Biomedical subjects

A Iwama

Publications and source records attributed to A Iwama.

At least 37 records · Page 2Linked to original sources

PubMed1998

Physiology and morphology of olfactory neurons associating with the protocerebral lobe of the honeybee brain.

Physiology and morphology of olfactory neurons associated with the protocerebral lobe around the alpha-lobe of the mushroom body were studied in the brain of the honeybee Apis mellifera using intracellular recording and staining techniques. The responses of neurons to behaviorally relevant odorants (a blend, and components of the Nasonov pheromone, and some other non-pheromonal odors) were recorded. Different response patterns were observed within different neurons, and often within the same neuron, in response to different stimuli. All the neurons stained had innervations in the protocerebral lobe. The cell profiles varied from cells connecting the antennal lobe with both the protocerebral and lateral protocerebral lobes (projection neurons), cells linking the pedunculus of the mushroom body with both the protocerebral and lateral protocerebral lobes (PE1 neurons), cells linking the alpha-lobe and protocerebral lobe with the calyces of the mushroom body (feedback neurons), and cells linking the alpha-lobe and protocerebral lobe with the antennal lobe (recurrent neurons), to cells connecting the protocerebral lobe with the contralateral protocerebrum (bilateral neurons). These findings suggest that the protocerebral lobe acts as an olfactory center associating with other centers, and provides multi-layered recurrent networks within the protocerebrum and between the deutocerebrum and the protocerebrum in honeybee olfactory pathways.

Journal Article↗

PubMed1998

Cytotoxic effect of endodontic bacteria on periapical fibroblasts.

This study was conducted to investigate the effects of sonicated bacterial extracts (SBEs) from anaerobic Gram-negative bacteria on periapical fibroblast obtained from the apical portion of human periodontal ligaments. Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum were chosen from among the endodontic bacteria isolated from root canals having a periapical lesion and compared in terms of their cytotoxicity. The purpose of this study was to examine which bacteria are involved in the development of periapical inflammation. The anaerobes were cultured under strict anaerobic conditions, and the bacterial cells were then harvested by centrifugation after incubation. The concentrated cell suspensions were sonicated and subsequently centrifuged. An SBE was made of each of the filtered supernatants. Each SBE was added to cultures of periapical fibroblasts. The cell growth and proliferation were measured by the MTT method after 3, 5, and 7 days. The SBEs from P. endodontalis, P. gingivalis, and F. nucleatum inhibited the growth of the fibroblasts, whereas the SBE from P. intermedia did not inhibit it. The SBEs from P. gingivalis and F. nucleatum inhibited the fibroblast growth more strongly than did the P. endodontalis, P. gingivalis, and F. nucleatum may participate in the development of periapical lesions.

Bacterial Proteins↗

PubMed1998

Characterization of TEK receptor tyrosine kinase and its ligands, Angiopoietins, in human hematopoietic progenitor cells.

TEK, or TIE-2, is a receptor tyrosine kinase (RTK) that is known as a functioning molecule of vascular endothelial cells. TEK comprises a subfamily of RTK with TIE, and these two receptors play critical roles in vascular maturation, maintenance of integrity and remodeling. We generated mAb against the extracellular domain of human TEK protein to elucidate its expression pattern in human hematopoietic cells. Flow cytometric analysis of bone marrow cells revealed that TEK was expressed in 27% of CD34+ cells, 20% of c-KIT+ cells and 26% of CD34+CD38- cells, indicating that TEK is expressed in a subset of primitive hematopoietic stem cells (HSC). TEK was also expressed in 20% of CD19+ B lymphocytes but not in other lineage-committed cells. Progenitor assays in methylcellulose culture showed that CD34+TEK+ cells formed significantly less BFU-E and CFU-Mix than CD34+TEK- cells, but there was no difference in the number of CFU-GM between these two populations. Two recently identified TEK ligands, termed Angiopoietin-1 and -2, bound to TEK with similar affinities, and Angiopoietin-1 effectively induced TEK phosphorylation in hematopoietic cells. Angiopoietin-2 also induced a low level of TEK phosphorylation and weakened the phosphorylation induced by Angiopoietin-1, suggestive of an elaborate regulator of the TEK-TEK ligand signaling pathway. Although neither ligands affected the proliferation of TEK-transfected hematopoietic cells or the colony formation of CD34+TEK+ bone marrow cells, both promoted the adhesion of TEK-transfected hematopoietic cells to a collagen matrix or a layer of bone marrow stromal cells. These findings indicate that the TEK-TEK ligand signaling pathway is regulated in a refined manner and is involved in hematopoietic cell-microenvironment interaction.

Angiopoietin-1↗

PubMed1998

Macrophage-stimulating protein (MSP) and its receptor, RON, stimulate human osteoclast activity but not proliferation: effect of MSP distinct from that of hepatocyte growth factor.

Stem cell-derived tyrosine kinase (STK) is a member of the hepatocyte growth factor (HGF) receptor family. The ligand for STK, macrophage-stimulating protein (MSP), is a serum protein activated by members of the coagulation cascade. The RON gene is a human homolog of the murine STK. In this study we examined the role of MSP-RON in the signal pathway of human osteoclasts. Using anti-RON antibody, we detected RON expressed in multinucleated osteoclast-like cells (OCLs) formed in cultures of human bone marrow cells. To determine bone resorption, we placed OCLs on thin films of ceramic calcium phosphate formed on quartz plate-coated slides (Millenium Biologix) and measured pit formation. MSP stimulated pit formation by OCLs in a dose-dependent manner. MSP (50 ng/mL) caused a fourfold increase in pit area compared with the control. Furthermore, we examined the effects of MSP and HGF on OCL formation by purified populations of hematopoietic progenitors. OCLs were phenotypically identified by their cross-reactivity with 23c6, a monoclonal antibody that preferentially binds to osteoclasts. HGF (50 ng/mL) stimulated the differentiation of progenitors to 23c6-positive OCLs but did not enhance bone absorption. In contrast, MSP did not affect proliferation of osteoclast precursors but stimulated bone resorption by OCLs. We conclude that the MSP signal transduction pathway plays a role in bone resorption that is distinct from that of HGF.

Bone Marrow Cells↗

PubMed1997

Expression and function of murine receptor tyrosine kinases, TIE and TEK, in hematopoietic stem cells.

Two highly related receptor tyrosine kinases, TIE and TEK, comprise a family of endothelial cell-specific kinase. We established monoclonal antibodies against them and performed detailed analyses on their expression and function in murine hematopoietic stem cells (HSCs). TIE and TEK were expressed on 23.7% and 33.3% of lineage marker-negative, c-Kit+ and Sca-1+ (Lin- c-Kit+ Sca-1+) HSCs that contain the majority of day-12 colony-forming units-spleen (CFU-S) and long-term reconstituting cells, but not committed progenitor cells. Lin- c-Kit+ Sca-1+ cells were further divided by the expression of TIE and TEK. TIE+ and TEK+ HSCs as well as each negative counterpart contained high proliferative potential colony-forming cells and differentiated into lymphoid and myeloid progenies both in vitro and in vivo. However, day-12 CFU-S were enriched in TIE+ and TEK+ HSCs. Our findings define TIE and TEK as novel stem cell marker antigens that segregate day-12 CFU-S, and provide evidence of novel signaling pathways that are involved in the functional regulation of HSCs at a specific stage of differentiation, particularly of day-12 CFU-S.

Animals↗

PubMed1997

Selective expression of the receptor tyrosine kinase, HTK, on human erythroid progenitor cells.

HTK is a receptor tyrosine kinase of the Eph family. To characterize the involvement of HTK in hematopoiesis, we generated monoclonal antibodies against HTK and investigated its expression on human bone marrow cells. About 5% of the bone marrow cells were HTK+, which were also c-Kit+, CD34(low), and glycophorin A(-/low). Assays of progenitors showed that HTK+ c-Kit+ cells consisted exclusively of erythroid progenitors, whereas HTK- c-Kit+ cells contained progenitors of granulocytes and macrophages as well as those of erythroid cells. Most of the HTK+ erythroid progenitors were stem cell factor-dependent for proliferation, indicating that they represent mainly erythroid burst-forming units (BFU-E). During the erythroid differentiation of cultured peripheral CD34+ cells, HTK expression was upregulated on immature erythroid cells that corresponded to BFU-E and erythroid colony-forming units and downregulated on erythroblasts with high levels of glycophorin expression. These findings suggest that HTK is selectively expressed on the restricted stage of erythroid progenitors, particularly BFU-E, and that HTK is the first marker antigen that allows the purification of erythroid progenitors. Furthermore, HTKL, the ligand for HTK, was expressed in the bone marrow stromal cells. Our findings provide a novel regulatory system of erythropoiesis mediated by the HTKL-HTK signaling pathway.

Antibodies, Monoclonal↗

PubMed1997

Role of macrophage-stimulating protein and its receptor, RON tyrosine kinase, in ciliary motility.

Macrophage-stimulating protein (MSP) is an 80-kD serum protein with homology to hepatocyte growth factor (HGF). Its receptor, RON tyrosine kinase, is a new member of the HGF receptor family. The MSP-RON signaling pathway has been implicated in the functional regulation of mononuclear phagocytes. However, the function of this pathway in other types of cells has not been elucidated. Here we show that in contrast to the HGF receptor, which was expressed at the basolateral surface, RON was localized at the apical surface of ciliated epithelia in the airways and oviduct. In addition, MSP was found in the bronchoalveolar space at biologically significant concentrations. MSP bound to RON on normal human bronchial epithelial cells with a high affinity (Kd = 0.5 nM) and induced autophosphorylation of RON. Activation of RON by MSP led to a significant increase in ciliary beat frequency of human nasal cilia. These findings indicate that the ciliated epithelium of the mucociliary transport apparatus is a novel target of MSP. Ciliary motility is critical for mucociliary transport. Our findings suggest that the MSP-RON signaling pathway is a novel regulatory system of mucociliary function and might be involved in the host defense and fertilization.

Animals↗

PubMed1997

Quantitative assays for hepatitis C virus in serum as predictors of the long-term response to interferon.

BACKGROUND/AIMS: Interferon therapy has a beneficial effect in patients with chronic hepatitis C who have a low viral load. The aim of this study was to compare the core protein level with HCV RNA levels and to analyze whether virus quantitation predicts the efficacy of interferon therapy. METHODS: HCV core protein level assessed by the recently developed assay was compared with HCV RNA levels measured by three different methods (Amplicor-HCV monitor, competitive RT(CRT)-PCR, and bDNA probe assay) in 352 patients with chronic hepatitis C in relation to viral serotype. RESULTS: From 91% (320/352) to 93% (299/322) of patients with viremia were detected by Amplicor-monitor and CRT-PCR, in contrast to 60% (187/312) and 74% (191/258) by bDNA and HCV core protein assay, respectively. The HCV core protein level was positively correlated with HCV RNA levels measured by the three assays (r = 0.680 to 0.731). Serum HCV RNA and core protein levels were significantly lower in patients with serotype 2 than in those with serotype 1. Viral eradication after interferon therapy was observed in 60-70% of the patients with < 1 x 10(4) copies/ml of HCV RNA by Amplicor-monitor assay, < 2 x 10(5) copies/ml by CRT-PCR, < 0.5 Meq/ml by bDNA assay, and < 20 pg/ml of core protein by HCV core protein assay. Viral eradication was uncommon (< 11%) among the patients with higher viral loads. Bivariate analysis revealed that the outcome of interferon therapy was more closely associated with both HCV core protein and RNA levels than the HCV serotype. CONCLUSIONS: Quantitation of HCV core protein and HCV RNA in useful for prediction of the interferon response.

Adolescent↗

PubMed1997

Deregulated inflammatory response in mice lacking the STK/RON receptor tyrosine kinase.

Immune and inflammatory responses must be rightly regulated to maintain a homoeostatic balance between an effective immune response and tissue damage to the host. NO is a principal mediator of many of the cytokine-inducible macrophage activities during a normal cell-mediated immune response. STK, the murine homologue of the human RON receptor tyrosine kinase, is expressed on murine resident peritoneal macrophages. The ligand for STK, macrophage-stimulating protein (MSP), is a serum protein that is activated by members of the coagulation cascade in response to tissue damage. In addition to its potential to induce chemotaxis and phagocytosis of C3bi-coated erythrocytes, MSP has an inhibitory effect on the production of NO by activated peritoneal macrophages in vitro. Here we demonstrate that peritoneal macrophages from mice lacking STK produce elevated levels of NO in response to interferon (IFN)-gamma in a dose-dependent manner, without the need for a co-stimulus. However, production of pro-inflammatory cytokines by activated macrophages from stk -/- mice is unaltered. In vivo, stk -/- mice exhibit increased inflammation in an IFN-gamma-mediated delayed-type hypersensitivity reaction and increased susceptibility to lipopolysaccharide (LPS)-induced endotoxic shock. Furthermore, the levels of NO in the serum of mice injected with LPS are significantly higher than those in control littermates. Nevertheless, the serum levels of IFN-gamma and the intermediate cytokines generated by the inflammatory response, which have previously been shown to play a role in septicaemic shock, do not differ significantly from controls. These data suggest that the STK receptor suppresses NO production, therefore ameliorating the potentially tissue-damaging effects of a cell-mediated immune response, through negative regulation of the IFN-gamma signalling pathway.

Animals↗

PubMed1997

Receptor tyrosine kinases involved in hematopoietic progenitor cells.

To analyze the molecular mechanisms of the proliferation and differentiation of hematopoietic cells, we have cloned PTKs from sorted stem cells. We discuss the expression and function of receptor tyrosine kinases, STK/RON, TIE, TEK and HTK which have been cloned from these cells. STK and its ligand, MSP contributed to the motility and phagocytosis of peritoneal macrophages and bone absorption of osteoclasts. Apoptosis was induced in an erythroid cell line by the binding of MSP(MacrophageStimulating Protein). TIE, TEK and HTK were interestingly expressed in the subpopulations of stem cells and related to the myeloid differentiation. These study will indicate the heterogeneity of stem cells and their diverse differentiation.

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

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

Macrophage-stimulating protein activates STK receptor tyrosine kinase on osteoclasts and facilitates bone resorption by osteoclast-like cells.

Recently we cloned a novel receptor tyrosine kinase, STK. STK belongs to the hepatocyte growth factor receptor family and was identified as the receptor for macrophage-stimulating protein (MSP). STK is expressed on a restricted, macrophage population such as peritoneal macrophages, but not on mononuclear phagocytes of peripheral blood, bone marrow, or alveoli. Using an anti-STK monoclonal antibody, we observed STK expression on multinuclear osteoclast-like cells (OCLs) formed by murine bone marrow cultures in the presence of 1,25-dihydroxyvitamin D3, and interleukin-3. The OCLs expressed both the calcitonin receptor and STK. We also detected STK expression in bone-derived mouse osteoclasts. The addition of MSP to OCLs induced rapid morphologic changes such as cytoplasmic contraction and formation of ruffled border. In addition, MSP caused rapid redistribution of src to the borders of cytoplasm. These phenomena were associated with enhanced bone resorption. MSP caused a threefold increase in pit formation compared with control OCLs. These findings suggest that by involving src kinase, the MSP/STK signal transduction pathway induces rapid cytoskeletal reorganization in osteoclasts and facilitates bone resorption by osteoclasts.

Animals↗

PubMed1996

Predominant expression of a receptor tyrosine kinase, TIE, in hematopoietic stem cells and B cells.

A receptor tyrosine kinase (RTK), TIE (tyrosine kinase that contains immunoglobulin-like loops and epidermal growth factor [EGF] homology domains), is expressed in vascular endothelial and hematopoietic cells. We generated monoclonal antibodies (MoAbs) against the extracellular domain of TIE and a polyclonal antibody against the TIE carboxyterminus and used them to analyze expression of TIE in hematopoietic cells. Western blotting detected two forms of TIE protein with a molecular mass of 135 and 130 kD in hematopoietic and endothelial cells. Northern blotting analysis revealed that TIE was expressed preferentially in undifferentiated cell lines, especially when megakaryocytic, but not erythroid differentiation was induced. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that TIE was predominantly expressed in the human hematopoietic progenitor fraction, CD34+ cells. Fluorescence-activated cell sorting (FACS) showed that 42% of CD34+ and 17% of KIT-positive (KIT+) cells were TIE-positive (TIE+). The majority (81%) of the primitive hematopoietic stem cells, CD34+CD38- cells, were TIE+. Assays of progenitor cells and long-term culture-initiating cells (LTC-IC) showed that the TIE+ fraction contained more primitive cells than the TIE- fraction. Some TIE+ cells were in the CD34- fraction, which were CD19+ and CD20+ (B cells). These findings indicate that TIE has a unique spectrum of expression in primitive hematopoietic stem cells and B cells. Although its ligand has not been identified, TIE and its ligand may establish a novel regulatory pathway not only in early hematopoiesis, but also in the differentiation and/or proliferation of B cells.

Animals↗

PubMed1996

The receptor tyrosine kinase, Cek8, is transiently expressed on subtypes of motoneurons in the spinal cord during development.

Receptor tyrosine kinases (RTKs) play important roles in cellular proliferation, differentiation, and survival. We performed reverse transcriptase-polymerase chain reactions (RT-PCR) from enriched embryonic day 5 (E5) chick motoneurons by panning to identify RTKs involved in the early development of motoneuron. In situ hybridization revealed that Cek8, a member of the eph family, was specifically expressed on motoneurons at the brachial and lumbar segments of the spinal cord which innervate limb muscles, and disappeared after the naturally occurring cell death period (E6-E11). Immunohistochemistry using an anti-Cek8 monoclonal antibody showed the localization of Cek8 protein at the cell bodies and axonal fibers of motoneurons and muscles. The unique expression of Cek8 suggests its involvement in cellular survival or cell-cell interactions for specific subpopulations of developing motoneurons.

Amino Acid Sequence↗

PubMed1995

Terminal differentiation of murine resident peritoneal macrophages is characterized by expression of the STK protein tyrosine kinase, a receptor for macrophage-stimulating protein.

STK, a new member of the hepatocyte growth factor receptor family, is the receptor for macrophage-stimulating protein (MSP), which acts on murine resident peritoneal macrophages. We established polyclonal and monoclonal antibodies against STK and characterized the structure of STK protein and STK expression on cells of the mononuclear phagocyte system. Western blotting showed that the STK transcript is translated into a single-chain precursor and then cleaved into a 165-kD disulfide-linked heterodimer composed of a 35-kD alpha-chain and a 144-kD beta-chain. Western blotting detected STK protein on resident peritoneal macrophages, a target of MSP, and showed that it was autophosphorylated in cells stimulated by MSP. By flow cytometric analysis using a monoclonal anti-STK antibody, we showed that STK protein is expressed on restricted macrophage populations such as resident peritoneal macrophages, but not on exudate peritoneal macrophages or mononuclear phagocytes of the bone marrow, peripheral blood, spleen, or alveoli. Resident peritoneal macrophages were classified into two fractions according to their reactivity with an anti-STK antibody and a marker antibody for macrophages: STKhigh-F4/80high cells and STKnegative-F4/80low cells. Acute exudative macrophages were all STKnegative-F4/80low, but they gradually became predominantly STKhigh-F4/80high several days after entrance into the peritoneal cavity. These results showed that after monocytes migrate into the peritoneal cavity, they undergo terminal differentiation in the peritoneal microenvironment. This is the first evidence of tissue-specific terminal differentiation of peritoneal macrophages, and this terminal differentiation can be characterized by the expression of STK receptor tyrosine kinase.

Animals↗