Biomedical subjects

M Noda

Publications and source records attributed to M Noda.

At least 217 records · Page 12Linked to original sources

PubMed1998

Enhancement of gene expression under hypoxic conditions using fragments of the human vascular endothelial growth factor and the erythropoietin genes.

PURPOSE: Selective gene expression in response to tumor hypoxia may provide new avenues, not only for radiotherapy and chemotherapy, but also for gene therapy. In this study, we have assessed the extent of hypoxia responsiveness of various DNA constructs by the luciferase assay to help design vectors suitable for cancer therapy. MATERIALS AND METHODS: Reporter plasmids were constructed with fragments of the human vascular endothelial growth factor (VEGF) and the erythropoietin (Epo) genes encompassing the putative hypoxia-responsive elements (HRE) and the pGL3 promoter vector. Test plasmids and the control pRL-CMV plasmid were cotransfected into tumor cells by the calcium phosphate method. After 6 h hypoxic treatment, the reporter assay was performed. RESULTS: The construct pGL3/VEGF containing the 385 bp fragment of the 5' flanking region in human VEGF gene showed significant increases in luciferase activity in response to hypoxia. The hypoxic/aerobic ratios were about 3-4, and 8-12 for murine and human tumor cells, respectively. Despite the very high degree of conservation among the HREs of mammalian VEGF genes, murine cells showed lower responsiveness than human cells. We next tested the construct pGL3/Epo containing the 150 bp fragment of the 3' flanking region in the Epo gene. Luciferase activity of pGL3/Epo was increased with hypoxia only in human cell lines. The insertion of 5 copies of the 35-bp fragments derived from the VEGF HREs and 32 bp of the E1b minimal promoter resulted in maximal enhancement of hypoxia responsiveness. CONCLUSIONS: The constructs with VEGF or Epo fragments containing HRE may be useful for inducing specific gene expression in hypoxic cells. Especially, the application of multiple copies of the HREs and an E1b minimal promoter appears to have the advantage of great improvement in hypoxia responsiveness.

Animals↗

PubMed1998

Regulation of matrix metalloproteinase-9 and inhibition of tumor invasion by the membrane-anchored glycoprotein RECK.

A human fibroblast cDNA expression library was screened for cDNA clones giving rise to flat colonies when transfected into v-Ki-ras-transformed NIH 3T3 cells. One such gene, RECK, encodes a membrane-anchored glycoprotein of about 110 kDa with multiple epidermal growth factor-like repeats and serine-protease inhibitor-like domains. While RECK mRNA is expressed in various human tissues and untransformed cells, it is undetectable in tumor-derived cell lines and oncogenically transformed cells. Restored expression of RECK in malignant cells resulted in suppression of invasive activity with concomitant decrease in the secretion of matrix metalloproteinase-9 (MMP-9), a key enzyme involved in tumor invasion and metastasis. Moreover, purified RECK protein was found to bind to, and inhibit the proteolytic activity of, MMP-9. Thus, RECK may link oncogenic signals to tumor invasion and metastasis.

3T3 Cells↗

PubMed1998

In vivo phosphorylation of poly(ADP-ribose) polymerase is independent of its activation.

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme, which is activated by DNA strand breaks. Although PARP is known to be cleaved by the cysteine protease, caspase-3/CPP32, during apoptosis, signal cascade which regulates the PARP activity has not been fully understood. In this study, we investigated post-translational modification of PARP. We found that PARP was phosphorylated by a serine kinase in vivo. PARP was activated temporarily and extensive auto-modification occurred on PARP, possibly by the fragmented DNA during apoptosis induced by etoposide in Jurkat cells. However, the phosphorylation level was not changed for up to 6 h, after PARP cleavage began in apoptosis by the treatment with etoposide. Furthermore, we showed the presence of a PARP-associated kinase in nuclear extracts of the HTLV-I infected T-cell lines but not in uninfected T-cell lines, whereas this kinase did not inhibit the PARP activity even in the presence of ATP. Taken together, in vivo phosphorylation of PARP might be independent of the activation or cleavage of PARP.

Cell Line↗

PubMed1998

Cloning and characterization of the gene encoding the murine glycoprotein V: the conserved thrombin-cleavable protein on platelet surface.

Glycoprotein V (GPV) is a platelet membrane protein present as a subunit of the GPIb/V/IX complex, a major receptor for von Willebrand factor, and is specifically cleaved by thrombin. In this study, we have cloned and characterized murine GPV gene. The entire coding sequence of murine GPV consisted of 1704 nucleotides and coded 567 amino acids, which were 70% identical with human GPV. Fifteen leucine-rich tandem repeats were present and the consensus sequence of the repeats was completely matched with that of human GPV. The thrombin-cleavage site was also conserved exactly at the same position. In Northern blot, murine GPV mRNA was specifically expressed in murine platelets, bone marrow cells and megakaryocytic cell lines. In the survey of other organs, GPV was not expressed at all. These results demonstrate that GPV is highly conserved, thrombin-cleavable protein beyond the species, and is a specific protein in the platelet-megakaryocyte lineage.

Amino Acid Sequence↗

PubMed1998

Fibroblast growth factor downregulates expression of a basic helix-loop-helix-type transcription factor, scleraxis, in a chondrocyte-like cell line, TC6.

Scleraxis is a basic helix-loop-helix-type transcription factor that is expressed in sclerotome. Fibroblast growth factor (FGF) is one of the cytokines produced by the cells in skeletal tissues and is a potent modulator of skeletogenesis. The aim of this study was to examine the effects of FGF on the expression of scleraxis in chondrocyte-like cells, TC6. In these cells, scleraxis mRNA was constitutively expressed as a 1 .2 kb message at a high level in contrast to its low levels of expression in fibroblast-like cells or osteoblast-like cells. Upon treatment with FGF, scleraxis mRNA level was decreased within 12 h. This effect was at its nadir at 24 h and the scleraxis mRNA level returned to its base line level by 48 h. The FGF effect was maximal at 1 ng/ml. FGF effects on scleraxis were blocked by actinomycin D but not by cycloheximide, suggesting the involvement of transcriptional events that do not require new protein synthesis. The FGF effects on scleraxis were blocked by genistein, suggesting the involvement of tyrosine kinase in the post-receptor signaling. TGFbeta treatment of TC6 cells enhanced scleraxis mRNA expression; however, combination of the saturation doses of FGF and TGFbeta resulted in suppression of scleraxis mRNA level. BMP2 also suppressed scleraxis mRNA expression in TC6 cells and no further suppression was observed in combination with FGF. These results indicate that scleraxis is expressed in chondrocyte-like TC6 cells and it is one of the targets of FGF action in these cells.

Basic Helix-Loop-Helix Proteins↗

PubMed1998

Ethidium bromide-induced inhibition of mitochondrial gene transcription suppresses glucose-stimulated insulin release in the mouse pancreatic beta-cell line betaHC9.

Recently, a mitochondrial mutation was found to be associated with maternally inherited diabetes mellitus (Kadowaki, T., Kadowaki, H., Mori, Y., Tobe, K., Sakuta, R., Suzuki, Y., Tanabe, Y, Sakura, H., Awata, T., Goto, Y., Hayakawa, T., Matsuoka, K., Kawamori, R., Kamada, T., Horai, S., Nonaka, I., Hagura, R., Akanuma, Y., and Yazaki, Y. (1994) N. Engl. J. Med. 330, 962-968). In order to elucidate its etiology, we have investigated the involvement of mitochondrial function in insulin secretion. Culture of the pancreatic beta-cell line, betaHC9, with low dose ethidium bromide (EB) (0.4 microg/ml) for 2-6 days resulted in a substantial decrease in the transcription level of mitochondrial DNA (to 10-20% of the control cells) without changing its copy number, whereas the transcription of nuclear genes was grossly unaffected. Electron microscopic analysis revealed that treatment by EB caused morphological changes only in mitochondria and not in other organelles such as nuclei, endoplasmic reticula, Golgi bodies, or secretory granules. When the cells were treated with EB for 6 days, glucose (20 mM) could no longer stimulate insulin secretion, while glibenclamide (1 microM) still did. When EB was removed after 3- or 6-day treatment, mitochondrial gene transcription recovered within 2 days, and the profiles of insulin secretion returned to normal within 7 days. Studies with fura-2 indicated that in EB-treated cells, glucose (20 mM) failed to increase intracellular Ca2+, while the effect of glibenclamide (1 microM) was maintained. Our system provides a unique way to investigate the relationship between mitochondrial function and insulin secretion.

Animals↗

PubMed1998

Transcription factors and osteoblasts.

Transcription factors play a key role in determination of the fate of the cells in osteoblastic and chondrocytic lineage. A runt family member, Cbfa, is indispensable for osteoblastic differentiation. Sox 9 and scleraxis are involved in the phenotypic expression in chondrocytes and the cells of early stage connective tissues. These transcription factors will give us a clue to unravel interaction of these known and yet unknown transcription factors to fully understand the mechanisms of skeletal cells ' differentiation and regulation of their functions.

Animals↗

PubMed1998

Involvement of receptor-like protein tyrosine phosphatase zeta/RPTPbeta and its ligand pleiotrophin/heparin-binding growth-associated molecule (HB-GAM) in neuronal migration.

Pleiotrophin/heparin-binding growth-associated molecule (HB-GAM) is a specific ligand of protein tyrosine phosphatase zeta (PTPzeta)/receptor-like protein tyrosine phosphatase beta (RPTPbeta) expressed in the brain as a chondroitin sulfate proteoglycan. Pleiotrophin and PTPzeta isoforms are localized along the radial glial fibers, a scaffold for neuronal migration, suggesting that these molecules are involved in migratory processes of neurons during brain development. In this study, we examined the roles of pleiotrophin-PTPzeta interaction in the neuronal migration using cell migration assay systems with glass fibers and Boyden chambers. Pleiotrophin and poly-L-lysine coated on the substratums stimulated cell migration of cortical neurons, while laminin, fibronectin, and tenascin exerted almost no effect. Pleiotrophin-induced and poly-L-lysine-induced neuronal migrations showed significant differences in sensitivity to various molecules and reagents. Polyclonal antibodies against the extracellular domain of PTPzeta, PTPzeta-S, an extracellular secreted form of PTPzeta, and sodium vanadate, a protein tyrosine phosphatase inhibitor, added into the culture medium strongly suppressed specifically the pleiotrophin-induced neuronal migration. Furthermore, chondroitin sulfate C but not chondroitin sulfate A inhibited pleiotrophin-induced neuronal migration, in good accordance with our previous findings that chondroitin sulfate constitutes a part of the pleiotrophin-binding site of PTPzeta, and PTPzeta-pleiotrophin binding is inhibited by chondroitin sulfate C but not by chondroitin sulfate A. Immunocytochemical analysis indicated that the transmembrane forms of PTPzeta are expressed on the migrating neurons especially at the lamellipodia along the leading processes. These results suggest that PTPzeta is involved in the neuronal migration as a neuronal receptor of pleiotrophin distributed along radial glial fibers.

Animals↗

PubMed1998

The winged-helix transcription factor CWH-3 is expressed in developing neural crest cells.

Neural crest cells are a migratory population that finally give rise to peripheral neurons and numerous other differentiated cell types. We report here that chicken winged-helix 3 (CWH-3), a winged-helix transcription factor, is specifically expressed in the early premigratory and migrating neural crest cells at all axial levels in chick embryos. CWH-3-positive cells first appeared in the neural plate at the early primitive-streak stage when commitment to the neural crest cell fate occurs, and the expression declined after they arrived at their final destinations. These observations suggested that CWH-3 is a determinant for neural crest differentiation at the most dorsal part of the neural tube, reminiscent of HNF-3beta, another winged-helix factor, which determines the ventral part of the neural tube.

Animals↗

PubMed1998

Inhibition of M-type K+ current by linopirdine, a neurotransmitter-release enhancer, in NG108-15 neuronal cells and rat cerebral neurons in culture.

The effect of linopirdine, a neurotransmitter-release enhancer, on the M-type K+-current, IK(M), was examined in NGPM1-27 cells, mouse neuroblastomaxrat glioma NG108-15 cells transformed to express m1-muscarinic acetylcholine (ACh) receptors, using the nystatin-perforated patch-recording mode under voltage-clamp conditions. The application of linopirdine induced the inward current associated with an inhibition of IK(M), which mimics an excitatory part of the ACh-induced responses in NGPM1-27 cells. The affinity of linopirdine for the inhibition of IK(M) was 24.7 microM in NGPM1-27 cells. In the presence of linopirdine, ACh failed to evoke a further inward current, but ACh still elicited an outward current, thus suggesting that the Ca2+-dependent K+ current is rather insensitive to linopirdine. Linopirdine also inhibited another voltage-gated potassium current (IK(V)) at the concentration of 72.3 microM. Finally, the inhibitory effect of linopirdine on IK(M) was confirmed in pyramidal neurons acutely dissociated from the rat cerebral cortex at 35.8 microM. The results suggest that linopirdine is thus considered to be an inhibitor of some type of K+ channels in both NGPM1-27 cells and the rat cerebral neurons.

Acetylcholine↗

PubMed1998

Neurons as well as astrocytes express proteoglycan-type protein tyrosine phosphatase zeta/RPTPbeta: analysis of mice in which the PTPzeta/RPTPbeta gene was replaced with the LacZ gene.

PTPzeta/RPTPbeta is a receptor-like protein tyrosine phosphatase expressed as a chondroitin sulfate proteoglycan. We generated mice in which the PTPzeta gene was replaced by the LacZ gene by gene targeting. Analysis of heterozygous PTPzeta-targeted mice allowed us to identify PTPzeta-producing cells during development by examining expression of the LacZ gene. LacZ expression was detected only in the central nervous system throughout development from embryonic day 8.5. In the postnatal period, subsets of neurons and astrocytes in the brain, including pyramidal cells and astrocytes in the hippocampus, expressed LacZ. Primary cultures of cells from the cerebral cortex of embryonic day 16 mice also indicated that both neurons and astrocytes were positive for LacZ. These results indicated that neurons and astrocytes express PTPzeta.

Animals↗

PubMed1998

Cystic fibrosis transmembrane conductance regulator mediates sulphonylurea block of the inwardly rectifying K+ channel Kir6.1.

1. Recombinant ATP-sensitive K+ channels (KATP channels) were heterologously expressed in the NIH3T3 mouse cell line, and the electrophysiological properties were studied using patch-clamp techniques. 2. The NIH3T3 cell lines transfected with the inwardly rectifying K+ channel Kir6.1 alone or with both Kir6.1 and cystic fibrosis transmembrane conductance regulator (CFTR) exhibited time-independent K+ currents with weak inward rectification. In contrast, no measurable K+ conductance was observed in mock-transfected cells or in cells transfected with CFTR alone. Regardless of co-transfection with Kir6.1, the transfection with CFTR produced a Cl- conductance that was activated by cell dialysis with cAMP (1 mM). The conductance was reversibly suppressed by glibenclamide (30 microM). 3. Whole-cell currents at +60 mV were blocked in a concentration-dependent manner by Ba2+ ions with similar IC50 values: 89.3 +/- 23.3 microM (Kir6.1 alone) and 67.3 +/- 24.9 microM (Kir6.1-CFTR). 4. The currents recorded from Kir6. 1-transfected cells were not affected by glibenclamide, whereas glibenclamide did inhibit the conductance expressed in cells co-transfected with CFTR (IC50 = 35.9 +/- 6.6 microM). 5. In the cell-attached mode with a 150 mM K+ pipette solution, both Kir6.1- and Kir6.1-CFTR-transfected cells displayed a class of K+ channels showing weak inward rectification and a slope conductance of 50.7 +/- 1.0 and 52.4 +/- 4.9 pS, respectively. 6. In the inside-out mode, the single-channel currents recorded from both types of cells were not inhibited by intracellular ATP (1 mM). However, glibenclamide was found to block the single-channel activities in the co-transfected cells.

3T3 Cells↗

PubMed1998

Muscarinic acetylcholine receptor subtypes in the human iris.

Employing subtype-specific antisera, we measured the relative immunoreactivity of five muscarinic acetylcholine receptor (mAChR) subtype proteins (m1-m5) in the human iris. The most intensive FITC immunofluorescence was detected by the anti-m3 antibody, followed by anti-m1 and -m5 antisera, in the iris sphincter muscle cells. Only very weak fluorescence was obtained by anti-m2 and -m4 antibodies. In dilator muscle cells, weak but not consistent immunoreactivity was found by anti-m1 and -m5 antibodies. The results suggest that the m3 muscarinic receptor is the predominant subtype in sphincter muscle cells of the human iris.

Adult↗

PubMed1998

Intestinal trefoil factor controls the expression of the adenomatous polyposis coli-catenin and the E-cadherin-catenin complexes in human colon carcinoma cells.

Intestinal trefoil factor 3 (TFF3) is a member of the trefoil family of peptides, small molecules constitutively expressed in epithelial tissues, including the gastrointestinal tract. TFF3 has been shown to promote migration of intestinal epithelial cells in vitro and to enhance mucosal healing and epithelial restitution in vivo. In this study, we evaluated the effect of recombinant TFF3 (rTFF3) stimulation on the expression and cellular localization of the epithelial (E)-cadherin-catenin complex, a prime mediator of Ca2+ dependent cell-cell adhesion, and the adenomatous polyposis coli (APC)-catenin complex in HT29, HCT116, and SW480 colorectal carcinoma cell lines. Stimulation by rTFF3 (10(-9) M and 10(-8) M) for 20-24 hr led to cell detachment and to a reduction in intercellular adhesion in HT29 and HCT116 cells. In both cell lines, E-cadherin expression was down-regulated. The expression of APC, alpha-catenin and beta-catenin also was decreased in HT29 cells, with a translocation of APC into the nucleus. No change in either cell adhesion or in the expression of E-cadherin, the catenins, and APC was detected in SW480 cells. In addition, TFF3 induced DNA fragmentation and morphological changes characteristic of apoptosis in HT29. Tyrphostin, a competitive inhibitor of protein tyrosine kinases, inhibited the effects of TFF3. Our results indicate that by perturbing the complexes between E-cadherin, beta-catenin, and associated proteins, TFF3 may modulate epithelial cell adhesion, migration, and survival.

Adenomatous Polyposis Coli Protein↗

PubMed1998

Characterization of the internal promoter of human T-cell leukemia virus type I.

The HTLV-I provirus contains two different promoters: the classical retroviral promoter in the 5' long terminal repeat (LTR) and our previously identified second promoter in the pol gene just upstream of the ATG codon of the tax gene. Here, we demonstrated that the internal promoter expresses the gene for Tax but not Rex. As the deletion of upstream of the transcriptional initiation site (nt 5130) caused down-regulation of the promoter activity, we termed the region HTLV-I internal regulatory element (HIRE). We found a cellular sequence-specific DNA binding protein which binds to HIRE. Furthermore, we demonstrated that the 3' LTR regulates Tax expression from the internal promoter. These findings may shed light on a novel mechanism for gene expression in complex retroviruses of the HTLV family.

Amino Acid Sequence↗

PubMed1998

Expression of catenins and E-cadherin during epithelial restitution in inflammatory bowel disease.

Catenins are cytoplasmic proteins associated with E-cadherin, the prime mediator of cell-cell adhesion. Perturbation in any of these molecules results in altered intercellular adhesion, cell differentiation, and increased migration. In this study, the expression and cellular localization of catenins and E-cadherin in inflammatory bowel disease were examined. The expression of E-cadherin; alpha-, beta-, and gamma-catenin; and p120 was evaluated immunohistochemically in 31 paraffin-embedded colonic specimens from 21 patients with ulcerative colitis and Crohn's disease. Loss of normal membranous E-cadherin and alpha-catenin staining was detected at the mucosal edges around epithelial ulcerations in all cases of active ulcerative colitis and in 50 per cent of cases with active Crohn's disease. Reduced expression of p120 protein was also found at the margins of ulcerated mucosa in all cases of active ulcerative colitis and in 75 per cent of those with active Crohn's disease. There was a statistically significant correlation between the expression of E-cadherin, alpha-catenin and p120 and disease activity. There were no changes in beta- and gamma-catenin expression in either ulcerative colitis on Crohn's disease. These findings indicate that altered expression of E-cadherin, alpha-catenin, and p120 occurs during mucosal ulceration in inflammatory bowel disease. These changes may be involved in promoting cell migration during epithelial restitution of the gastrointestinal mucosa.

Cadherins↗

PubMed1998

IgG anti-GM1 antibody is associated with reversible conduction failure and axonal degeneration in Guillain-Barré syndrome.

To investigate the pathophysiological role of anti-GM1 antibody in Guillain-Barré syndrome (GBS), we reviewed sequential nerve conduction studies of 345 nerves in 34 GBS patients. Statistically significant correlation between IgG anti-GM1 antibodies and electrodiagnoses was found. Sixteen IgG anti-GM1-positive patients were classified as having acute motor or acute motor sensory axonal neuropathy (AMAN or AMSAN) (12 patients), as having acute inflammatory demyelinating polyneuropathy (AIDP) (3 patients), or as undetermined (1 patient) by electrodiagnostic criteria. Besides axonal features, there was rapid resolution of conduction slowing and block. In 3 patients initially diagnosed as having AIDP, conduction slowing was resolved within days, and 1 of them and 3 AMAN patients showed markedly rapid increases in amplitudes of distal compound muscle action potentials that were not accompanied by prolonged duration and polyphasia. The time courses of conduction abnormalities were distinct from those in IgG anti-GM1-negative AIDP patients. Rapid resolution of conduction slowing and block, and the absence of remyelinating slow components, suggest that conduction failure may be caused by impaired physiological conduction at the nodes of Ranvier. Reversible conduction failure as well as axonal degeneration constitutes the pathophysiological mechanisms in IgG anti-GM1-positive GBS. In both cases, immune-mediated attack probably occurs on the axolemma of motor fibers.

Adult↗

PubMed1998

Identification of septins in neurofibrillary tangles in Alzheimer's disease.

Septins are evolutionarily conserved cytoskeletal GTPases that can form heteropolymer complexes involved in cytokinesis and other cellular processes. We detected expression of the human septin genes Nedd5, H5, Diff6, and hCDC100 in postmortem brain tissues using the reverse transcription-coupled polymerase chain reaction and their products by immunoblot analysis. Four antibodies directed against three septins, Nedd5, H5, and Diff6, consistently labeled neurofibrillary tangles, neuropil threads, and dystrophic neurites in the senile plaques in brains affected by Alzheimer's disease but did not label obvious structures in young control brains. Immunoelectron microscopy revealed that Nedd5 localized to the paired helical filaments. Pre-tangles, the precursory granular deposits that accumulate in the neuronal cytoplasm, also were labeled with the antibodies. These findings suggest that at least the three septins are associated with tau-based paired helical filament core, and may contribute to the formation of neurofibrillary tangle as integral constituents of paired helical filaments.

Adult↗