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Biomedical subjects

T Fujita

Publications and source records attributed to T Fujita.

At least 109 records · Page 6Linked to original sources
PubMed2001

Smad7 is induced by norepinephrine and protects rat hepatocytes from activin A-induced growth inhibition.

Activin A induces growth arrest of rat hepatocytes in vitro and in vivo. The alpha(1)-adrenergic agonist, norepinephrine (NE), enhances epidermal growth factor-stimulated DNA synthesis and inhibits activin A-induced growth inhibition, but the mechanisms of these actions are unclear. Smad proteins have recently been identified as intracellular signaling mediators of transforming growth factor-beta family members. In the present study, we explored how NE modulates the Smad signaling pathway in rat cultured hepatocytes. We demonstrate that NE inhibits activin A-induced nuclear accumulation of Smad2/3 and that NE rapidly induces inhibitory Smad7 mRNA expression. Infection of Smad7 adenovirus into rat hepatocytes inhibited activin A-induced nuclear accumulation of Smad2/3, enhanced epidermal growth factor-stimulated DNA synthesis, and abolished the growth inhibitory effect of activin A. We also demonstrated that the induction of Smad7 by NE is dependent on nuclear factor-kappa B (NF-kappa B). The amount of active NF-kappa B complex rapidly increased after NE treatment. Preincubation of the cells with an NF-kappa B pathway inhibitor N-tosyl-l-phenylalanine chloromethyl ketone or infection of the cells with an adenovirus expressing an I kappa B super-repressor (Ad5I kappa B) abolished the NE-induced Smad7 expression. These results indicate a mechanism of transmodulation between the Smad and trimeric G protein signaling pathways in rat hepatocytes.

Activins↗
PubMed2001

Puromycin aminonucleoside induces apoptosis and increases HNE in cultured glomerular epithelial cells(1).

Puromycin aminonucleoside induces apoptosis and increases 4-hydroxy-2-nonenal (HNE) in cultured glomerular epithelial cells. We have previously reported the detachment of cultured glomerular epithelial cells (GECs) from their substrata by puromycin aminonucleoside (PAN) treatment. In this study we explored whether or not apoptosis was involved in the mechanisms of the detachment. DNA fragmentation on gel electrophoresis was clearly shown by 10(-3) M PAN treatment of GECs. Nuclear staining by Hoechst 33342 indicated the greatest number of apoptotic cells at 10(-3) M PAN for 48 h treatment. Similarly, TUNEL methods revealed maximal apoptotic cells at 10(-3) M PAN for 48 h treatment. Caspase-3 (like) protease activity increased at 10(-3) M PAN, and decreased at 2 x 10(-3) M PAN for 48 h treatment as well as at 10(-3) M PAN for 60 h treatment. Pretreatment with 2'-deoxycoformycin (DCF), inhibitor of adenosine deaminase, abolished these effects of PAN on cultured GECs. PAN treatment increased HNE, a lipid peroxide adduct, modified protein in cultured GECs, which was also prevented by pretreatment by DCF. These results for the first time indicate that the PAN-induced detachment of GECs from culture substrata is mediated at least in part through apoptosis via oxidative stresses by adenosine deaminase activity.

Adenosine Deaminase Inhibitors↗
PubMed2001

Increased susceptibility of decay-accelerating factor deficient mice to anti-glomerular basement membrane glomerulonephritis.

To prevent complement-mediated autologous tissue damage, host cells express a number of membrane-bound complement inhibitors. Decay-accelerating factor (DAF, CD55) is a GPI-linked membrane complement regulator that is widely expressed in mammalian tissues including the kidney. DAF inhibits the C3 convertase of both the classical and alternative pathways. Although DAF deficiency contributes to the human hematological syndrome paroxysmal nocturnal hemoglobinuria, the relevance of DAF in autoimmune tissue damage such as immune glomerulonephritis remains to be determined. In this study, we have investigated the susceptibility of knockout mice that are deficient in GPI-anchored DAF to nephrotoxic serum nephritis. Injection of a subnephritogenic dose of rabbit anti-mouse glomerular basement membrane serum induced glomerular disease in DAF knockout mice but not in wild-type controls. When examined at 8 days after anti-glomerular basement membrane treatment, DAF knockout mice had a much higher percentage of diseased glomeruli than wild-type mice (68.8 +/- 25.0 vs 10.0 +/- 3.5%; p < 0.01). Morphologically, DAF knockout mice displayed increased glomerular volume (516 +/- 68 vs 325 +/- 18 x 10(3) microm(3) per glomerulus; p < 0.0001) and cellularity (47.1 +/- 8.9 vs 32.0 +/- 3.1 cells per glomerulus; p < 0.01). Although the blood urea nitrogen level showed no difference between the two groups, proteinuria was observed in the knockout mice but not in the wild-type mice (1.4 +/- 0.7 vs 0.02 +/- 0.01 mg/24 h albumin excretion). The morphological and functional abnormalities in the knockout mouse kidney were associated with evidence of increased complement activation in the glomeruli. These results support the conclusion that membrane C3 convertase inhibitors like DAF play a protective role in complement-mediated immune glomerular damage in vivo.

Animals↗
PubMed2001

PPARgamma ligands inhibit TNF-alpha-induced LOX-1 expression in cultured endothelial cells.

Endothelial dysfunction or activation, elicited by oxidized low-density lipoprotein (OxLDL), has been implicated in the initiation and progression of atherosclerosis. We elucidated whether tumor necrosis factor-alpha (TNF-alpha)-induced endothelial OxLDL receptor, lectin-like OxLDL receptor-1 (LOX-1), mRNA expression is modified by peroxisome proliferator-activated receptor (PPAR) activators in cultured bovine aortic endothelial cells (BAEC). We confirmed that both PPARalpha and PPARgamma were expressed in BAEC by reverse transcription-polymerase chain reaction analysis. Natural PPARgamma ligand 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) and the thiazolidinediones, pioglitazone and troglitazone, decreased TNF-alpha-induced LOX-1 mRNA expression in BAEC. LOX-1 expression induced by phorbol 12-myristrate 13-acetate was also inhibited by 15d-PGJ(2). In contrast, PPARalpha ligands, Wy14643 and fenofibric acid, did not alter TNF-alpha-induced LOX-1 expression. TNF-alpha-induced immunohistochemical staining of LOX-1 was suppressed by 15d-PGJ(2) but not Wy14643. Taken together, PPARgamma activators inhibit TNF-alpha-induced LOX-1 expression in cultured BAEC, which may beneficially influence inflammatory responses in atherosclerosis.

Animals↗
PubMed2001

Effects of lipofectin-antigen complexes on major histocompatibility complex class I-restricted antigen presentation pathway in murine dendritic cells and on dendritic cell maturation.

We previously reported that exogenous antigens complexed with the cationic liposome lipofectin (LF) were efficiently presented via major histocompatibility complex (MHC) class I molecules on pulsed dendritic cells (DCs) in vitro. In the present study, we demonstrated that MHC class I-restricted antigen presentation on DC2.4 cells, a murine immature DC line, treated with LF-antigen complexes was remarkably suppressed through the inhibition of endocytosis, proteasome catalysis, and Golgi transport. We also found that LF did not influence expression of interleukin-12 p40 mRNA, MHC molecules, or co-stimulatory molecules in DC2.4 cells. These findings suggest that an antigen-loading procedure using LF could enhance delivery of exogenous antigens to the classical MHC class I pathway in DCs, but it does not initiate DC maturation.

Acetylcysteine↗
PubMed2001

Quasisteady high-confinement reversed shear plasma with large bootstrap current fraction under full noninductive current drive condition in JT-60U.

A quasisteady reversed shear plasma with a large bootstrap current fraction ( approximately 80%) has been obtained for the first time in the JT-60U tokamak. The shrinkage of reversed shear region was suppressed by the bootstrap current peaked at the internal transport barrier (ITB) layer and the ITBs at a large radius were sustained, which, by combination with an H-mode edge pedestal, resulted in a high confinement or 2.2 times the H-mode scaling for 6 times energy confinement time or 2.7 s. Furthermore, a full noninductive current drive was obtained by the bootstrap current and the beam driven current.

Journal Article↗
PubMed2001

Administration route-dependent vaccine efficiency of murine dendritic cells pulsed with antigens.

Dendritic cells (DCs) loaded with tumour antigens have been successfully used to induce protective tumour immunity in murine models and human trials. However, it is still unclear which DC administration route elicits a superior therapeutic effect. Herein, we investigated the vaccine efficiency of DC2.4 cells, a murine dendritic cell line, pulsed with ovalbumin (OVA) in the murine E.G7-OVA tumour model after immunization via various routes. After a single vaccination using 1 x 10(6)OVA-pulsed DC2.4 cells, tumour was completely rejected in the intradermally (i.d.; three of four mice), subcutaneously (s.c.; three of four mice), and intraperitoneally (i.p.; one of four mice) immunized groups. Double vaccinations enhanced the anti-tumour effect in all groups except the intravenous (i.v.) group, which failed to achieve complete rejection. The anti-tumour efficacy of each immunization route was correlated with the OVA-specific cytotoxic T lymphocyte (CTL) activity evaluated on day 7 post-vaccination. Furthermore, the accumulation of DC2.4 cells in the regional lymph nodes was detected only in the i.d.-and s.c.-injected groups. These results demonstrate that the administration route of antigen-loaded DCs affects the migration of DCs to lymphoid tissues and the magnitude of antigen-specific CTL response. Furthermore, the immunization route affects vaccine efficiency.

Animals↗
PubMed2001

Role of galactosylation in the renal pathogenicity of murine immunoglobulin G3 monoclonal cryoglobulins.

Cryoglobulin activity associated with murine immunoglobulin G3 (IgG3) has been shown to play a significant role in the development of murine lupuslike glomerulonephritis. A fraction, but not all, IgG3 monoclonal antibodies are capable of inducing a severe acute lupuslike glomerulonephritis as a result of direct localization of IgG3 cryoglobulins, suggesting the importance of qualitative features of cryoglobulins in their nephritogenic activities. Here a remarkable difference is shown in the renal pathogenicity of 2 murine IgG3 monoclonal cryoglobulins, identical in the amino acid sequences of their heavy and light chains but different in galactosylation patterns of oligosaccharide side chains because of their synthesis in different myeloma cells. The antibody lacking the capacity to induce severe glomerulonephritis displayed an increased proportion of galactosylated heavy chains. Changes in conformation, as revealed by gel filtration analysis, reduced cryoglobulin activity, and accelerated clearance could account for the lack of the renal pathogenicity of the more galactosylated variant. This observation provides a direct demonstration for the role of IgG galactosylation in the pathogenic potential of cryoglobulins. (Blood. 2001;97:3537-3543)

Animals↗
PubMed2001

Depth of invasion parallels increased cyclooxygenase-2 levels in patients with gastric carcinoma.

BACKGROUND: Nonsteroidal anti-inflammatory drugs may reduce the incidence of intestinal carcinoma, presumably through inhibition of cyclooxygenase-2 (COX-2). The authors correlated tumor expression of COX-2 with clinicopathologic features in tissues from patients with gastric carcinoma. METHODS: Thirty-three surgical specimens, including carcinomas and corresponding noncancerous mucosa, were sampled. Reverse transcription-polymerase chain reaction analysis was performed concomitantly for COX-1 and COX-2. A COX-2 index was determined from the band density ratio of COX-2 to constitutively expressed COX-1. Immunohistochemical staining with COX-2 antibody and routine histologic assessment were performed in the same specimens. RESULTS: The COX-2 index in gastric carcinoma was significantly higher than in normal mucosa (3.4 +/- 0.7 vs. 2.2 +/- 0.7; P < 0.05). COX-2 indices were significantly higher in gastric carcinoma tissues with deep invasion; indices for pT1, pT2, pT3, and pT4 carcinomas were 0.8 +/- 0.3, 2.8 +/- 0.5, 4.3 +/- 1.0, and 8.8 +/- 5.5, respectively (P < 0.05). Immunohistochemistry demonstrated COX-2 protein diffusely in the cytoplasm of tumor cells but not in surrounding stroma or in noncancerous mucosa. CONCLUSIONS: COX-2 mRNA expression in gastric carcinoma tissue is correlated closely with depth of invasion, indicating that COX-2 is involved in the growth of gastric carcinoma.

Adenocarcinoma↗
PubMed2001

Mutant G protein alpha subunit activated by Gbeta gamma: a model for receptor activation?

How receptors catalyze exchange of GTP for GDP bound to the Galpha subunit of trimeric G proteins is not known. One proposal is that the receptor uses the G protein's betagamma heterodimer as a lever, tilting it to pull open the guanine nucleotide binding pocket of Galpha. To test this possibility, we designed a mutant Galpha that would bind to betagamma in the tilted conformation. To do so, we excised a helical turn (four residues) from the N-terminal region of alpha(s), the alpha subunit of G(S), the stimulatory regulator of adenylyl cyclase. In the presence, but not in the absence, of transiently expressed beta(1) and gamma(2), this mutant (alpha(s)Delta), markedly stimulated cAMP accumulation. This effect depended on the ability of the coexpressed beta protein to interact normally with the lip of the nucleotide binding pocket of alpha(s)Delta. We substituted alanine for an aspartate in beta(1) that binds to a lysine (K206) in the lip of the alpha subunit's nucleotide binding pocket. Coexpressed with alpha(s)Delta and gamma(2), this mutant, beta(1)-D228A, elevated cAMP much less than did beta(1)-wild type; it did bind to alpha(s)Delta normally, however, as indicated by its unimpaired ability to target alpha(s)Delta to the plasma membrane. We conclude that betagamma can activate alpha(s) and that this effect probably involves both a tilt of betagamma relative to alpha(s) and interaction of beta with the lip of the nucleotide binding pocket. We speculate that receptors use a similar mechanism to activate trimeric G proteins.

Amino Acid Sequence↗
PubMed2001

Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia.

Tumor-induced osteomalacia (TIO) is one of the paraneoplastic diseases characterized by hypophosphatemia caused by renal phosphate wasting. Because removal of responsible tumors normalizes phosphate metabolism, an unidentified humoral phosphaturic factor is believed to be responsible for this syndrome. To identify the causative factor of TIO, we obtained cDNA clones that were abundantly expressed only in a tumor causing TIO and constructed tumor-specific cDNA contigs. Based on the sequence of one major contig, we cloned 2,270-bp cDNA, which turned out to encode fibroblast growth factor 23 (FGF23). Administration of recombinant FGF23 decreased serum phosphate in mice within 12 h. When Chinese hamster ovary cells stably expressing FGF23 were s.c. implanted into nude mice, hypophosphatemia with increased renal phosphate clearance was observed. In addition, a high level of serum alkaline phosphatase, low 1,25-dihydroxyvitamin D, deformity of bone, and impairment of body weight gain became evident. Histological examination showed marked increase of osteoid and widening of growth plate. Thus, continuous production of FGF23 reproduced clinical, biochemical, and histological features of TIO in vivo. Analyses for recombinant FGF23 products produced by Chinese hamster ovary cells indicated proteolytic cleavage of FGF23 at the RXXR motif. Recent genetic study indicates that missense mutations in this RXXR motif of FGF23 are responsible for autosomal dominant hypophosphatemic rickets, another hypophosphatemic disease with similar features to TIO. We conclude that overproduction of FGF23 causes TIO, whereas mutations in the FGF23 gene result in autosomal dominant hypophosphatemic rickets possibly by preventing proteolytic cleavage and enhancing biological activity of FGF23.

Alanine↗
PubMed2001

Expression of IL-1 beta and IL-8 by human gingival epithelial cells in response to Actinobacillus actinomycetemcomitans.

The interaction between epithelial cells and microorganisms is the most important step in bacterial infections. Epithelial cells in response to exposure to pathogenic bacteria produce cytokines that initiate inflammation. However, little is known about the cytokine response of gingival epithelial cells to periodontopathogenic bacteria. Actinobacillus actinomycetemcomitans is thought to play a significant role in the initiation of periodontitis because of its bacteriological characteristics. In the present study, we investigated the cytokine induction by human gingival epithelial cells (HGEC) following exposure to A. actinomycetemcomitans in comparison with human gingival fibroblasts (HGF) in culture. Northern blot analysis showed that mRNAs of interleukin 1beta (IL-1beta) and IL-8, but not IL-6, in HGEC were induced in response to A. actinomycetemcomitans. Secretion of IL-8 by HGEC was also increased following A. actinomycetemcomitans challenge, whereas production of IL-1beta could not be detected. The levels of IL-8 and its mRNA were increased depending on the concentration of A. actinomycetemcomitans. The co-culture with HGF and A. actinomycetemcomitans resulted in an increase in the levels of IL-6 and IL-8 mRNA in HGF. However, HGF exposed to A. actinomycetemcomitans, showed no expression of IL-1beta mRNA. These findings demonstrated that HGEC and HGF stimulated with A. actinomycetemcomitans have different profiles in cytokine mRNA expression. Furthermore, A. actinomycetemcomitans may play an important role in amplifying the local immune response and in initiating inflammatory reaction through release of IL-8 from gingival epithelial cells.

Aggregatibacter actinomycetemcomitans↗
PubMed2001

Involvement of transporter recruitment as well as gene expression in the substrate-induced adaptive regulation of amino acid transport system A.

We investigated the molecular mechanism involved in the adaptive regulation of the amino acid transport system A, a process in which amino acid starvation induces the transport activity. These studies were done with rat C6 glioma cells. System A activity in these cells is mediated exclusively by the system A subtype, amino acid transporter A2 (ATA2). The other two known system A subtypes, ATA1 and ATA3, are not expressed in these cells. Exposure of these cells to an amino acid-free medium induces system A activity. This process consists of an acute phase and a chronic phase. Laser-scanning confocal microscopic immunolocalization of ATA2 reveals that the acute phase is associated with recruitment of preformed ATA2 from an intracellular pool to the plasma membrane. In contrast, the chronic phase is associated with an induction of ata2 gene expression as evidenced from the increase in the steady-state levels of ATA2 mRNA, restoration of the intracellular pool of ATA2 protein, and blockade of the induction by cycloheximide and actinomycin D. The increase in system A activity induced by amino acid starvation is blocked specifically by system A substrates, including the non-metabolizable alpha-(methylamino)isobutyric acid.

Adaptation, Physiological↗
PubMed2001

PACT, a double-stranded RNA binding protein acts as a positive regulator for type I interferon gene induced by Newcastle disease virus.

Virus infection triggers innate responses to host cells including production of type I interferon (IFN). Since IFN production is also induced by treatment with poly(I:C), viral double-stranded (ds) RNA has been postulated to play a direct role in the process. In the present study, we investigated the effect of dsRNA binding proteins on virus-induced activation of the IFN-beta gene. We found that PACT, originally identified as protein activator for dsRNA-dependent protein kinase (PKR) and implicated in the regulation of translation, augmented IFN-beta gene activation induced by Newcastle disease virus. Concomitantly with the augmented activity of IFN-beta enhancer, increased activity of NF-kappaB and IRF-3 and IRF-7 was observed. For the observed effect, the dsRNA-binding activity of PACT was essential. We identified residues of PACT that interact with a presumptive target molecule to exert its function. Furthermore, PACT colocalized with viral replication complex in the infected cells. Thus the observed effect of PACT is novel and PACT is involved in the regulation of viral replication and results in a marked increase of cellular IFN-beta gene expression.

Amino Acid Sequence↗
PubMed2001

Efficient gene delivery into dendritic cells by fiber-mutant adenovirus vectors.

Recent studies have demonstrated the usefulness of dendritic cells (DCs) genetically modified by adenovirus vectors (Ad) to immunotherapy, while sufficient gene transduction into DCs is required for high doses of Ad. The RT-PCR analysis revealed that the relative resistance of DCs to Ad-mediated gene transfer is due to the absence of Coxsackie-adenovirus receptor expression, and that DCs expressed adequate alpha(v)-integrins. Therefore, we investigated whether fiber-mutant Ad containing the Arg-Gly-Asp (RGD) sequence in the fiber knob can efficiently transduce and express high levels of the LacZ gene into DCs. The gene delivery by fiber-mutant Ad was more efficient than that by conventional Ad in both murine DC lines and normal human DCs (NHDC). Furthermore, NHDC transduced with fiber-mutant Ad and conventional Ad at 8000-vector particles/cell resulted in a 70-fold difference in beta-galactosidase activity. We propose that alpha(v)-integrin-targeted Ad is a very powerful tool with which to implement DC-based vaccination strategies.

Adenoviridae↗
PubMed2001

Cloning and characterization of novel ficolins from the solitary ascidian, Halocynthia roretzi.

Ficolins are animal lectins with collagen-like and fibrinogen-like domains. They are involved in the first line of host defense against pathogens. Human ficolin/P35 as well as mannose-binding lectin (MBL) activates the complement lectin pathway in association with MBL-associated serine proteases. To elucidate the origin and evolution of ficolins, we separated approximately 40 kDa (p40) and approximately 50 kDa (p50) N-acetylglucosamine-binding lectins from hemolymph plasma of the solitary ascidian. Binding assays revealed that p40 recognizes N-acetyl groups in association with a pyranose ring and that p50 recognizes N-acetylglucosamine alone. Based on the amino acid sequences of the proteins, we isolated two clones each of p40 and p50 from the ascidian hepatopancreas cDNA and determined the entire coding sequences of these clones. Because all of the clones contained both collagen-like and fibrinogen-like domains, we concluded that these were homologs of the mammalian ficolin family and designated ascidian ficolins (AsFCNs). The fibrinogen-like domain of the AsFCNs shows 45.4-52.4% amino acid sequence identity with the mammalian ficolin family. A phylogenetic tree of the fibrinogen-like sequences shows that all the fibrinogen-like domains may have evolved from a common ancestor that branched off an authentic fibrinogen. These results suggest that AsFCNs play an important role with respect to ascidian hemolymph lectin activity and the correlation of different functions with binding specificity.

Amino Acid Sequence↗
PubMed2001

Palladium-catalyzed allylic alkylation using chiral hydrazones as ligands.

Palladium-catalyzed asymmetric allylic alkylation of 1,3-diphenyl-2-propenyl acetate (4) with a dimethyl malonate-BSA-LiOAc system and its derivatives has been successfully carried out in the presence of a new chiral hydrazone ligands such as 2-(diphenylphosphino)benzaldehyde SAMP hydrazone (DPPBA-SAMP) (3a) in high yields with high enantioselectives.

Journal Article↗