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T Fujita

Publications and source records attributed to T Fujita.

At least 19 recordsLinked to original sources
PubMed1992

Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease.

Serum mannose-binding protein (MBP) is a C-type lectin that binds to terminal mannose and N-acetylglucosamine moieties present on surfaces of certain pathogens and activates the classical complement pathway. In the present study, we describe the mechanism underlying the activation triggered by MBP. The human serum MBP fraction was obtained by sequential affinity chromatography on mannan-Sepharose, anti-IgM-Sepharose and anti-MBP-Sepharose in the presence of calcium ions. This fraction contained a C1s-like serine protease as assessed by C4 consumption. The C1s-like serine protease, designated MBP-associated serine protease (MASP), was separated from MBP by rechromatography on anti-MBP-Sepharose in the presence of ethylenediaminetetraacetic acid. MASP exhibited both C4- and C2-consuming activities. The molecular mass of MASP was estimated to be 83 kD with two polypeptides of heavy (66 kD) and light (L) (31 kD) chains linked by disulfide bonds. The serine residue responsible for protease activity is located on the L chain. Reconstitution experiments using MASP and MBP revealed that combination of the two components restores C4- and C2-activating capacity on mannan. Based on analyses of molecular size, antigenicity, and 11 NH2-terminal amino acid sequences of the L chain, we conclude that MASP is a novel protein different from C1r or C1s. Our findings are not in accord with a proposed mechanism by which MBP utilizes the C1r2-C1s2 complex to initiate the classical complement pathway.

Amino Acid Sequence↗
PubMed1992

Signal transduction through decay-accelerating factor. Interaction of glycosyl-phosphatidylinositol anchor and protein tyrosine kinases p56lck and p59fyn 1.

Decay-accelerating factor (DAF or CD55) is a 70-kDa glycosyl-phosphatidylinositol (GPI)-anchored protein that protects cells from complement-mediated lysis by either preventing the formation of or dissociating C3 convertases. Cross-linking of DAF on human peripheral T cells by polyclonal antibodies has previously been reported to lead to lymphocyte proliferation. Two mAb, both mapping to the third short consensus repeat region of DAF, were able to trigger proliferation of human peripheral T cells. To determine the role of the GPI anchor in cell activation, we transfected EL-4 murine thymoma cells with cDNA encoding either DAF or a transmembrane form of DAF (DAF-TM). The DAF-transfected cells were able to transduce late activation events as evidenced by IL-2 production, whereas DAF-TM transfected cells were unable to do so. The GPI-anchored DAF was able to transduce early activation events leading to the tyrosine phosphorylation of a 40-kDa protein and several proteins in the 85-95 kDa range--an event absent in DAF-TM-transfected cells. Furthermore, anti-DAF immunoprecipitates of DAF-transfected cells contain tyrosine kinase activity leading to the phosphorylation of 40-, 56-60-, and 85-kDa proteins, whereas anti-DAF immunoprecipitates of DAF-TM-transfected cells did not have an associated kinase activity. Both p56lck and p59fyn were associated with DAF in DAF-transfected EL-4 cells. In HeLa cells transfected with fyn, DAF associated with p59fyn. This complex of DAF with src family protein tyrosine kinases requires the GPI anchor and suggests a pathway for signaling through GPI-anchored membrane proteins.

Antigens, CD↗
PubMed1992

Therapeutic effects of superoxide dismutase derivatives modified with mono- or polysaccharides on hepatic injury induced by ischemia/reperfusion.

Therapeutic effects of four types of recombinant superoxide dismutase (SOD) derivatives, conjugates with polysaccharides, carboxymethyl (SOD-CMD) and diethylaminoethyl (SOD-DEAED) dextrans and galactosylated (Gal-SOD) and mannosylated (Man-SOD) derivatives, on hepatic ischemia/reperfusion injury were studied in rats. Hepatic injury induced by transient occlusion and subsequent reflow of hepatic blood was evaluated by the analysis of biliary excretion of bromosulfophthalein (BSP) injected intravenously. At a dose of 10000 units/kg, native SOD and SOD-DEAE did not show any significant effect and SOD-CMD showed slight effect. On the other hand, Gal-SOD and Man-SOD, targeted to the liver parenchymal and nonparenchymal cells, respectively, by a receptor-mediated endocytosis, exhibited superior inhibitory effects. These results demonstrated that these glycosylated SOD derivatives were useful for the prevention of hepatic ischemia/reperfusion injury.

Animals↗
PubMed1992

Mapping of epitopes, glycosylation sites, and complement regulatory domains in human decay accelerating factor.

Decay accelerating factor (DAF, CD55) is a glycophospholipid-anchored membrane protein that protects cells from complement-mediated damage by inhibiting the formation and accelerating the decay of C3/C5 convertases. DAF deletion mutants lacking each of the four short consensus repeats (SCR) or the serine/threonine-rich region (S/T) were created by site-directed mutagenesis. These deletion mutants were expressed by stable transfection in Chinese hamster ovary cells for the purpose of mapping important structural and functional sites in DAF. The epitopes on DAF for 16 murine mAb were mapped by immunoprecipitation studies as follows: SCR1, 6; SCR2, 3; SCR3, 3; SCR4, 3; S/T, 1. Testing of 13 mAb showed complete blocking of DAF function only by 1C6 and 1H4, both directed at SCR3. The single N-linked glycosylation site was confirmed at a location between SCR1 and SCR2, and the multiple O-linked oligosaccharides were localized to the S/T region. Functional activity of DAF mutants was assessed by the ability of these transfected constructs to protect Chinese hamster ovary cells from cytotoxicity induced by rabbit antibody plus human complement. Removal of SCR1 had no effect on DAF function, but individual deletion of SCR2, SCR3, or SCR4 totally abolished DAF function. Surprisingly, deletion of the S/T region totally abrogated DAF function, but this could be restored by a fusion construct placing the four SCR domains of DAF onto the HLA-B44 molecule, implying that the O-glycosylated S/T region serves as an important but nonspecific spacer projecting the DAF functional domains above the plasma membrane. Overall, the creation of DAF deletion mutants has elucidated important structure-function relations in the DAF molecule.

Animals↗
PubMed1992

Isolation of cDNA clone encoding rat senescence marker protein-30 (SMP30) and its tissue distribution.

We have isolated and characterized two cDNA clones encoding senescence marker protein-30 (SMP30), the amounts of which are known to decrease androgen-independently with aging in the livers of rats. Of these cDNA clones, one consisted of 1588 bp nucleotides and the other of 1195 bp nucleotides generated by alternative polyadenylation. These two cDNA clones shared the same open reading frame, but the larger species had 393 bp nucleotides of 3' untranslated region in addition to the first polyadenylation site of smaller species. Northern hybridization analysis showed that two species of mRNA (1.7 kb and 1.4 kb) located in the liver and kidney were consistent with these short and long forms of cDNA. The open reading frame, 897 bp could encode 299 amino acids. The estimated molecular weight and pI of the deduced polypeptide were 33,387 and 5.1, respectively. Furthermore, immunohistochemical analysis confirmed that SMP30 was preferentially localized in the hepatocytes and renal proximal tubular epithelium. Genomic Southern hybridization analysis demonstrated that SMP30 was widely conserved among higher animals. A computer-assisted homology analysis of nucleic acid and protein databases revealed no remarkable homology with other known proteins. Therefore, SMP30 seems to be a novel protein. In addition, the existence of putative A-U rich mRNA degradation signals and protein degradation signals (PEST sequence) in the structure of SMP30 may suggest important regulatory function of this unique protein manifested by changes in its concentrations.

Aging↗
PubMed1992

Insulin inhibits norepinephrine overflow from peripheral sympathetic nerve ending.

The effects of insulin on peripheral nervous system are unknown. We therefore studied the effects of insulin on sympathetic nerve activity in isolated mesenteric arteries of Sprague-Dawley rats. The overflow of norepinephrine (NE) by electrical stimulation was used as the index of sympathetic nervous system activity. Insulin (0.5 to 1U/l) decreased the NE release in a dose-dependent fashion. This inhibitory effect was, however, reversed by either 5 x 10(-5)M cocaine or 5 x 10(-4)M ouabain treatment. Thus, we postulate that insulin attenuates NE overflow from peripheral sympathetic nerve endings, probably due to enhanced NE reuptake.

Animals↗
PubMed1992

Decay-accelerating factor functions as a signal transducing molecule for human monocytes.

Decay-accelerating factor (DAF) is a glycosylphosphatidylinositol-anchored membrane protein that protects cells from damage by autologous complement activation. Of the four mAb against DAF prepared in our laboratory, 1C6 completely blocked DAF function, whereas 5B2 partially blocked it. Using these mAb, we investigated whether human monocytes were activated via DAF molecules. When monocytes were incubated with 1C6 alone, glucose was consumed in significant amounts and phagocytosis of latex beads was enhanced, indicating that the monocytes had been activated. However, 1C6 did not enhance the production of monokines, TNF-alpha, and IL-1 alpha and -beta. The F(ab')2 fragment of 1C6 also activated monocytes, whereas 5B2 and the Fab fragment of 1C6 could not. To further examine monocyte activation, these cells were treated with phosphatidylinositol-specific phospholipase C. Increased glucose consumption and enhanced phagocytic activity by 1C6 were considerably reduced in monocytes treated with phosphatidylinositol-specific phospholipase C. In addition, we found that 1C6 stimulated the generation of inositol trisphosphate. These results demonstrate that the signal transmitted via the DAF molecule is capable of stimulating monocytes.

Antibodies, Monoclonal↗
PubMed1992

Inhibitory effects of dihydroagarofuran sesquiterpenes on Epstein-Barr virus activation.

To search for possible antitumor promoters, we carried out a primary screening of thirty-seven dihydroagarofuran sesquiterpenes from Tripterygium wilfordii Hook fil. var. regelii Makino and Euonymus sieboldianus Blume, using their possible inhibitory effects on the Epstein-Barr virus early antigen (EBV-EA) activation which is induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Some of these sesquiterpenes, triptofordin F-2 (Takaishi et al., 1988), 1,2,6,8,15-pentaacetoxy-9-benzoyloxy-4-hydroxy-beta-dihydroagarofuran and triptogelin A-1 (Takaishi et al., 1990) were observed to significantly inhibit the EBV-EA activation at low doses. Based on the results, the structural requirements for the activity of these compounds were discussed [corrected].

Anticarcinogenic Agents↗
PubMed1992

Protective effect of NGF atelocollagen mini-pellet on the hippocampal delayed neuronal death in gerbils.

Very recently, contradictory results were presented as to the effects of exogenous nerve growth factor (NGF) on the hippocampal delayed neuronal necrosis following transient ischemia. In the present study, we administered a large amount of NGF with the atelocollagen mini-pellet system, measured the local NGF contents, and evaluated the effect of this neurotrophic factor on the postischemic hippocampal pyramidal cells in gerbils. We concluded that the exogenous NGF, when given continuously at sufficient concentrations, prevents pyramidal cell damage. The possible cause of discrepancy in previous studies is discussed.

Animals↗
PubMed1992

Studies on antidiabetic agents. 11. Novel thiazolidinedione derivatives as potent hypoglycemic and hypolipidemic agents.

In the course of further chemical modification of the novel antidiabetic pioglitazone (AD-4833, U-72,107), a series of 5-[4-(2- or 4-azolylalkoxy)benzyl- or -benzylidene]-2,4-thiazolidinediones was prepared and evaluated for hypoglycemic and hypolipidemic activities in insulin-resistant, genetically obese, and diabetic KKA(y) mice. Replacement of the 2-pyridyl moiety of pioglitazone by a 2- or 4-oxazolyl or a 2- or 4-thiazolyl moiety greatly enhanced in vivo potency. The corresponding 5-benzylidene-type compounds, in which a methine was used as a linker between the benzene ring and the thiazolidinedione ring, also had potent biological activity. Among the compounds synthesized, 5-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]benzyl]-2,4- thiazolidinedione (18) exhibited the most potent activity, more than 100 times that of pioglitazone. The synthesis and structure-activity relationships for this novel series of derivatives are detailed.

Animals↗
PubMed1992

Destructive arthritis without lymphocyte infiltration in H2-c-fos transgenic mice.

H2-c-fos transgenic (c-fos+) mice are characterized by the inability to raise specific IgG antibodies against immunizing Ag. To examine the contribution of Ag-specific IgG antibody to the development of arthritis, Ag-induced arthritis was produced in c-fos+ mice and their control littermates (c-fos- mice). Intra-articular injection of OVA into c-fos- mice hyperimmunized with OVA induced destructive arthritis with massive lymphocyte infiltration. The c-fos+ mice also developed destructive arthritis comparable in degree with that seen in c-fos- mice. However, joints from the c-fos+ mice had few or no infiltrating lymphocytes. The majority of cells invading the extensively eroded collagenous tissue in the c-fos+ mice had a mesenchymal appearance. These cells, producing excess amounts of c-Fos protein, adhered to and invaded the cartilage matrix when cultured on cartilage slices. These cells, thus, appear to directly cause joint destruction in c-fos+ mice.

Animals↗
PubMed1992

Purification of senescence marker protein-30 (SMP30) and its androgen-independent decrease with age in the rat liver.

Age-associated changes in the soluble proteins from rat liver were examined by a newly developed two-dimensional cellulose acetate membrane electrophoresis (2D-CAME). We detected and isolated a novel rat liver protein, the amounts of which decreased androgen-independently with aging. We designated this protein, whose molecular mass was 30 kDa and pI value was 4.9, as senescence marker protein-30 (SMP30). The expression of SMP30 was not modified by castration or treatment with testosterone propionate after castration. Age-associated decrease of SMP30 level was also recognized in aged female. We noted another protein with a pI value of 7.3 that decreased with aging. Its expression seemed to be androgen-dependent and it was markedly expressed only in young and adult male rat livers. We then purified SMP30 and prepared an anti-serum to SMP30. Immunohistochemical analysis showed that the localization of SMP30 was restricted to liver and kidney among numerous organs tested. Although the amount of SMP30 in the liver was relatively large and the tissue distribution was characteristic, no known protein corresponds to this protein.

Aging↗
PubMed1992

Human mannose-binding protein is identical to a component of Ra-reactive factor.

Human Ra-reactive factor (RaRF) and mannose-binding protein (MBP) were isolated from sera by utilizing their affinity to Ra chemotype Salmonella typhimurium and yeast mannan, respectively. A predominant polypeptide of human RaRF with an Mr of 32 kDa (P32) has the same mobility as human MBP on SDS-PAGE gels. A monoclonal antibody against P32 of human RaRF, 3E7, was found to react not only with P32 but also with human MBP, as assessed by immunoblotting. In addition, 3E7 blocked the complement-activating capacity of human MBP. The NH2-terminal amino acid sequence of P32 of human RaRF was determined and found to coincide with that of human MBP. Based on these results, it is clear that human MBP is identical to a component of RaRF.

Antibodies, Monoclonal↗
PubMed1992

Biochemical characterization of nerve growth cones isolated from both fetal and neonatal rat forebrains: the growth cone particle fraction mainly consists of axonal growth cones in both stages.

Nerve growth cones are responsible for the exact pathway finding, and for the establishment of neurocytoarchitecture. To elucidate the developmental changes of biochemical characteristics of nerve growth cones, growth cone particle (GCP) fractions were isolated biochemically from embryonal day 17 (E17) rat forebrain and from postnatal day 5 (P5). There were no significant differences in protein phosphorylation pattern in a Ca(2+)-dependent manner between E17-GCP fraction and that of P5. As for the membrane lipid composition, molar ratios of cholesterol to total phospholipids were well conserved during these ages. The immunoreactivity to anti-synaptophysin monoclonal antibody as a marker of mature synaptic elements could not be detected either in E17-GCP or P5-GCP fractions. To exclude the possibility of the contamination of dendritic elements, RNA contents and immunoreactivity to anti-high molecular weight microtubule-associated protein 2 (MAP2) monoclonal antibody were examined. RNA contents of the GCP fractions were extremely low compared to those of other subcellular fractions both in E17 and P5. No immunoreactivities to anti-MAP2 antibody were observed in either GCP fraction. Our results suggest that the GCP fractions, isolated from forebrains of E17 to P5 rat, are free from the contamination of the synaptic elements, and that the GCP fractions are mainly composed of axonal growth cones.

Aging↗
PubMed1992

Calcitonin inhibits the growth of human gastric carcinoma cell line KATO III.

Calcitonin has a wide variety of actions on gastrointestinal function. In this study, we investigated the effects of calcitonin on the growth of human gastric carcinoma cell line KATO III in comparison with those of calcitonin gene-related peptide (CGRP). Calcitonin, but not CGRP, significantly and dose-dependently inhibited the growth of KATO III cells. This inhibition of cell growth was accompanied by an increase in cyclic AMP production. The proliferation of KATO III cells was also inhibited by forskolin and dibutyryl cyclic AMP, although agents which do not stimulate cyclic AMP production had no effect. Furthermore, in the presence of GTP, calcitonin stimulated adenylate cyclase activity in KATO III cell membranes, and this increase was reduced in the absence of GTP. On the other had, neither calcitonin nor CGRP enhanced the turnover of inositolphospholipid or the intracellular Ca2+ level. In addition, 125I-labeled human calcitonin was specifically bound to KATO III cell membranes, and this binding was dose-dependently displaced by unlabeled calcitonin but not CGRP. Furthermore, the specific binding of 125I-labeled human calcitonin to KATO III cell membranes was significantly reduced by addition of GTP but not ATP. These results suggest that calcitonin inhibits the growth of human gastric carcinoma cell line KATO III by stimulating cyclic AMP production via a GTP-dependent process coupled to specific calcitonin receptors.

Adenylyl Cyclases↗
PubMed1992

Constitutive expression of c-fos gene inhibits type 1 collagen synthesis in transfected osteoblasts.

To clarify the contribution of c-fos DNA to bone formation, the effect of constitutive expression of the c-fos gene in collagen synthesis was examined by introducing c-fos DNA into osteoblastic MC3T3-E1 cells. The [3H] proline incorporation into the collagenase digestible protein(CDP) and the percent collagen synthesis were significantly decreased in the c-fos transfectants which constitutively express c-fos mRNA as compared with control transfectants. Transcription of type 1(alpha 1) collagen gene was also specifically decreased in the c-fos transfectants. This indicates that constitutive expression of c-fos DNA interferes with bone formation by inhibiting collagen synthesis in osteoblasts.

Animals↗