated sialyl-Tn antigens.

The sialyl-Tn (sTn) antigen is a well known cancer-associated antigen, the expression of which is related to the prognosis of cancer patients. We aimed to isolate a human gene encoding an N -acetylgalactosamine alpha2,6-sialyltransferase which synthesizes sTn antigen, and to characterize the enzyme. Degenerate primers encoding sialyl motifs were used for the polymerase chain reaction to amplify complementary DNAs prepared from RNAs of human pyloric mucosae with intestinal metaplasia, which abundantly expressed sTn antigen, followed by screening of full-length cDNAs using the amplified DNA fragment as a probe. We isolated two human cDNA clones, long-form (2.46 kb) and short-form (2.23 kb) cDNAs. The former encodes an active enzyme with a predicted 600 amino acid sequence. The latter, a splice-variant of the long-form, encodes an inactive enzyme. HCT15 human colorectal cancer cells stably expressing the long-form cDNA expressed sTn epitopes on O -glycans. The long form cDNA was considered to encode a human homologue of chick ST6GalNAc I for the following reasons: (1) the putative amino acid sequence showed greater homology to that of chick ST6GalNAc I (55%) compared to other sialyltransferases, (2) it encodes the extraordinarily long stem region that is a typical feature of chick ST6GalNAc I, and (3) the substrate specificity was very similar to that of chick ST6GalNAc I. In situ hybridization demonstrated that the localization of transcripts correlated well with that of sTn antigen in gastric cancer cells and Goblet cells in intestinal metaplastic glands. Thus, we determined that the long-form cDNA of the human ST6GalNAc I gene encodes the probable candidate for the human sTn synthase(s).

Acetylglucosamine↗

PubMed1999

A neurological disease caused by an expanded CAG trinucleotide repeat in the TATA-binding protein gene: a new polyglutamine disease?

To investigate whether the expansion of CAG repeats of the TATA-binding protein (TBP) gene is involved in the pathogenesis of neurodegenerative diseases, we have screened 118 patients with various forms of neurological disease and identified a sporadic-onset patient with unique neurologic symptoms consisting of ataxia and intellectual deterioration associated with de novo expansion of the CAG repeat of the TBP gene. The mutant TBP with an expanded polyglutamine stretch (63 glutamines) was demonstrated to be expressed in lymphoblastoid cell lines at a level comparable with that of wild-type TBP. The CAG repeat of the TBP gene consists of impure CAG repeat and the de novo expansion involves partial duplication of the CAG repeat. The present study provides new insights into sporadic-onset trinucleotide repeat diseases that involve de novo CAG repeat expansion.

Adolescent↗

PubMed1999

Adenovirus-mediated expression of mutant DRPLA proteins with expanded polyglutamine stretches in neuronally differentiated PC12 cells. Preferential intranuclear aggregate formation and apoptosis.

To investigate the molecular mechanisms of neurodegeneration caused by expanded CAG repeats in dentatorubral-pallidoluysian atrophy (DRPLA), an autosomal dominant neuro degrees enerative disorder caused by unstable expansion of a CAG trinucleotide repeat in the DRPLA gene on 12p13.31, we established an efficient expression system for truncated and full-length DRPLA proteins with normal or expanded polyglutamine stretches in neuronally differentiated PC12 cells and fibroblasts using an adenovirus expression system. Although aggregate body formation was observed both in neuronally differentiated PC12 cells and in fibroblasts expressing truncated DRPLA proteins with Q82, >97% ( n = 3) of neuronally differentiated PC12 cells showed intra-nuclear inclusions, while only 31 21% ( n = 3) of fibro-blasts had intranuclear inclusions at 3 days after infection. The percentage of apoptotic cells was significantly higher in neuronally differentiated PC12 cells expressing the truncated DRPLA protein with Q82 than in fibroblasts, suggesting the possibility that intranuclear aggregate bodies are formed preferentially in neuronally differentiated PC12 cells and that these cells are more vulnerable than fibroblasts to the toxic effects of expanded polyglutamine stretches in the DRPLA protein. When the full-length DRPLA protein with Q82 was expressed, aggregate bodies were found exclusively in the nuclei of the neuronally differentiated PC12 cells, while they were found in the cytoplasm of fibroblasts. Despite the presence of aggregate bodies, apoptosis was not induced by expression of the full-length DRPLA protein with Q82 in either neuronally differentiated PC12 cells or fibroblasts, suggesting that the presence of intranuclear aggregate bodies is in itself not necessarily toxic to cells.

Adenoviridae↗

PubMed1999

Identification of an alpha2,6-sialyltransferase induced early after lymphocyte activation.

We have used mRNA differential display PCR to search for genes induced in activated T cells and we identified a gene encoding an alpha2,6-sialyltransferase (ST6GalNAc IV) that is rapidly induced in lymphocytes after antigen or mitogen stimulation. The 3.6 kb full-length cDNA clone (MK45) obtained contained a single open reading frame encoding a 302 amino acid protein and a 2.5 kb 3' untranslated region. MK45 expression in in vivo-activated CD8 T cells reached the highest level 4 h after antigen triggering and then declined rapidly to nearly base levels within 45 h. Northern blot analysis further revealed that MK45 expression was also induced in LPS-activated B cells and antigen-triggered CD4 T cells in vitro. MK45 expression was low or undetectable in most other mouse tissues examined, when compared to activated lymphocytes. Importantly, the mRNA expression level of other sialyltransferases remained largely unchanged during the early stage of lymphocyte activation. Finally, increased ecto-sialyltransferase activity and an altered sialylation pattern were demonstrated on the cell surface of early activated CD8 T cells. Our report identifies a candidate sialyltransferase gene that is involved in the early alteration of the sialylation pattern of cell surface molecules in activated lymphocytes.

Amino Acid Sequence↗

PubMed1999

The relationship between ST6Gal I Golgi retention and its cleavage-secretion.

The ST6Gal I is a sialyltransferase that modifies N-linked oligosaccharides of glycoproteins. Previous results suggested a role for luminal stem and active domain sequences in the efficiency of ST6Gal I Golgi retention. Characterization of a series of STtyr isoform deletion mutants demonstrated that the stem is sensitive to proteases and that preventing cleavage in this region leads to increased cell surface expression. A mutant lacking amino acids 32-104 (STDelta4) is not active or cleaved and secreted like the wild type STtyr, but does exhibit increased cell surface expression. It is probable that the STDelta4 mutant lacks the stem region and some amino acids of the active domain because the STDelta5 mutant lacking amino acids 86-104 is also not active but is cleaved and secreted. In contrast, deletion of stem amino acids between residues 32 and 86 in the STDelta1, STDelta2, and STDelta3 mutants does not inactive these enzyme forms, eliminate their cleavage and secretion, or increase their cell surface expression. Surprisingly, cleavage occurs even though the previously identified Asn63-Ser 64 cleavage site is missing. Further evaluation demonstrated that a cleavage site between Lys 40 and Glu 41 is used in COS cells. Mutagenesis of Lys 40 significantly decreased, but did not eliminate cleavage, suggesting that there are additional secondary sites of cleavage in the ST6Gal I stem.

Amino Acid Sequence↗

PubMed1999

Reversible cerebral hypoperfusion observed with Tc-99m HMPAO SPECT in reversible dementia caused by hypothyroidism.

A 69-year-old man had hypothyroid dementia as a result of I-131 therapy and an overdose of methimazole. Tc-99m HMPAO SPECT revealed diffuse cerebral hypoperfusion. The findings of brain SPECT normalized with the disappearance of symptoms and a return to the euthyroid state. There was a 25% or 26% reduction of the mean cerebral blood flow during dementia. This may be the first report in which SPECT brain imaging revealed reversible hypoperfusion associated with reversible hypothyroid dementia.

Aged↗

PubMed1999

Effect of ticlopidine on exercise-induced platelet aggregation and exercise tolerance time in patients with ischemic heart disease.

Platelet aggregation is one of the most important mechanisms for acute myocardial infarction during exercise. We sought to evaluate the effect of ticlopidine (TP) on platelet aggregation (PA) during exercise in patients with ischemic heart disease (IHD). We studied 38 patients with IHD, 26 patients with effort angina pectoris, and 12 patients with a previous myocardial infarction. In protocol I, subjects were divided into two groups. Drugs altering platelet aggregation were withheld 2-4 weeks before the study in 25 patients (control group). TP (200 mg/day) was administered for 7 days in 13 patients (ticlopidine group). A symptom-limited modified Bruce protocol treadmill exercise test was performed. PA was measured at rest and after exercise by using optical densitometry induced by adenosine diphosphate (ADP). PA ratio (percentage of maximum) was compared. In protocol II, in 12 patients, treadmill exercise test and PA measurement were performed with and without TP. PA significantly increased after exercise in control (from 51.7+/-23.3% to 64.4+/-27.7%, p < 0.01) and ticlopidine (from 31.9+/-10.5% to 42.0+/-20.4%, p < .01) groups; however, its grade was lower in the ticlopidine group than in the control group. After exercise, PA was lower in the ticlopidine group than in control group (42.0+/-20.4% vs. 64.4+/-27.7%; p < 0.01). In the same patients, PA was lower with TP than without TP after exercise. Treadmill exercise-tolerance time was greater in the ticlopidine group than in the control group, but not statistically significant (762.3+/-139.2 vs. 711.6+/-169.6 s; NS). Exercise-tolerance time was significantly greater with TP than without TP in same patient (791.7+/-98.9 vs. 733.3+/-152.8 s; p < .05). TP suppressed the increase of PA during exercise and increased the exercise-tolerance time in patients with IHD.

Adenosine Diphosphate↗

PubMed1999

Cyclooxygenase-2 overexpression enhances lymphatic invasion and metastasis in human gastric carcinoma.

OBJECTIVE: Recent epidemiological studies indicate that there is reduced risk of all digestive carcinomas in patients who regularly take nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin. Cyclooxygenase (COX) is a target enzyme for NSAIDs. We investigated the role of two isoforms, COX-1 and COX-2, in the development and metastasis of gastric carcinoma. METHODS: Fifteen gastric carcinoma tissue specimens and accompanying adjacent mucosa specimens were obtained from surgical resections. COX-1 and COX-2 protein expression were evaluated using Western blotting analysis, and their relative band densities were semi quantified using standard densitometry scanning techniques. RESULTS: Compared with paired noncancerous specimens, COX-2 was overexpressed in 10 of 15 carcinoma tissue specimens (66.7%). Overall, COX-2 levels in carcinoma tissue were significantly higher. Two early carcinomas (confined to the mucosa and submucosa) and three of 13 advanced carcinomas (extended below the submucosa into the muscular wall) had weak or similar COX-2 expression in paired tissue specimens. COX-2 overexpression in tumors significantly correlated with tumor invasion into the lymphatic vessels in the gastric wall and metastasis to the lymph nodes. Furthermore, the stage grouping in the TNM classification significantly correlated with COX-2 overexpression. In contrast, COX-2 overexpression did not correlate with histopathological grading, surface size, and venous vessel invasion of the tumors. COX-1 levels were similar between paired tissues. CONCLUSION: COX-2 overexpression might enhance lymphatic invasion and metastasis in patients with gastric carcinoma, implicating a poor prognosis. Therefore, the use of COX-2-specific inhibitor to suppress lymphatic metastasis in humans should be investigated.

Adenocarcinoma↗

PubMed1999

A non-familial Huntington's disease patient with grumose degeneration in the dentate nucleus.

We report a non-familial Huntington's disease (HD) patient presenting with increased levels of protein and IgG in his cerebrospinal fluid (CSF), antineuronal antibody in his serum and CSF, Purkinje cell and granule cell degeneration in the cerebellum, and grumose degeneration in the dentate nucleus, in addition to typical HD findings. This patient showed an expanded CAG repeat in the HD gene, and provides new information on the clinical and neuropathologic varieties of HD.

Aged↗

PubMed1999

Molecular detection of EWS-FLI1 chimeric transcripts in Ewing family tumors by nested reverse transcription-polymerase chain reaction: application to archival paraffin-embedded tumor tissues.

Chromosomal translocations generating unique chimeric genes are highly characteristic of specific sarcomas, and their use as diagnostic markers has been suggested. From a diagnostic pathologic point of view, detection of such cytogenetic or molecular aberrations applicable to routinely processed archival tissue specimens is considered a powerful tool for tumor diagnosis. To assess the feasibility and reliability of the molecular detection of the transcript originating from the chimeric gene in paraffin-embedded tumor specimens, we performed a nested reverse transcription-polymerase chain reaction (RT-PCR)-based assay to detect the EWS-FLI1 chimeric message in a series of Ewing family tumors. Of 24 paraffin-embedded tumor specimens from 23 cases analyzed, the chimeric message was detectable in 20 (83%) specimens from 20 cases (87%) by this nested RT-PCR assay, whereas none of 7 small round cell tumors not from this family (3 alveolar rhabdomyosarcomas, 2 neuroblastomas, 2 malignant lymphomas) showed detectable chimeric messages. In the sequence analysis of the PCR products, the amplified chimeric messages contained the junctions between exon 7 of the EWS gene and any one of exons 5, 6 and 8 of the FLI1 gene. The detection process was usually completed within 3 days, except for the subseqent sequence analysis. Our results endorse the use of this molecular assay as an ancillary technique in the diagnosis of Ewing family tumors using paraffin-embedded material.

Adolescent↗