Biomedical subjects

T Fujii

Publications and source records attributed to T Fujii.

At least 289 records · Page 16Linked to original sources

PubMed1999

Transforming growth factor-beta(1) induces apoptosis via connective tissue growth factor in human aortic smooth muscle cells.

We examined the possible involvement of connective tissue growth factor (CTGF) in the apoptosis induced by transforming growth factor-beta(1) (TGF-beta(1)) in human aortic vascular smooth muscle cells (HASC). In quiescent HASC, TGF-beta(1) induced the mRNA and protein of CTGF. A CTGF antisense oligonucleotide inhibited this induction. TGF-beta(1) significantly reduced cell viability and induced DNA fragmentation, and the CTGF antisense oligonucleotide reversed these effects. Moreover, TGF-beta(1) activated caspase 3 in HASC, and the CTGF antisense oligonucleotide reduced this activation. These findings show that CTGF plays a key role in the TGF-beta(1)-induced apoptosis in HASC.

Aorta↗

PubMed1999

Overexpression of connective tissue growth factor gene induces apoptosis in human aortic smooth muscle cells.

BACKGROUND: Connective tissue growth factor (CTGF) is expressed at very high levels particularly in the shoulder of human atherosclerotic lesions but not in normal blood vessels. Thus, CTGF may be important in the regulation of vascular smooth muscle cell function in atherosclerosis, but its precise role remains elusive. METHODS AND RESULTS: Full-length CTGF cDNA driven by a cytomegalovirus promoter was transiently transfected into cultured human aortic smooth muscle cells (HASCs). Northern and Western analysis demonstrated that CTGF was overexpressed in these cells 48 hours after transfection. The effects of CTGF overexpression on cell proliferation were evaluated by [(3)H]thymidine uptake and cell count in quiescent HASCs or those stimulated with platelet-derived growth factor (PDGF). Although mock transfection showed no effect, CTGF overexpression significantly inhibited cell proliferation in cells stimulated by PDGF. Moreover, CTGF overexpression, but not mock transfection, significantly increased apoptosis as assessed by DNA fragmentation associated with histone, TdT-mediated dUTP biotin nick end-labeling, and appearance of hypodiploid cells by flow cytometry. CONCLUSIONS: Our results for the first time demonstrate that CTGF can also act as a growth inhibitor in human aortic smooth muscle cells at least in part by inducing apoptosis. This may be important for the formation and composition of lesions and plaque stability in atherosclerosis.

Aorta↗

PubMed1999

Second transmembrane segment of FtsH plays a role in its proteolytic activity and homo-oligomerization.

The FtsH (HflB) protein of Escherichia coli is a membrane-bound ATP-dependent zinc protease. The role(s) of the N-terminal membrane-anchoring region of FtsH were studied by fusion with a maltose-binding protein (MBP) at five different N-termini of FtsH. The MBP-FtsH fusions were expressed in the cytoplasm of E. coli, and were purified as soluble proteins. The four longer constructs, which have a second transmembrane segment and the C-terminal cytoplasmic region in common, retained ATP-dependent protease activity toward heat-shock transcription factor sigma(32), and were found to be homo-oligomers. In contrast, the shortest construct which has the C-terminal cytoplasmic region but not the second transmembrane segment showed neither protease activity nor oligomerization. Therefore, the second transmembrane segment, which neighbors the C-terminal cytoplasmic region of the FtsH, participates in not only its membrane-anchoring, but also its protease activity and homo-oligomerization.

ATP-Binding Cassette Transporters↗

PubMed1999

Antinociceptive properties of FR140423 mediated through spinal delta-, but not mu- and kappa-, opioid receptors.

We investigated the antinociceptive effect of FR140423, 3-(difluoromethyl)-1-(4-methoxyphenyl)-5-[4-(methylsulfinyl)phenyl] pyrazole, in the tail-pinch test in mice, and evaluated the mechanism of action using various opioid receptor antagonists. P.o. and i.t. injection of FR140423 exerted dose-dependent antinociceptive activities with ED50 values of 21 mg/kg and 3.1 microg/mouse, respectively. However, i.c.v. injection of FR140423 did not show an antinociceptive effect. The antinociceptive effects of FR140423 were completely abolished by naloxone and naltrindole but not by naloxonazine, beta-funaltrexamine and nor-binaltorphimine. FR140423 did not affect any opioid receptor binding in mouse spinal membranes at concentrations up to 100 microM in vitro. Naloxone-induced jumping and diarrhea tests for morphine-like physical dependence of FR140423 gave negative results. These results suggest that FR140423 can induce antinociception by acting on the spinal but not the supraspinal site, and that spinal delta-opioid systems indirectly play a role in the antinociception produced by FR140423 in mice.

Administration, Oral↗

PubMed1999

Molecular cloning of haploid germ cell-specific tektin cDNA and analysis of the protein in mouse testis.

Tektins are a class of proteins that form filamentous polymers in the walls of ciliary and flagellar microtubules. We report here the molecular cloning of a new member of the tektin family, tektin-t, identified from a mouse haploid germ cell-specific cDNA library. Tektin-t mRNA encodes a protein of 430 deduced amino acids possessing RSNVELCRD, the conserved sequence of tektin family proteins. Western blotting showed a single band having a molecular weight of 86 kDa in the mouse testis. Immunohistochemistry of the testis showed that tektin-t is localized in the flagella of elongating spermatids from developmental step 15 to maturity.

Amino Acid Sequence↗

PubMed1999

Nerve growth factor increases the synthesis and release of acetylcholine and the expression of vesicular acetylcholine transporter in primary cultured rat embryonic septal cells.

Acetylcholine (ACh) is synthesized by choline acetyltransferase (ChAT) in the cytoplasm of cholinergic nerve terminals and transported into synaptic vesicles by vesicular ACh transporter (VAChT). The genes encoding ChAT and VAChT are colocalized within the genome, and their products are known to be coregulated by various neurotrophic factors. In the present study, nerve growth factor (NGF; 100 ng/ml) was shown to enhance expression of VAChT and ChAT mRNA in primary cultured rat embryonic septal cells. By using a radioimmunoassay, we also found that NGF increased both neuronal content and spontaneous release of ACh, which were first detected on day 2 of culture and time-dependently increased up to day 10. Stimulated release of ACh elicited by high K+ (50 mM KCl) was also significantly greater in NGF-treated cells than in control cells. NGF enhanced immunoreactivity to antiserum against VAChT, indicating that the augmented responses were due to, at least in part, increased expression of VAChT protein. In contrast, the numbers of immunocytochemically positive cells were unaffected. Thus, NGF appears to augment ACh synthesis, its transport into synaptic vesicles, and its subsequent release. The data also suggest that NGF facilitates growth and development of cholinergic neurons, but not their survival.

Acetylcholine↗

PubMed1999

Simultaneous determination of the urinary metabolites of toluene, xylene and styrene using high-performance capillary electrophoresis. Comparison with high-performance liquid chromatography.

A simple and rapid method using high-performance capillary electrophoresis (HPCE) for the simultaneous determination of the urinary metabolites of toluene, xylene and styrene, plus creatinine and uric acid in human urine specimens and standard solutions is described. The compounds were well separated from each other on a fused-silica capillary utilizing a 20 mM sodium tetraborate buffer (pH 9.65) with 15 mM beta-cyclodextrin and UV detection at 200 and 225 nm. The total analysis time was less than 6 min per sample. The capillary zone electrophoresis (CZE) method shows a good correlation with the high-performance liquid chromatography (HPLC) method with respect to urinary hippuric acid concentrations in the urine specimens of subjects exposed to the vapors of a solvent mixture of toluene and xylene. In comparing these two techniques, HPCE was found to be superior to HPLC because the analysis time is shorter, and the separation of m-MHA and p-MHA takes a long time with HPLC.

Calibration↗

PubMed1999

Functional expression of rat thioredoxin reductase: selenocysteine insertion sequence element is essential for the active enzyme.

Mammalian thioredoxin reductase (TR) is a flavoprotein catalysing reduction of oxidized thioredoxin in an NADPH-dependent manner, and contains a selenocysteine (Sec) residue near the C-terminus. We observed that TR activity was decreased in A549 cells by the lowering of the fetal bovine serum content in the culture medium and was recovered by the addition of selenium. To study the role of Sec in TR activity, we have isolated a full-length clone of the rat TR cDNA (3.3 kb) and have expressed it in COS-1 cells in a transient-expression system. TR activities in COS-1 cells expressing rat TR were increased in accordance with supplemented sodium selenite concentrations, whereas levels of TR protein, examined by Western blotting, were not affected by sodium selenite concentrations. We introduced various deletions into the 3'-untranslated region of the TR cDNA to localize and examine the role of a Sec insertion-sequence (SECIS) element in the functional expression of TR. TR activities were observed only in COS-1 cells transfected with the TR cDNAs containing the putative SECIS element located between 1856 and 1915 bp in the correct orientation. We also carried out radiolabelling of proteins by incubation of the cDNA-transfected cells with sodium [75Se]selenite. 75Se was incorporated into the expressed TR protein of the cells transfected with the SECIS element-containing cDNAs, but not into those without the SECIS element or with an inverted SECIS element. These data clearly showed a requirement of selenium for the formation of functional TR protein.

3' Untranslated Regions↗

PubMed1999

A nifS-like gene, csdB, encodes an Escherichia coli counterpart of mammalian selenocysteine lyase. Gene cloning, purification, characterization and preliminary x-ray crystallographic studies.

Selenocysteine lyase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the exclusive decomposition of L-selenocysteine to L-alanine and elemental selenium. An open reading frame, named csdB, from Escherichia coli encodes a putative protein that is similar to selenocysteine lyase of pig liver and cysteine desulfurase (NifS) of Azotobacter vinelandii. In this study, the csdB gene was cloned and expressed in E. coli cells. The gene product was a homodimer with the subunit Mr of 44,439, contained 1 mol of PLP as a cofactor per mol of subunit, and catalyzed the release of Se, SO2, and S from L-selenocysteine, L-cysteine sulfinic acid, and L-cysteine, respectively, to yield L-alanine; the reactivity of the substrates decreased in this order. Although the enzyme was not specific for L-selenocysteine, the high specific activity for L-selenocysteine (5.5 units/mg compared with 0.019 units/mg for L-cysteine) supports the view that the enzyme can be regarded as an E. coli counterpart of mammalian selenocysteine lyase. We crystallized CsdB, the csdB gene product, by the hanging drop vapor diffusion method. The crystals were of suitable quality for x-ray crystallography and belonged to the tetragonal space group P43212 with unit cell dimensions of a = b = 128.1 A and c = 137.0 A. Consideration of the Matthews parameter Vm (3.19 A3/Da) accounts for the presence of a single dimer in the crystallographic asymmetric unit. A native diffraction dataset up to 2.8 A resolution was collected. This is the first crystallographic analysis of a protein of NifS/selenocysteine lyase family.

Animals↗

PubMed1999

Diversity of mRNA expression for muscarinic acetylcholine receptor subtypes and neuronal nicotinic acetylcholine receptor subunits in human mononuclear leukocytes and leukemic cell lines.

Previously, we reported that various levels of acetylcholine (ACh), currently known as a neurotransmitter, are detectable in the blood of several mammals including humans and that most blood ACh originates from T-lymphocytes. To investigate whether ACh in the blood acts on lymphocytes and participates in the modulation of immune responses, we have analyzed the expression of mRNA for muscarinic (Ms) ACh receptor subtypes and nicotinic (Nc) ACh receptor subunits using reverse transcription-polymerase chain reaction (RT-PCR) methods. The cells tested were human peripheral mononuclear leukocytes (MNLs) from seven healthy donors and eight leukemic cell lines, as models of lymphocytes. We detected mRNA expression for various neuronal Nc receptor subunits and Ms receptor subtypes in all of the MNL samples and in all of the cell lines tested. However, the expression pattern of mRNA for neuronal Nc receptor subunits (alpha2-alpha7 and beta2-beta4) and Ms receptor subtypes (m1-m5) varied among the individuals and cell lines. No expression of mRNA for three muscle-type Nc receptor subunits (alpha1, beta1 and epsilon) was observed in the MNLs and cell lines. These results indicate that both neuronal-type Nc and Ms ACh receptors are present on the surface of lymphocytes.

Adult↗

PubMed1999

Structure of the glucan-binding sugar chain of Tip1p, a cell wall protein of Saccharomyces cerevisiae.

Tip1p is one of the major cell wall mannoproteins of Saccharomyces cerevisiae and is presumed to be synthesized as a glycosylphosphatidylinositol (GPI)-anchored form. We purified Tip1p from a glucanase extract of yeast cell walls and analyzed the sugar chain involved in the cell wall linkage. One mol of glucanase-extracted Tip1p contained 7.5 mol of glucose derived from glucan and 1 mol of ethanolamine, a component of the GPI anchor. One mol of the C-terminal peptide of Tip1p digested with Achromobacter protease I also contained 7.9 mol of glucose and 1 mol of ethanolamine. On the other hand, Tip1p contained no glucosamine, which is a component of the GPI anchor. The glucan-binding sugar chain of Tip1p was released by hydrazinolysis and isolated. This sugar chain contained ethanolamine with a free amino group and a glucose reducing end, but no mannose reducing end. Phosphodiesterase treatment eliminated the free amino group from this sugar chain, suggesting that a phosphodiester bond exists between the ethanolamine and the glucan remnant. These results indicate (1) the glucan-binding sugar chain of Tip1p is a GPI derivative, and (2) the GPI anchor is cleaved at the glycosyl moiety, and the resultant mannose reducing end is probably used to link Tip1p to cell wall glucan.

Amino Acid Sequence↗

PubMed1999

Enhancement of cortical and hippocampal cholinergic neurotransmission through 5-HT1A receptor-mediated pathways by BAY x 3702 in freely moving rats.

Involvement of serotonin (5-HT) in the regulation of cholinergic neuronal activity by modulation of acetylcholine (ACh) release has been reported for various regions of the brain. Cortical and hippocampal cholinergic neurotransmission is of particular importance in the mechanisms of attention as well as learning and memory. In the present study, we investigated the effect of R-(-)-2-[4-[(chroman-2-yl-methyl)-amino-butyl]-1,1-dioxo-benzo[d]++ +isothiazolone hydrochloride (BAY x 3702), a novel, high-affinity 5-HT1A receptor agonist, on ACh release in the cerebral cortex and hippocampus of freely moving rats using an in vivo microdialysis technique. Acetylcholine efflux from the cortex and hippocampus was measured every 30 m using a sensitive and specific radioimmunoassay and was stable for at least 5 h. The ACh efflux from the cortex and hippocampus was increased significantly by BAY x 3702 (0.3 mg/kg, i.p.) compared with saline administration. WAY-100635 (0.6 mg/kg, s.c.), a selective 5-HT1A receptor antagonist, eliminated the augmentation of ACh efflux induced by BAY x 3702 in both brain regions. These results demonstrate that stimulation by BAY x 3702 enhanced ACh release in both the cortex and hippocampus through 5-HT1A receptor-mediated pathways.

Acetylcholine↗

PubMed1999

Histochemical studies of glycosaminoglycans in developing periodontal ligaments of ICR mice.

Although the periodontal ligament (PL) contains an abundance of glycosaminoglycans (GAGs), there are only a few histochemical studies describing GAGs in the developing PL. In the present study, the relationship between the formation of principal fibers and the molecular species of GAGs in the developing PL was examined by light microscopic histochemistry. Jcl:ICR mice were killed on day 0 to day 28 after birth. Paraffin-embedded tissue sections were routinely made and stained with hematoxylin-eosin (H&E), Azan, or the sensitized high iron diamine (S-HID) procedure combined with enzyme digestions. Before tooth eruption, thin threads of collagen fibers in the PL assembled and constructed principal fibers, which projected from both the side of the alveolar bone and the root of the tooth. After tooth eruption, the principal fibers from both sides were tightly entangled. In the developing PL, the molecular species of GAGs was mainly dermatan sulfate. Moreover, the relative amount of dermatan sulfate increased together with the maturation of the principal fibers, while the principal fibers adjacent to the alveolar bone and cementum contained chondroitin sulfate. These results suggest that dermatan sulfate contributes to collagen fiber assembly in the PL and that chondroitin sulfate relates to PL adhesion to the alveolar bone and to the cementum of the root.

Animals↗

PubMed1999

Sperizin is a murine RING zinc-finger protein specifically expressed in Haploid germ cells.

Through the preparation of a subtracted cDNA library, we have extensively isolated genes whose expression is induced in mouse spermatogenic cells. One of the genes encoded a protein with a RING zinc-finger motif, which we termed sperizin (spermatid-specific ring zinc finger). Transcription of the sperizin gene was not observed in prepubertal testis, but became detectable at day 23. Northern and in situ hybridization analyses indicated that the sperizin gene was exclusively expressed in the round spermatid. The sperizin gene is intronless, and GFP-tagged sperizin was found to be localized in the cytoplasm of ectopically expressed somatic cultured cells. We assigned the chromosomal localization of the sperizin gene to chromosome 17 using an interspecific backcross mapping panel. The data suggest that we have identified a new member of the RING-finger family of proteins that may be involved in spermatogenesis.

Animals↗

PubMed1999

Proper name anomia after left temporal lobectomy: a patient study.

A patient with a selective deficit in retrieving proper names after left temporal lobectomy is reported. He showed proper name anomia in conversation, in response to photographs, and in verbal descriptions, despite being able to provide semantic information about the people he was unable to name. This report provides evidence that the rostral part of the left temporal lobe plays a crucial role in processing proper names without involvement of other verbal functions.

Anomia↗

PubMed1999

Genetic polymorphisms of drug-metabolizing enzymes and susceptibility to head-and-neck squamous-cell carcinoma.

We have investigated the association between the polymorphisms of drug-metabolizing enzymes and susceptibility to head-and-neck squamous-cell carcinoma (HNSCC). PCR-based analysis was performed on 145 Japanese patients and 164 healthy Japanese controls to determine genotypes of polymorphisms in CYP1A1, CYP2E1, GSTM1, GSTP1, and NAT2. Patients and controls were compared by multivariate analysis. The CYP1A1 Val/Val genotype was seen more frequently in patients than in controls [odds ratio (OR) 4.1, p = 0.038). The frequency of the slow plus intermediate NAT2 genotypes was also higher in patients (OR 2.0, p = 0.039). When we analyzed the distributions of the genotypes in 69 laryngeal and 45 pharyngeal cancer patients, laryngeal cancer patients had a higher frequency of NAT2 slow or intermediate genotype (OR 2.7, p = 0.011) and GSTP1 AA genotype (OR 2.4, p = 0.047) than controls. Pharyngeal cancer patients had a higher frequency of the CYP1A1 Val/Val genotype than controls (OR 5.7, p = 0.034), suggesting that different organs may be responsive to different chemicals from the environment. Furthermore, 23 patients who developed multiple cancers (HNSCC plus other) were compared with 115 patients with HNSCC alone. There was no significant difference in the polymorphisms between the 2 groups, though excessive alcohol consumption (more than 50 g/day of ethanol) appeared to be a risk factor for multiple cancers (p = 0.053).

Aged↗

PubMed1999

p16 inactivation in small-sized lung adenocarcinoma: its association with poor prognosis.

p16, an inhibitor of cell cycle machinery, is frequently inactivated in non-small cell carcinoma of the lung (NSCCL). To clarify the significance of p16 inactivation in the progression of lung adenocarcinoma, we immunohistochemically evaluated p16 protein status and Rb, p53 and cyclin D1 expression in 51 surgically resected adenocarcinomas that were less than 3 cm in diameter (median follow-up period: 52.5 months). Twenty-one of 51 adenocarcinomas showed negative immunostaining for p16. Twenty adenocarcinomas were also negative for Rb, while 31 and 13 were positive for p53 and cyclin D1, respectively. Loss of p16 expression was significantly correlated with scar grade, lymphatic permeation, lymph node metastasis and clinical stage. Rb protein expression was also inversely correlated with scar grade, pleural involvement and vascular invasion. When the cases were stratified according to the expression of both proteins, the Rb-/p16- subset (7/51) consisted of poorly differentiated adenocarcinoma with a higher grade of invasion. While Rb, p53 and cyclin D1 protein status showed no significant correlations with prognosis, p16 inactivation was significantly correlated with poor prognosis, and the prognosis of Rb-/p16- was the worst among the 4 subsets. Inactivation of p16 may play a role in accelerating scar formation and lymph node metastasis, and may contribute through these mechanisms to poor prognosis in patients with small-sized lung adenocarcinoma.

Adenocarcinoma↗

PubMed1999

The Dependence of the Apparent Contact Angles on Gravity.

We have studied theoretically the effect of gravity on the rough solid-liquid interface and have shown that its tension is enhanced by gravity when gas is adsorbed at it. As a result, the apparent contact angle on rough surfaces, which has been considered not to be influenced by gravity so far, can be raised by gravity. The calculated dependence of contact angles on gravity under the ordinary conditions of the sessile drop method is large enough to detect by experiment. The observed asymmetrical deviations from Wenzel's contact angle caused by the gas adsorption at the solid-liquid interface and by the liquid adsorption at the solid-gas interface are explained in terms of this gravitational effect. Copyright 1999 Academic Press.

Journal Article↗