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Biomedical subjects

Y Kitamura

Publications and source records attributed to Y Kitamura.

At least 199 records · Page 11Linked to original sources
PubMed1998

The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract.

Vascularization of organs generally occurs by remodelling of the preexisting vascular system during their differentiation and growth to enable them to perform their specific functions during development. The molecules required by early vascular systems, many of which are receptor tyrosine kinases and their ligands, have been defined by analysis of mutant mice. As most of these mice die during early gestation before many of their organs have developed, the molecules responsible for vascularization during organogenesis have not been identified. The cell-surface receptor CXCR4 is a seven-transmembrane-spanning, G-protein-coupled receptor for the CXC chemokine PBSF/SDF-1 (for pre-B-cell growth-stimulating factor/stromal-cell-derived factor), which is responsible for B-cell lymphopoiesis, bone-marrow myelopoiesis and cardiac ventricular septum formation. CXCR4 also functions as a co-receptor for T-cell-line tropic human immunodeficiency virus HIV-1. Here we report that CXCR4 is expressed in developing vascular endothelial cells, and that mice lacking CXCR4 or PBSF/SDF-1 have defective formation of the large vessels supplying the gastrointestinal tract. In addition, mice lacking CXCR4 die in utero and are defective in vascular development, haematopoiesis and cardiogenesis, like mice lacking PBSF/SDF-1, indicating that CXCR4 is a primary physiological receptor for PBSF/SDF-1. We conclude that PBSF/SDF-1 and CXCR4 define a new signalling system for organ vascularization.

Animals↗
PubMed1998

Systematic method to obtain novel genes that are regulated by mi transcription factor: impaired expression of granzyme B and tryptophan hydroxylase in mi/mi cultured mast cells.

The mi locus encodes a member of the basic-helix-loop-helix-leucine zipper protein family of transcription factors (hereafter called MITF). We have reported that the expression of several genes was impaired in cultured mast cells (CMCs) of mi/mi genotype, and demonstrated the involvement of MITF in the transcription of these genes. To obtain new genes whose transcription may be regulated by MITF, we prepared a subtracted cDNA library using +/+ and mi/mi CMCs. We found two clones carrying the granzyme (Gr) B and tryptophan hydroxylase (TPH) cDNAs in the subtracted library. The expression of the Gr B and TPH genes decreased in mi/mi CMCs, and recovered to nearly normal level by the overexpression of normal (+) MITF but not of mutant (mi) MITF. The +-MITF bound three and one CANNTG motifs in the Gr B and TPH promoters, respectively, and transactivated these two genes, indicating the involvement of +-MITF in their expression. Because TPH is the rate-limiting enzyme for serotonin synthesis, we examined the serotonin content of +/+ and mi/mi CMCs. The serotonin content was significantly smaller in mi/mi CMCs than in +/+ CMCs. The introduction of +-MITF but not of mi-MITF normalized the serotonin content in mi/mi CMCs.

Animals↗
PubMed1998

Retroviral vector targeting human cells via c-Kit-stem cell factor interaction.

Targeted gene transfer into hematopoietic stem cells by retroviral vectors would greatly facilitate the development of in vivo strategies for stem cell gene therapy. We engineered a recombinant retroviral vector that can target human cells expressing a c-Kit receptor via a ligand-receptor interaction. The ecotropic (Moloney murine leukemia virus) envelope protein was modified by insertion of a sequence encoding the N-terminal 161 amino acids of murine stem cell factor (mSCF), the ligand for murine c-Kit. The chimeric envelope protein was correctly processed and incorporated into viral particles as efficiently as the wild-type envelope protein. Virions pseudotyped with the chimeric envelope proteins bound to 293 cells expressing murine c-Kit (293KIT) preferentially; however, they could not transduce any c-Kit-positive cells under conventional conditions. They could transduce 293KIT cells in the presence of chloroquine, and HEL cells expressing human c-Kit on a fibronectin fragment (CH296)-coated dish. The fact that recombinant mSCF in the medium at the time of transduction greatly reduced the efficiency of both gene deliveries implies that the vector utilized the mSCF-c-Kit interaction for the initial step of transduction in either case. The vector may prove useful for targeting cells expressing c-Kit on their surface.

Cells, Cultured↗
PubMed1998

Delayed onset of hemolytic anemia in CBA-Pk-1slc/Pk-1slc mice with a point mutation of the gene encoding red blood cell type pyruvate kinase.

The Pk-1slc gene encodes a mutant red blood cell (RBC) type pyruvate kinase (PK), and adult CBA-Pk-1slc/Pk-1slc mice show a severe nonspherocytic hemolytic anemia. However, the number of RBCs and the proportion of reticulocytes were comparable between neonatal CBA-Pk-1slc/Pk-1slc mice and control -+/+ mice. Since the age-dependent increase of RBCs was much greater in CBA-+/+ mice than in CBA-Pk-1slc/Pk-1slc mice, significant anemia was observed in the latter mice on day 14 after birth. The increase of RBCs in CBA-+/+ mice was due to the prolongation of their survival time. The half life of RBCs increased in CBA-+/+ mice with ages, but it decreased in CBA-Pk-1slc/Pk-1slc mice. The relatively longer half life of RBCs in neonatal CBA-Pk-1slc/Pk-1slc mice appeared to be due to the delayed switching from M2-type PK that are expressed by undifferentiated erythroid precursor cells to RBC-type PK that are expressed by mature RBCs.

Age of Onset↗
PubMed1998

Strand specificity and absence of hot spots for p53 mutations in ultraviolet B-induced skin tumors of XPA-deficient mice.

We examined the spectrum of p53 mutations found in 40 UV-induced skin tumors of xeroderma pigmentosum group A gene (XPA)-deficient mice. p53 mutations were detected in 48% of the tumors. Nearly all of the mutations were induced at dipyrimidine sites. Ninety-three % of the mutations were G.C-->A.T transitions at dipyrimidine sites, including tandem transitions (CC-->TT), which are the hallmark of the UVB-induced mutation. Seventy-two % of the mutations at dipyrimidine sites could be ascribed to damage on the transcribed strand. In addition, no evident mutational hot spots were detected. This is in contrast to the UVB-induced skin tumors of normal mice, in which 92% of p53 mutations occurred as a result of DNA damage on the nontranscribed strand, and clear hot spots were observed. Thus, XPA-deficient mice showed significant mutation features that might be characteristic of the absence of nucleotide excision repair and may provide a good animal model for the analysis of the high incidence of skin cancer in xeroderma pigmentosum group A patients.

Animals↗
PubMed1998

Metabolism of ferulic acid to vanillin. A bacterial gene of the enoyl-SCoA hydratase/isomerase superfamily encodes an enzyme for the hydration and cleavage of a hydroxycinnamic acid SCoA thioester.

A gene encoding a novel enoyl-SCoA hydratase/lyase enzyme for the hydration and nonoxidative cleavage of feruloyl-SCoA to vanillin and acetyl-SCoA was isolated and characterized from a strain of Pseudomonas fluorescens. Feruloyl-SCoA is the CoASH thioester of ferulic acid (4-hydroxy-3-methoxy-trans-cinnamic acid), an abundant constituent of plant cell walls and a degradation product of lignin. The gene was isolated by a combination of mutant complementation and biochemical approaches, and its function was demonstrated by heterologous expression in Escherichia coli under the control of a T7 RNA polymerase promoter. The gene product is a member of the enoyl-SCoA hydratase/isomerase superfamily.

Acetyl Coenzyme A↗
PubMed1998

Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the human digestive tract, but their molecular etiology and cellular origin are unknown. Sequencing of c-kit complementary DNA, which encodes a proto-oncogenic receptor tyrosine kinase (KIT), from five GISTs revealed mutations in the region between the transmembrane and tyrosine kinase domains. All of the corresponding mutant KIT proteins were constitutively activated without the KIT ligand, stem cell factor (SCF). Stable transfection of the mutant c-kit complementary DNAs induced malignant transformation of Ba/F3 murine lymphoid cells, suggesting that the mutations contribute to tumor development. GISTs may originate from the interstitial cells of Cajal (ICCs) because the development of ICCs is dependent on the SCF-KIT interaction and because, like GISTs, these cells express both KIT and CD34.

Amino Acid Sequence↗
PubMed1998

Alteration of proteins regulating apoptosis, Bcl-2, Bcl-x, Bax, Bak, Bad, ICH-1 and CPP32, in Alzheimer's disease.

Recently, apoptosis has been implicated in the selective neuronal loss of Alzheimer's disease (AD). Apoptosis is regulated by the B cell leukemia-2 gene product (Bcl-2) family (Bcl-2, Bcl-x, Bax, Bak and Bad) and the caspase family (ICH-1 and CPP32), with apoptosis being prevented by Bcl-2 and Bcl-x, and promoted by Bax, Bak, Bad, ICH-1 and CPP32. In the present study, we examined the levels of these proteins in the membranous and cytosolic fractions of temporal cortex in AD and control brain. In the membranous fraction, the levels of Bcl-2 alpha, Bcl-xL, Bcl-x beta, Bak and Bad were increased in AD. In the cytosolic fractions, the level of Bcl-x beta was increased, while Bcl-xL, Bax, Bak, and Bad and ICH-1L were unchanged. CPP32 was not detected in AD or control brain. These findings demonstrate a differential involvement of cell death-regulatory proteins in AD and suggest that Bak, Bad, Bcl-2 and Bcl-x are upregulated in AD brains.

Age Factors↗
PubMed1998

Bafilomycin A1 induces apoptosis in the human pancreatic cancer cell line Capan-1.

Bafilomycin A1, a specific inhibitor of vacuolar type H(+)-ATPase, can inhibit the growth of a variety of cultured cells in a dose-dependent manner, but its mechanism is unclear. The aim of this study was to examine whether bafilomycin A1 inhibits the growth of Capan-1 human pancreatic cancer cells through apoptosis. The effect of bafilomycin A1 on tumour growth in vitro and in vivo was examined using an MTT assay and an in vivo tumour model. The presence or absence of apoptosis was determined by morphology and DNA analysis of tumour cells. The concentration of bafilomycin A1 for 50 per cent inhibition of cell viability during 72 h by the MTT assay was 5 nm. In DNA analysis, a ladder of fragmented DNA was detected in Capan-1 cells treated with bafilomycin A1 at concentrations greater than 10 nm for 24 h. Nude mice bearing a xenografted Capan-1 cell line tumour received 4 weeks of bafilomycin A1 (1.0 mg/kg per day). This treatment significantly inhibited tumour growth compared with controls after 21 days (P < 0.05). Histopathological examination of tumour cells in the treated group demonstrated signs of apoptosis with chromatin condensation and cell shrinkage. These observations suggest that bafilomycin A1 inhibits the growth of Capan-1 human pancreatic cancer cells through apoptosis.

Adenocarcinoma↗
PubMed1998

Mechanism of selective motor neuronal death after exposure of spinal cord to glutamate: involvement of glutamate-induced nitric oxide in motor neuron toxicity and nonmotor neuron protection.

In this study, we analyzed the mechanism of selective motor neuronal death, a characteristic of amyotrophic lateral sclerosis, using embryonic rat spinal cord culture. When dissociated cultures were exposed to low-level glutamate (Glu) coadministered with the Glu transporter inhibitor L-trans-pyrrolidine-2,4-decarboxylate (PDC) for 24 hours, motor neurons were selectively injured through N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptors. Nitric oxide synthase (NOS) inhibitors attenuated this toxicity, and long-acting nitric oxide (NO) donors damaged motor neurons selectively. Nonmotor neurons survived after exposure to low-dose Glu/PDC, but Glu-induced toxicity was potentiated by coadministration of an NO-dependent guanylyl cyclase inhibitor. In addition, 8-bromo-cyclic GMP, a soluble cyclic GMP analogue, rescued nonmotor neurons, but not motor neurons, exposed to high-dose Glu/PDC. Twenty-four hours' incubation with PDC elevated the number of neuronal NOS-immunoreactive neurons by about twofold compared with controls, and a double-staining study, using the motor neuron marker SMI32, revealed that most of them were nonmotor neurons. These findings suggest that selective motor neuronal death caused by chronic low-level exposure to Glu is mediated by the formation of NO in nonmotor neurons, which inversely protects nonmotor neurons through the guanylyl cyclase-cyclic GMP cascade. Induction of neuronal NOS in nonmotor neurons might enhance both the toxicity of motor neurons and the protection of nonmotor neurons, which could explain the pathology of amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis↗
PubMed1998

Expression of E-cadherin, alpha-catenin, beta-catenin, and CD44 (standard and variant isoforms) in human cholangiocarcinoma: an immunohistochemical study.

Immunolocalization of E-cadherin (E-cad), alpha-catenin, beta-catenin, and CD44 has rarely been investigated in human cholangiocarcinoma (CC). We, therefore, immunohistochemically examined the expression of E-cad, alpha-catenin, beta-catenin, CD44 standard (CD44s), and CD44 variants (CD44v) including CD44v5, CD44v6, CD44v7-8, and CD44v10 in normal adult livers and in 47 cases of CC; and the results were then correlated with tumor grade, vascular invasion, metastasis, p53 expression, proliferative fraction (Ki-67 labeling), and c-erbB2 expression. In normal livers, E-cad, alpha-catenin and beta-catenin, but not CD44s, CD44v5, CD44v6, CD44v7-8, and CD44v10, were expressed at the cell membrane of normal intrahepatic bile ducts. In CC, membranous expression of E-cad, alpha-catenin, and beta-catenin was the same or reduced when compared with non-cancerous bile ducts in the majority of CC. We found that the down-regulation of E-cad, alpha-catenin, and beta-catenin expression significantly correlated with tumor high grade, but not with vascular invasion, metastasis, p53 expression, Ki-67 labeling, or c-erbB2 expression, except for beta-catenin, the down-regulation of which was associated with c-erbB2 down-regulation. CD44s, CD44v5, CD44v6, CD44v7-8 and CD44v10 were frequently expressed at the membrane of CC cells. There were, however, no significant correlations between these aberrant CD44 expression and tumor grade, metastasis, vascular invasion, p53 expression, Ki-67 labeling, or c-erbB2 expression, with a few exceptions of CD44s and CD44v5. We found that CD44s aberrant expression significantly correlated with absence of metastasis and vascular invasion, and that CD44v5 aberrant expression significantly correlated with p53 under-expression. These results suggest that membranous expression of E-cad, alpha-catenin, and beta-catenin is reduced in a majority of CC and this down-regulation correlates with CC high grade, and that beta-catenin down-regulation is associated with c-erbB2 down-regulation. The data also suggested that CD44s, CD44v5, CD44v6, CD44v7-8, and CD44v10 may be neoexpressed during carcinogenesis of CC but this neoexpression does not correlate with tumor progression in CC, with the exception of CD44s and CD44v5.

Adult↗
PubMed1998

Differential in situ expression of alpha2(XI) collagen mRNA isoforms in the developing mouse.

Type XI collagen is an essential structural component of the extracellular matrix of cartilage and plays a role in collagen fibril formation and skeletal morphogenesis. The expression of all three type XI collagen genes is not restricted to cartilage. In addition, alternative exon usage seems to increase the structural diversity and functional potential of type XI collagen during development. In order to investigate type XI collagen expression during development, we have examined alpha2(XI) and alpha1(XI) collagen genes by in situ hybridization in mice. Transcripts of the alpha2(XI) collagen gene were first detected in the notochord of mouse embryos after 11.5 days of gestation. Subsequently, alpha2(XI) mRNA was mainly found in the cartilaginous tissues of the developing limbs and axial skeleton together with transcripts of the alpha1(XI) gene. The alpha2(XI) transcripts seemed to be alternatively spliced isoforms lacking exons 6-8, which code for an acidic domain. Expression of alpha2(XI) outside the cartilage was relatively restricted, whereas expression of the alpha1(XI) gene was widespread. However, expression of alpha2(XI) transcripts containing exons 6-8 was found in non-chondrogenic tissues, including the calvarium and periosteum where intramembranous ossification occurs. These results indicate that alpha2(XI) mRNA isoforms are differentially expressed in various tissues during development. In addition, alpha2(XI) mRNA isoforms containing alternative exons are present in osteogenic cells, and their expression may be closely related to the formation of bone or cartilage.

Alternative Splicing↗
PubMed1998

Expression of epithelial-cadherin, alpha-catenin and beta-catenin during human intrahepatic bile duct development: a possible role in bile duct morphogenesis.

BACKGROUND/AIMS: Cell adhesion molecules play an important role in the morphogenesis of developing organs. However, little is known about their expression during intrahepatic bile duct development. METHODS: We immunohistochemically investigated the expression of E-cadherin (E-Cad), alpha-catenin (A-Cat) and beta-catenin (B-Cat) during human intrahepatic bile duct development, using 31 fetal livers of various gestational ages. The developmental stages of bile ducts were classified into the ductal plate, migrating biliary cells (remodeling stage), and immature bile ducts (remodeled stage). RESULTS: E-Cad was broadly and strongly expressed in the ductal plate with a cytoplasmic pattern, heterogeneously and weakly expressed in the migrating biliary cells with a cytoplasmic pattern, and broadly and strongly expressed in immature bile ducts with a membranous pattern. A-Cat was broadly and strongly expressed in the ductal plate with a membrane pattern, broadly and moderately expressed in the migrating biliary cells with a membranous pattern, and broadly and strongly expressed in immature bile ducts with a membranous pattern. In contrast, expression of B-Cat was weak or slight in the ductal plate, but B-Cat was expressed broadly and strongly with a membranous pattern in migrating biliary cells and in immature bile ducts. Immature hepatocytes rarely expressed E-Cad and A-Cat, but expressed B-Cat with a membranous pattern throughout development. CONCLUSIONS: These results suggest that E-Cad, A-Cat and B-Cat are involved in the normal developmental morphogenesis of human intrahepatic bile ducts.

Bile Ducts, Intrahepatic↗
PubMed1998

Alteration of protease expression phenotype of mouse peritoneal mast cells by changing the microenvironment as demonstrated by in situ hybridization histochemistry.

Mouse mast cell protease (MMCP) mRNA expression was examined by in situ hybridization histochemistry. Peritoneal mast cells (PMCs) of WBB6F1-(+/+) mice expressed MMCP-2, MMCP-4, MMCP-5, and MMCP-6 mRNAs, but did not express MMCP-1 mRNA. When proliferation of PMCs was induced by culturing them in methylcellulose with T cell-derived cytokines, cells in mast cell colonies expressed MMCP-1 mRNA. These mast cells were transferred to a suspension culture to induce further proliferation. The phenotype of the resulting PMC-derived cultured mast cells was similar to that of bone marrow-derived cultured mast cells. When 10(5) PMC-derived cultured mast cells or 10(5) bone marrow-derived cultured mast cells were injected into the stomach wall of mast cell-deficient WBB6F1-W/Wv mice, mast cells that appeared in the mucosa and muscularis propria were similar to mast cells in the stomach of intact WBB6F1-(+/+) mice, indicating the injected cells adapted to a new tissue environment. In contrast, when 10(5) PMCs were injected into the stomach wall of WBB6F1-W/Wv mice, the injected PMCs did not adapt to the mucosa. When 20 PMCs were injected, they proliferated and adapted to the mucosal environment. The present results suggest that PMCs adapt to new environments when proliferation occurs before redifferentiation.

Animals↗
PubMed1998

A novel gain-of-function mutation of c-kit gene in gastrointestinal stromal tumors.

BACKGROUND & AIMS: The c-kit gene encodes a receptor tyrosine kinase (KIT). Recently, we found gain-of-function mutations of the c-kit gene in gastrointestinal stromal tumors (GISTs). All mutations were confined within the 11 amino acids (Lys-550 to Val-560) in the juxtamembrane domain, but one GIST showed a novel deletion-type mutation at codon 579 (Asp) in the juxtamembrane domain. The aim of this study was to clarify whether the mutation is activating. METHODS: Mutant c-kit cDNA was transfected into an interleukin 3 (IL-3)-dependent Ba/F3 murine lymphoid cell line, and the magnitude of autophosphorylation of the mutant KIT was examined with or without stem cell factor (SCF), a ligand of KIT. An in vitro kinase assay was also performed. The biological behavior of the transfectant was estimated by both an in vitro proliferation assay and in vivo transplantation to nude mice. RESULTS: The mutant KIT exhibited constitutive phosphorylation and strong kinase activity without SCF. The transfectant grew autonomously without IL-3 and SCF, and it formed tumors in nude mice. CONCLUSIONS: Deletion at codon 579 (Asp) in the juxtamembrane domain of the c-kit gene is a novel gain-of-function mutation other than the region between Lys-550 and Val-560.

Amino Acid Sequence↗
PubMed1998

Induction of inducible nitric oxide synthase and heme oxygenase-1 in rat glial cells.

Recent observations suggest a possible interaction between the nitric oxide (NO)/NO synthases and carbon monoxide (CO)/heme oxygenases systems. We examined the effects of lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), and NO donor such as S-nitroso-N-acetylpenicillamine (SNAP) on induction of inducible NO synthase (iNOS) and heme oxygenase-1 (HO-1) in mixed glial cells and in rat hippocampus. In in vitro glial cells, treatment with LPS induced the expression of 130-kDa iNOS after 6 h, and NO2- accumulation and enhancement of the protein level of 33-kDa HO-1 after 12 h. In addition, treatment with SNAP induced HO-1 expression after 6 h. Although a NOS inhibitor, such as N(G)-nitro-L-arginine (NNA), did not change LPS-induced iNOS expression, the inhibitor suppressed both NO2- accumulation and the enhancement of HO-1. Immunocytochemistry showed that LPS-treatment induced iNOS-immunoreactivity predominantly in microglia, while this treatment induced HO-1-immunoreactivity in both microglia and astrocytes. These results suggest that endogenous NO production by iNOS in microglia causes autocrine- and paracrine-induction of HO-1 protein in microglia and astrocytes in rat brain.

Animals↗