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Biomedical subjects

T Suda

Publications and source records attributed to T Suda.

At least 325 records · Page 18Linked to original sources
PubMed1996

A novel activity of HMG domains: promotion of the triple-stranded complex formation between DNA containing (GGA/TCC)11 and d(GGA)11 oligonucleotides.

The high mobility group protein (HMG)-box is a DNA-binding domain found in many proteins that bind preferentially to DNA of irregular structures in a sequence-independent manner and can bend the DNA. We show here that GST-fusion proteins of HMG domains from HMG1 and HMG2 promote a triple-stranded complex formation between DNA containing the (GGA/TCC)11 repeat and oligonucleotides of d(GGA)11 probably due to G:G base pairing. The activity is to reduce association time and requirements of Mg2+ and oligonucleotide concentrations. The HMG box of SRY, the protein determining male-sex differentiation, also has the activity, suggesting that it is not restricted to the HMG-box domains derived from HMG1/2 but is common to those from other members of the HMG-box family of proteins. Interestingly, the box-AB and box-B of HMG1 bend DNA containing the repeat, but SRY fails to bend in a circularization assay. The difference suggests that the two activities of association-promotion and DNA bending are distinct. These results suggest that the HMG-box domain has a novel activity of promoting the association between GGA repeats which might be involved in higher-order architecture of chromatin.

Base Composition↗
PubMed1996

Functional assessment of two vitamin D-responsive elements in the rat 25-hydroxyvitamin D3 24-hydroxylase gene.

Two vitamin D-responsive elements (VDRE-1 and VDRE-2) were recently identified in the 5'-upstream region of the rat 25-hydroxyvitamin D3 24-hydroxylase gene at -151/-137 and -259/-245, respectively. We studied the transcriptional regulation of this gene by vitamin D by means of mutational analysis. Introducing mutations into VDRE-1 and VDRE-2 in the native promoter -291/+9 reduced vitamin D-dependent chloramphenicol acetyltransferase activity by 86 and 41%, respectively. Mutation of the direct repeat -169/-155 located at 3 base pairs upstream of VDRE-1 also caused 50% decrease of chloramphenicol acetyltransferase activity. Connection of the element -169/-155 to VDRE-1 enhanced the vitamin D responsiveness of VDRE-1 5-fold through the heterologous beta-globin promoter. The fragment -291/-102 containing the two VDREs showed two shifted bands in the presence of the vitamin D receptor and retinoid X receptor in gel retardation analysis, and the appearance of the slower migrating band indicates that two sets of receptor complexes bind to this fragment simultaneously. These results demonstrate that VDRE-1 is a stronger mediator of vitamin D function than VDRE-2 due to the presence of the accessory element -169/-155 located adjacent to VDRE-1, although VDRE-2 exhibits a smaller dissociation constant for the vitamin D receptor-retinoid X receptor complex than VDRE-1.

Animals↗
PubMed1996

Apoptosis of mouse naive T cells induced by recombinant soluble Fas ligand and activation-induced resistance to Fas ligand.

We have investigated the susceptibility of mouse T cells at various maturation and activation stages to recombinant soluble mouse Fas ligand (termed WX1). CD4+CD8+ thymocytes were preferentially killed by WX1, although CD4+CD8- and CD4-CD8+ thymocytes were also killed at higher concentrations of WX1. The majority of CD4-CD8- thymocytes were resistant to the same doses of WX1. Naive peripheral T cells were also killed by WX1 at concentrations similar to those for single-positive thymocytes. Activation-induced cell death of previously activated T cells is known to be mediated by Fas and Fas ligand. However, the activation of naive splenic T cells by mAbs against CD3/TCR complex rather induced resistance against WX1. This resistance was induced in activated T cells but not in bystander T cells. These mechanisms seem to explain why activated T cells are not immediately killed and why bystander T cells are not activated in an Ag-nonspecific manner.

Animals↗
PubMed1996

STK/RON receptor tyrosine kinase mediates both apoptotic and growth signals via the multifunctional docking site conserved among the HGF receptor family.

STK/RON tyrosine kinase, a member of the hepatocyte growth factor (HGF) receptor family, is a receptor for macrophage-stimulating protein (MSP). To examine the STK/RON signalling pathway, we generated STK/ RON transfectants showing opposite features in growth. STK/RON-expressing Ba/F3 pro-B cells (BaF/STK) exhibited MSP-dependent growth, whereas STK/ RON-expressing mouse erythroleukaemia cells (MEL/ STK) displayed MSP-induced apoptosis. This apoptosis was accompanied by the prolonged activation of c-Jun N-terminal kinase (JNK), which has recently been implicated in the initiation of apoptosis. Co-immunoprecipitation analyses showed that autophosphorylated STK/RON associated with PLC-gamma, P13-kinase, Shc and Grb2 in both transfectants. However, major tyrosine-phosphorylated proteins, p61 and p65, specifically associated with STK/RON in MEL/STK cells. Mutations at two C-terminal tyrosine residues, Y1330 and Y1337, in the counterpart of the multifunctional docking site of the HGF receptor abolished both MSP-induced growth and apoptosis. Analyses of these mutants and in vitro association revealed that signalling proteins including p61 and p65 directly bound to the phosphotyrosines in the multifunctional docking site. These results demonstrate that positive or negative signals toward cell growth are generated through the multifunctional docking site and suggest the involvement of p61 and p65 as well as JNK in apoptosis. Our findings provide the first evidence for apoptosis via a receptor tyrosine kinase.

Amino Acid Sequence↗
PubMed1996

Molecular cloning of rat macrophage-stimulating protein and its involvement in the male reproductive system.

Macrophage-stimulating protein (MSP), a member of the hepatocyte growth factor family, is a ligand for receptor tyrosine kinase STK/RON. Here we isolated a full-length cDNA of rat MSP and a partial cDNA of rat STK/RON, then characterized their expression in the male reproductive system. In situ hybridization revealed that MSP mRNA was localized to spermatogonia and spermatocytes in the testis and the epithelium lining the lumen of the epididymis. On the other hand, RT-PCR analysis showed that STK/RON mRNA was expressed in sperm collected from both testis and epididymis. These findings suggest that locally produced MSP may play a vital role in germ cell-germ cell interaction during spermatogenesis as well as in the acquisition of sperm motility and/or fertilizing capacity in the epididymis. Our findings reveal new possible roles of the MSP-STK/RON signaling pathway.

Amino Acid Sequence↗
PubMed1996

Characterization of a ligand for receptor protein-tyrosine kinase HTK expressed in immature hematopoietic cells.

HTK is a receptor tyrosine kinase that belongs to the Eph subfamily. An extensive screening using BIAcore system revealed that a colon cancer cell line, C-1, expressed the ligand for HTK. From the conditioned medium of C-1 cells, a soluble form of ligand was purified by receptor affinity chromatography, and the isolation of full-length cDNA revealed that this ligand is identical to the human HTK ligand (HTKL) previously reported. HTK receptor tyrosine phosphorylation was induced by membrane-bound or clustered soluble HTKL but not by unclustered soluble HTKL, indicating that HTKL requires cell-to-cell interaction for receptor activation. Binding analysis demonstrated that HTKL binds to HTK with a much higher affinity (Kd: 1.23 nM) than the other transmembrane-type ligand for Eph family, LERK-2/ELKL (Kd: 135 nM). The expression of HTK in cord blood cells was upregulated after the culture in the presence of stem cell factor. Clustered soluble HTKL stimulated the proliferation of sorted HTK+ cord blood cells and a hematopoietic cell line, UT-7/EPO from which HTK was isolated. These findings suggest the involvement of HTK-HTKL system in the proliferation of HTK+ hematopoietic progenitor cells in the hematopoietic environment.

Amino Acid Sequence↗
PubMed1996

Swapping between Fas and granulocyte colony-stimulating factor receptor.

Fas belongs to the tumor necrosis factor/nerve growth factor receptor family. The Fas ligand binds to its receptor, Fas, and induces apoptosis in Fas-bearing cells. The granulocyte colony-stimulating factor receptor (G-CSFR) is a member of the hemopoietic growth factor receptor family. G-CSF induces its dimerization and regulates the proliferation and differentiation of neutrophilic granulocytes. We constructed hybrid receptors between Fas and G-CSFR and expressed them in the mouse T cell line WR19L or the mouse myeloid interleukin-3-dependent FDC-P1 cell line. The Fas ligand or an agonistic anti-Fas antibody stimulated proliferation of the FDC-P1 transformants expressing a chimera consisting of the Fas extracellular and G-CSFR cytoplasmic regions. On the other hand, G-CSF could not induce apoptosis in the transformants expressing the chimera consisting of the G-CSFR extracellular and Fas cytoplasmic regions, but these cells were killed by a polyclonal antibody against G-CSFR. These results indicated that receptors belonging to different receptor families can be functionally exchanged and confirm that a homodimer of G-CSFR can transduce the growth signal, whereas Fas must be oligomerized (probably trimerized) to transduce the apoptotic signal.

Animals↗
PubMed1996

Macrophage-stimulating protein induces proliferation and migration of murine keratinocytes.

Macrophage stimulating protein (MSP) is a chemotactic factor for murine peritoneal macrophages. The receptor for human MSP was recently identified as the ron gene product, a transmembrane protein tyrosine kinase cloned from a human keratinocyte cDNA library. Here we report that MSP induced proliferation of murine primary keratinocytes and established keratinocyte cell lines in a concentration-dependent manner. The growth efficacy of MSP was comparable to that of epidermal growth factor and keratinocyte growth factor. In three of four cell lines tested in a chemotaxis chamber, MSP also stimulated migration of keratinocytes on a collagen type IV substratum. The action of MSP was mediated by specific binding of MSP to the STK gene product, a murine homologue of the RON MSP receptor. Binding of MSP to keratinocyte STK induced phosphorylation of the 150 kDa STK beta chain. Herbimycin A, a protein tyrosine kinase inhibitor, blocked MSP-mediated phosphorylation of the STK receptor as well as proliferation of keratinocytes, suggesting the importance of tyrosine kinase activity for transduction of the message delivered by MSP. Previously, the only known target cell for MSP was the resident peritoneal macrophage. These studies establish the keratinocyte as a new target cell for MSP. The action of MSP on keratinocytes may have implications for tissue repair, wound healing, and tumor growth.

Animals↗
PubMed1996

Analysis of CSK homologous kinase (CHK/HYL) in hematopoiesis by utilizing gene knockout mice.

CHK/HYL is a non-receptor tyrosine kinase that belongs to CSK (C-terminal Src kinase) family. Northern blotting and RT-PCR analyses showed that CHK/HYL was expressed in large spectrum of hematopoietic cells except for erythroid cells and brain. To explore the function of CHK/HYL in hematopoietic cells, we generated CHK/HYL deficient mice. The mutant mice were apparently normal and fertile, while CSK knockout mice died until E11.5 from a defect in the neural tube formation. Hematological observations including blood counts and FACS analysis showed no significant abnormalities in CHK/HYL mutant mice. CHK/HYL did not affect the activity of Src, Hck, and Fgr in cultured bone marrow cells, although CSK negatively regulates Src family kinases. These results suggest that CHK/HYL might not have the same function as CSK.

Animals↗
PubMed1996

Impaired development of CD4+ CD8+ thymoyctes by csk-'knock-in' into fyn locus.

p59fyn is one of the Src-family kinases thought to play an important role in signaling through T cell receptor. However, Fyn deficiency has caused no overt defects in vivo on T cell development, nor has it caused any changes in the phosphorylation status of molecules such as ZAP-70 which have been proposed as p59fyn substrates. This could be explained as being due to compensation of Fyn deficiency by other Src-family kinases. Here, we have 'knocked-in' the csk gene, a negative regulator of Src-family kinases, into fyn locus to challenge the problem of redundant functions among Src-family kinases. The csk-'knock-in' mice displayed atrophy of the thymic cortex and impaired development of CD4+ CD8+ thymocytes. This was concomitant with decrease in tyrosine phosphorylation of ZAP-70 and p120cbl.

Animals↗
PubMed1996

Activation mediated by RP105 but not CD40 makes normal B cells susceptible to anti-IgM-induced apoptosis: a role for Fc receptor coligation.

Signals through the B cell antigen receptor lead to a variety of cellular events such as activation, anergy, and apoptosis. B cells select these outcomes to establish and maintain self-tolerance, and to mount adequate antibody responses. However, it is not fully understood how one and the same signal causes such different consequences. In the present study, we have studied the effect of activation signals on the outcome of responses to antigen receptor ligation. Two distinct growth-promoting signals were used to activate B cells. Ligation of either RP105, a newly discovered B cell surface molecule, or the CD40 molecule, drove B cells to proliferate. Resultant blastic cells were then exposed to anti-immunoglobulin M (IgM). Blast cells that had been stimulated with anti-RP105 ceased growing and underwent apoptosis after cross-linking of surface IgM. Coligation of the Fc gamma receptor IIB with surface IgM augmented, rather than aborted, this response. In contrast to RP105-activated B cells, blast cells that had been activated by CD40 ligation were unaltered by anti-IgM. On the other hand, CD40-activated B cells became extremely susceptible to Fas-mediated apoptosis, whereas RP105-activated B cells were much less sensitive. Anti-IgM-induced apoptosis in RP105 blasts was independent of Fas, because it was demonstrable with Fas-deficient MRL-lpr/lpr mice. These results demonstrate that the nature of an initial activation signal has a great influence on the fate of activated B cells after (re)engagement of the antigen receptor. RP105, as well as CD40, may be important in this life/death decision.

Animals↗
PubMed1996

Preferential hematopoiesis by paroxysmal nocturnal hemoglobinuria clone engrafted in SCID mice.

In paroxysmal nocturnal hemoglobinuria (PNH), little is known about the molecular events leading to the clinical manifestations except for the hemolysis. To unfold the complex pathophysiology, it is necessary to elucidate the nature of the PNH clone. PNH exhibits an acquired stem cell disorder, a clonal expansion of affected cells, concomitant depression of normal hematopoiesis in bone marrow (BM), and, although infrequently, the development of leukemia. The PNH clone is thus expected to exhibit some neoplastic features. We report here that CD34+ hematopoietic progenitor cells of PNH-BM yielded blood cells of three lineages with PNH phenotype alone when transplanted into sublethally irradiated severe combined immunedeficient mice. The hematopoiesis persisted for more than 10 months and did not always need human cytokines. In contrast, the hematopoiesis by control grafts obtained from healthy volunteers required an intense cytokine treatment. This in vivo model defines the preferential hematopoiesis of pluripotent PNH progenitor cells, indicating the intrinsic growth abnormality of PNH clone.

Adult↗
PubMed1996

Fas-mediated apoptosis of CD4+ and CD8+ T cells from human immunodeficiency virus-infected persons: differential in vitro preventive effect of cytokines and protease antagonists.

Human immunodeficiency syndrome (HIV) infection leads to a progressive loss of T-cell-mediated immunity associated with T-cell apoptosis. We report here that CD4+ and CD8+ T cells from HIV-1-infected persons are sensitive to Fas (CD95/APO-1)-mediated death induced either by an agonistic anti-Fas antibody or by the physiologic soluble Fas ligand, although showing no sensitivity to tumor necrosis factor alpha-induced death. CD4+ and CD8+ T-cell apoptosis induced by Fas ligation was enhanced by inhibitors of protein synthesis and was prevented either by a soluble Fas receptor decoy or an antagonistic anti-Fas antibody. Fas-mediated apoptosis could also be prevented in a CD4+ or CD8+ T-cell-type manner (1) by several protease antagonists, suggesting the involvement of the interleukin-1beta (IL-1beta)-converting enzyme (ICE)-related cysteine protease in CD4+ T-cell death and of both a CPP32-related cysteine protease and a calpain protease in CD8+ T-cell death; and (2) by three cytokines, IL-2, IL-12, and IL-10, that exerted their effects through a mechanism that required de novo protein synthesis. Finally, T-cell receptor (TCR)-induced apoptosis of CD4+ T cells from HIV-infected persons involved a Fas-mediated death process, whereas TCR stimulation of CD8+ T cells led to a different Fas-independent death process. These findings suggest that Fas-mediated T-cell death is involved in acquired immunodeficiency syndrome (AIDS) pathogenesis and that modulation of Fas-mediated signaling may represent a target for new therapeutic strategies aimed at the prevention of CD4+ T-cell death in AIDS.

Animals↗
PubMed1996

The role of gp130-mediated signals in osteoclast development: regulation of interleukin 11 production by osteoblasts and distribution of its receptor in bone marrow cultures.

Interleukin (IL)-11 is a multifunctional cytokine whose role in osteoclast development has not been fully elucidated. We examined IL-11 production by primary osteoblasts and the effects of rat monoclonal anti-mouse glycoprotein 130 (gp130) antibody on osteoclast formation, using a coculture of mouse osteoblasts and bone marrow cells. IL-1, TNF alpha, PGE2, parathyroid hormone (PTH) and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) similarly induced production of IL-11 by osteoblasts, but IL-6, IL-4, and TGF beta did not. Primary osteoblasts constitutively expressed mRNAs for both IL-11 receptor (IL-11R alpha) and gp130. Osteotropic factors did not modulate IL-11R alpha mRNA at 24 h, but steady-state gp130 mRNA expression in osteoblasts was upregulated by 1 alpha,25(OH)2D3, PTH, or IL-1. In cocultures, the formation of multinucleated osteoclast-like cells (OCLs) in response to IL-11, or IL-6 together with its soluble IL-6 receptor was dose-dependently inhibited by rat monoclonal anti-mouse gp130 antibody. Furthermore, adding anti-gp130 antibody abolished OCL formation induced by IL-1, and partially inhibited OCL formation induced by PGE2, PTH, or 1 alpha,25(OH)2D3. During osteoclast formation in marrow cultures, a sequential relationship existed between the expression of calcitonin receptor mRNA and IL-11R alpha mRNA. Osteoblasts as well as OCLs expressed transcripts for IL-11R alpha, as indicated by RT-PCR analysis and in situ hybridization. These results suggest a central role of gp130-coupled cytokines, especially IL-11, in osteoclast development. Since osteoblasts and mature osteoclasts expressed IL-11R alpha mRNA, both bone-forming and bone-resorbing cells are potential targets of IL-11.

Animals↗
PubMed1996

Bone morphogenetic protein-12 and -13 inhibit terminal differentiation of myoblasts, but do not induce their differentiation into osteoblasts.

Effects of bone morphogenetic protein (BMP)-12 and BMP-13, new members of the BMP family which belong to the transforming growth factor (TGF)-beta superfamily, on terminal differentiation of myoblasts were examined in C2C12 and L-6 myoblasts. When the myoblasts were cultured with BMP-12 or BMP-13, the expression of the myosin heavy chain and the formation of multinucleated myotubes mRNA in L-6 cells. The inhibitory effects of BMP-12 and BMP-13 on myogenic differentiation were similar to the effects of BMP-2, though their potencies were lower than BMP-2. Unlike BMP-2, neither BMP-12 nor BMP-13 induced alkaline phosphatase activity in C2C12 myoblasts. The differences in the biological activities of these new BMPs suggest that the intracellular signalling pathway used by BMP-12 and BMP-13 differs from that of BMP-2.

Alkaline Phosphatase↗
PubMed1996

Clinical significance of simultaneous determinations of alpha-fetoprotein and des-gamma-carboxy prothrombin in monitoring recurrence in patients with hepatocellular carcinoma.

BACKGROUND: Measurements of serum alpha-fetoprotein (AFP) concentration and plasma concentration of des-gamma-carboxy prothrombin (DCP) have been widely used for the early diagnosis of hepatocellular carcinoma (HCC). The two markers generally run parallel to each other. However, in our study, they sometimes fluctuated independently in response to tumor regression or recurrence. METHODS: A longitudinal series of concentrations of serum AFP and plasma DCP were determined simultaneously for 245 patients with HCC from the time of diagnosis to tumor recurrence after treatment. RESULTS: Positive reactions for AFP were noted in 168 patients (69%) and for DCP in 126 patients (51%). One hundred and ten of 245 patients with HCC (45%) were positive for both AFP and DCP. In 35 patients (14%), these 2 tumor markers fluctuated independently in response to tumor regression and recurrence. These patients were categorized into four groups as follows: Group 1 had elevated AFP only at diagnosis; it then decreased after treatment, but DCP was elevated at the time of tumor recurrence without AFP elevation (3 patients); Group 2 had elevated DCP at diagnosis and elevated AFP at tumor recurrence (4 patients); Group 3 had elevated AFP and DCP at diagnosis, but only AFP (8 patients) or DCP (7 patients) was elevated at tumor recurrence; Group 4 had only elevated AFP (2 patients) or DCP (11 patients) at diagnosis, but both AFP and DCP were elevated at tumor recurrence. CONCLUSIONS: The results of this study indicate that simultaneous determinations of AFP and DCP are useful for monitoring recurrence in patients with HCC after treatment, and that the decrease to normal levels of a single marker does not always indicate the absence of tumor recurrence.

Adolescent↗