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M Tada

Publications and source records attributed to M Tada.

At least 127 records · Page 7Linked to original sources
PubMed1998

Involvement of NF-Y in transcriptional regulation of the phospholamban gene.

To understand the transcriptional regulation of the phospholamban gene, we analyzed a 5'-upstream region of the gene. Using a series of deletion constructs, we demonstrated that the region from -96 bp to -78 bp, containing the CCAAT sequence, is essential for transcription of this gene. This region specifically bound to nuclear proteins extracted from rat hearts, and gel-shift assays using competitive oligonucleotides, antibodies and recombinant proteins showed that this region binds to the NF-YA and NF-YB, members of the CCAAT-binding nuclear protein family. This region-dependent transcription in cardiac myocytes transfected with antisense cDNAs encoding NF-YA and NF-YB was decreased to approximately 50% of that seen in cells transfected with the same sense cDNAs. We, therefore, conclude that the region from -96 bp to -78 bp plays a critical role in expression of the phospholamban gene, which is regulated by binding of the nuclear protein NF-Y.

Animals↗
PubMed1998

Immunohistochemical demonstration of carcinogen-DNA adducts in tissues of rats given 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP): detection in paraffin-embedded sections and tissue distribution.

A polyclonal antibody against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNA adducts was raised for their immunohistochemical demonstration in paraffin-embedded sections. Specificity of this antibody was confirmed by competitive ELISA. Positive signals were immunohistochemically detected in acetone-fixed but not in formalin- or ethanol-fixed sections from F344 rats treated by gavage with a single dose of PhIP at 37.5-300 mg/kg and killed at 1, 2, and 7 days thereafter. Dose-dependent positive staining was observed in almost all organs of both sexes, including the colon, prostate, and mammary gland but largely independent of the tumor response. Repair activity, judged by disappearance of adducts with time, differed according to the organ or cell type. One exception was hepatocytes, the liver incidentally being a nontarget organ. The results suggest that the generated antibody is applicable for detection of cells targeted by PhIP in paraffin-embedded sections and also for the investigation of the mechanisms of PhIP-carcinogenesis.

Administration, Oral↗
PubMed1998

Molecular regulation of phospholamban function and gene expression.

Ca-ATPase regulates intracellular Ca levels by pumping Ca into sarcoplasmic reticulum. Phospholamban (PLN) functions as an inhibitory cofactor for cardiac Ca-ATPase (SERCA2). To define the molecular mode of interaction between two proteins, interaction sites have been identified. Studies using photoactivated cross-linker and chimeric Ca-ATPase between SERCA2 and nonmuscle Ca-ATPase (SERCA3) indicated that potential binding residues are located just downstream of the active ATPase site (Asp351) of SERCA2. Site-directed mutagenesis study of this region showed that six residues, Lys-Asp-Asp-Lys-Pro-Val402, of SERCA2 are functionally important for the interaction. Further, mutagenesis study of PLN showed that the cytoplasmic region of PLN contains a potential binding site with SERCA2. The unique expression of PLN in cardiac cells has been analyzed by the transcriptional level of its gene using luciferase activity and Gel shift assays. CCAAT-box in the 5'-upstream region was found to be essential for its expression by associating with Y-box binding transcriptional factors.

Amino Acid Sequence↗
PubMed1998

Occult neurohypophyseal germinomas in patients presenting with central diabetes insipidus.

Although neurohypophyseal germinoma is known be a common initial symptom in cases of diabetes insipidus (DI), its radiological detection may take months or years even by a high-resolution magnetic resonance (MR) imaging. The term "occult neurohypophyseal germinoma" denotes such cases, but its clinical picture remains obscure. Of seven patients with neurohypophyseal germinoma presenting with DI during the last 5 years, three patients showed no evidence of tumor at the onset of DI and were treated as "idiopathic" DI. Neurohypophyseal germinoma was eventually diagnosed in these three patients as the tumor became evident on sequential MR imaging studies and the patients were successfully treated with chemotherapy and radiation therapy. To delineate the clinical features of the occult neurohypophyseal germinoma, the authors analyzed endocrinological aspects and MR images in these patients and compared them with those in two patients with true idiopathic DI and four patients with overt neurohypophyseal germinoma and DI. Nine previously reported cases in the literature were reviewed. During the stage at which the germinoma gave no notable change on MR images, patients often displayed anterior pituitary dysfunction, particularly growth hormone (GH) deficiency, or an elevation of serum or cerebrospinal fluid human chorionic gonadotropin-beta. Preceding the appearance of an obvious tumor mass, a slight swelling of the pituitary stalk with loss of normal hyperintensity of the posterior pituitary lobe was a common finding on MR imaging. Central DI associated either with an enlarged stalk, decreased GH secretion, or an elevated serum human chorionic gonadotropin-beta should prompt the diagnosis of an occult germinoma.

Journal Article↗
PubMed1998

Absence of p53 gene mutations in a tumor panel representative of pilocytic astrocytoma diversity using a p53 functional assay.

Although p53-gene mutations occur with significant frequency in diffuse low-grade and high-grade astrocytomas, and are postulated to play an important role in tumorigenesis in these cases, the role of the p53 gene in pilocytic astrocytomas remains unclear. Published data using DNA-based assays for p53-gene analysis in these tumors have shown contradictory results in mutation frequency (0-14%). It is not known whether these heterogeneous results stem from the biological diversity of this tumor group or from technical problems. To re-evaluate p53-gene status in pilocytic tumors, we analyzed 18 tumors chosen to represent the clinical and biological heterogeneity of this tumor type with respect to anatomical location, patient age, gender, ethnic origin (Caucasian or Japanese) and the concomitant occurrence of neurofibromatosis type 1 (NF1). All primary tumors were histologically diagnosed as pilocytic astrocytoma (WHO grade I), except for one anaplastic pilocytic astrocytoma (WHO grade III) which developed in an NF1 patient and recurred as glioblastoma multiforme (WHO grade IV). p53 mutations were detected using an assay in yeast which tests the transcriptional activity of p53 proteins synthesized from tumor mRNA-derived p53-cDNA templates. None of 18 tumors, including 3 NF1-related tumors, showed p53-gene mutations between and including exons 4 and 11. We conclude that p53-gene mutations are extremely rare findings in pilocytic astrocytomas, and are absent even in those exceptional cases in which malignant progression of such tumors has occurred.

Adolescent↗
PubMed1998

Phospholamban domain Ib mutations influence functional interactions with the Ca2+-ATPase isoform of cardiac sarcoplasmic reticulum.

Alanine-scanning mutagenesis of amino acids 21-30, forming cytoplasmic domain Ib in phospholamban (PLN), revealed that mutation to Ala of Asn27, Gln29, and Asn30 results in gain of inhibitory function. In an earlier study (Kimura, Y., Kurzydlowski, K., Tada, M. , and MacLennan, D. H. (1997) J. Biol. Chem. 272, 15061-15064), gain of function in PLN transmembrane domain II mutants was correlated with pentamer destabilization, leading to proposals that the PLN monomer is the active inhibitory species, that dissociation of the PLN pentamer is one determinant of PLN inhibitory function and that dissociation of the PLN.cardiac sarco(endo)plasmic Ca2+-ATPase isoform (SERCA2a) complex is a second determinant. Because each of the new domain Ib mutants contained a normal ratio of pentamer to monomer in SDS-polyacrylamide gel electrophoresis, gain of function must have resulted from mechanisms other than destabilization of pentameric structure. Evidence that domain Ib and domain II mutants act through different sites and different mechanisms was provided by a monomeric double mutant, N30A/I40A, in which the enhanced inhibitory function of each single mutant was additive. Evidence for an alteration in stability of the PLN/SERCA2a heterodimer was obtained in a study of double mutant N27A/N34A in which inhibitory function was regained by combining a gain of function, domain Ib mutation with a loss of function domain II mutation. These results support the proposal that PLN inhibition of SERCA2a involves, first, depolymerization of PLN and, second, the formation of inhibitory interactions between monomeric PLN and SERCA2a.

Animals↗
PubMed1998

Direct association of the gap junction protein connexin-43 with ZO-1 in cardiac myocytes.

The gap junction protein connexin-43 is normally located at the intercalated discs of cardiac myocytes, and it plays a critical role in the synchronization of their contraction. The mechanism by which connexin-43 is localized within cardiac myocytes is unknown. However, localization of connexin-43 likely involves an interaction with the cytoskeleton; immunofluorescence microscopy showed that in cardiac myocytes, connexin-43 specifically colocalizes with the cytoskeletal proteins ZO-1 and alpha-spectrin. In transfected HEK293 cells, immunoprecipitation experiments using coexpressed epitope-tagged connexin-43 and ZO-1 indicated that ZO-1 links connexin-43 with alpha-spectrin. The domains responsible for the protein-protein interaction between connexin-43 and ZO-1 were identified using affinity binding assays with deleted ZO-1 and connexin-43 fusion proteins. Immunoblot analysis of associated proteins showed that the C-terminal domain of connexin-43 binds to the N-terminal domain of ZO-1. The role of this linkage in gap junction formation was examined by a dominant-negative assay using the N-terminal domain of ZO-1. Overexpression of the N-terminal domain of ZO-1 in connexin-43-expressing cells resulted in redistribution of connexin-43 from cell-cell interfaces to cytoplasmic structures; this intracellular redistribution of connexin-43 coincided with a loss of electrical coupling. We therefore conclude that the linkage between connexin-43 and alpha-spectrin, via ZO-1, may serve to localize connexin-43 at the intercalated discs, thereby generating functional gap junctions in cardiac myocytes.

Amino Acid Sequence↗
PubMed1998

Selective sensitivity to radiation of cerebral glioblastomas harboring p53 mutations.

Recent studies suggest that a balance may exist between the cell cycle arrest and apoptosis-inducing functions of the p53 tumor suppressor gene. Adenoviral p21 transduction attenuates apoptosis, whereas deletion of the p21 gene promotes it, and p21-null xenografts respond better than isogenic p21-wild type tumors to irradiation. Hence, the role of p53 in dictating the clinical response to radiotherapy and chemotherapy may be more complex than previously thought. We have analyzed survival and radiation response (regrowth-free period) of 42 patients with glioblastomas whose p53 status was determined by a sensitive yeast functional assay. Multivariate analysis revealed that p53 mutation is associated with longer survival (P < 0.02). Among 36 radiation-treated patients, the regrowth-free period after treatment was significantly longer for tumors with p53 mutations (P < 0.0001), and p53 mutation was the sole independent factor predictive of radiotherapeutic response (P < 0.01). Survival time after regrowth was independent of p53 status, suggesting that the difference in survival was related to the treatment rather than to the intrinsic aggressiveness of the tumor. Thus, in this Northern Japanese population, p53 mutation is a marker for better radiation response in glioblastomas, and this results in significantly longer survival.

Adolescent↗
PubMed1998

Phospholamban domain I/cytochrome b5 transmembrane sequence chimeras do not inhibit SERCA2a.

A series of chimeras between the transmembrane domains of phospholamban (PLN) and cytochrome b5 were coexpressed with the Ca2+-ATPase of cardiac sarcoplasmic reticulum (SERCA2a). The chimeric molecules were not inhibitory, in line with our view that inhibitory PLN/SERCA2a interactions occur in transmembrane sequences, while cytoplasmic interactions regulate the inhibitory interactions in a four-base circuit.

Animals↗
PubMed1998

Identification of a human homolog of the Drosophila neuralized gene within the 10q25.1 malignant astrocytoma deletion region.

The loss of chromosome 10 is the most frequent genetic alteration found in malignant astrocytomas. In particular, the long arm of chromosome 10 was previously reported to have two or more common deletion regions where tumor suppressor genes may be located. In this study, we performed deletion mapping of 44 malignant astrocytomas using 12 microsatellite markers on chromosome 10q and demonstrated that the minimal common region of loss of heterozygosity (LOH) was present between D10S192 and D10S566 localized at 10q25.1. Subsequently, we have identified a novel gene, termed h-neu, within the region frequently deleted and found that h-neu encodes a protein with strong homology to the Drosophila neuralized (D-neu) protein. Northern blot and RT-PCR analyses revealed that h-neu mRNA was expressed at very low levels in human malignant astrocytoma tissues and the majority of glioma cell lines examined, while normal brains expressed h-neu transcript. Furthermore, DNA sequencing analysis of the h-neu transcript revealed one of the glioma cell lines, U251MG, had a single nucleotide substitution which resulted in an amino acid change from glycine (GGC) to serine (AGC) at codon 253. The D-neu gene is known to serve a critical function in neurogenesis in Drosophila, and loss-of-function mutations produce hyperplasia of primitive neuronal cells. These observations led us to hypothesize that h-neu gene plays a role in determination of cell fate in the human central nervous system and may act as a tumor suppressor whose inactivation could be associated with malignant progression of astrocytic tumors.

Amino Acid Sequence↗
PubMed1998

Short alternative splice transcripts of the mdm2 oncogene correlate to malignancy in human astrocytic neoplasms.

The mdm2 oncogene encodes a 90-kDa nuclear phosphoprotein that binds and inhibits the function of the p53 tumor suppressor protein. It was recently reported that the expression of alternatively spliced variants of mdm2 correlated with malignancy in ovarian tumors and bladder carcinomas. We analyzed the presence of alternatively spliced mdm2 variants and studied their correlation to p53 status in a total of 66 human astrocytic tumors, including 32 glioblastomas multiforme, 17 anaplastic astrocytomas, 12 astrocytomas, and 5 pilocytic astrocytomas, using a specific nested reverse transcription-PCR technique. The full-length mdm2 transcript was demonstrated in all of the cases. Multiple-sized PCR products were found in 29 cases. Two of 5 pilocytic astrocytomas (40%), none of 12 astrocytomas, and 5 of 17 anaplastic astrocytomas (29%) showed alternative splice variants. In contrast, 22 of 32 glioblastomas (69%) showed the presence of splice variants, demonstrating a significantly higher frequency than in lower-grade astrocytomas (P < 0.0003). A majority of the splice variants were 707 base-type (mdm2-b), which was confirmed by sequence analysis. There was no apparent correlation of the presence of mdm2 splice variants with p53 gene status. These results suggest a new role for mdm2, independent of p53 gene status, as an oncogene in the development of malignant astrocytic tumors.

Alternative Splicing↗
PubMed1998

Intercellular calcium signaling via gap junction in connexin-43-transfected cells.

In excitable cells, intracellular Ca2+ is released via the ryanodine receptor from the intracellular Ca2+ storing structure, the sarcoplasmic reticulum. To determine whether this released Ca2+ propagates through gap junctions to neighboring cells and thereby constitutes a long range signaling network, we developed a cell system in which cells expressing both connexin-43 and ryanodine receptor are surrounded by cells expressing only connexin-43. When the ryanodine receptor in cells was activated by caffeine, propagation of Ca2+ from these caffeine-responsive cells to neighboring cells was observed with a Ca2+ imaging system using fura-2/AM. Inhibitors of gap junctional communication rapidly and reversibly abolished this propagation of Ca2+. Together with the electrophysiological analysis of transfected cells, the observed intercellular Ca2+ wave was revealed to be due to the reconstituted gap junction of transfected cells. We next evaluated the functional roles of cysteine residues in the extracellular loops of connexin-43 in gap junctional communication. Mutations of Cys54, Cys187, Cys192, and Cys198 to Ser showed the failure of Ca2+ propagation to neighboring cells in accordance with the electrical uncoupling between transfected cells, whereas mutations of Cys61 and Cys68 to Ser showed the same pattern as the wild type. [14C]Iodoacetamide labeling of free thiols of cysteine residues in mutant connexin-43s showed that two pairs of intramolecular disulfide bonds are formed between Cys54 and Cys192 and between Cys187 and Cys198. These results suggest that intercellular Ca2+ signaling takes place in cultured cells expressing connexin-43, leading to their own synchronization and that the extracellular disulfide bonds of connexin-43 are crucial for this process.

Calcium↗
PubMed1998

DNA adduct formation in human and rat mammary epithelium by N-hydroxy derivatives of 2-aminofluorene and 4-aminobiphenyl.

Primary cultures of rat mammary epithelium and the human mammary cell line MCF-12 were incubated with 10 microM N-formyl-, N-acetyl-, or N-propionyl-derivatives of N-hydroxy-2-aminofluorene (N-OH-AF) or N-formyl-, or N-acetyl derivatives of N-hydroxy-4-aminobiphenyl (N-OH-ABP), in the medium with or without 100 microM paraoxon, for 3 h. Carcinogen-DNA adducts in the nuclei were detected with an immunohistochemical method using polyclonal antibodies against N-(deoxyguano-8-yl)-2-aminofluorene and ABP-DNA adducts. The relative amounts of adducts per nucleus were determined by image analysis. After treatment, more than 90% of the cells that were attached on the coverslip were alive, as determined by the trypan blue exclusion. All carcinogens produced adducts in both human and rat cells. Adduct formation by the formyl, but not the acetyl or porpionyl, derivatives was inhibited up to 65% by paraoxon. These results demonstrate that both acetyl and propionyl derivatives are primarily activated by cytosolic acetyltransferases and the formyl derivatives may be equally activated by the acetyltransferases and microsomal carboxylesterases. Additionally, the results suggest that exposure to aromatic amines may be a risk factor for human breast cancer.

Aminobiphenyl Compounds↗
PubMed1998

Rare occurrence of inactivating p53 gene mutations in primary non-astrocytic tumors of the central nervous system: reappraisal by yeast functional assay.

While it is established that p53 mutation plays a critical role in the carcinogenesis of astrocytic brain tumors, its role remains to be clarified for other types of tumors in the central nervous system (CNS). Using a yeast-based assay which tests the ability of human p53 to activate transcription, we analyzed p53 mutations in 85 non-astrocytic CNS tumors, including 4 benign neuronal tumors (3 central neurocytomas and 1 pineocytoma), 12 primitive neuroectodermal tumors, 14 germ cell tumors (7 germinomas, 7 non-germinomatous tumors), 4 craniopharyngiomas, 14 ependymomas, 22 schwannomas, 10 primary brain lymphomas in immunocompetent patients, and 5 bone tumors of the skull. The only tumors found to contain p53 mutations were 3 malignant lymphomas. The presence of mutations in these cases was confirmed by DNA sequencing. Given the high accuracy and sensitivity of the yeast assay and previous negative results using conventional techniques, this indicates that p53 mutation is a rare event in non-astrocytic CNS tumor types examined here.

Adolescent↗
PubMed1998

Expression of oncogenic molecules in primary central nervous system lymphomas in immunocompetent patients.

We studied overexpression of p53, Bcl-2, Bcl-6, c-Myc and Mdm2 proteins by immunohistochemistry for a total of 27 primary central nervous system B cell lymphomas (CNS lymphomas) in immunocompetent patients and one CNS lymphoma in an AIDS patient. The expression of Epstein-Barr (EB) virus-encoded small RNA-1 (EBER-1) was also analysed using in situ hybridisation. Overexpression (more than 20% of cells stained) of p53 protein was detected in 8 of 27 immunocompetent cases (30%); 6 cases showed a nuclear stain and 2 cases showed cytoplasmic stain (nuclear exclusion). Strong Bcl-2 or Bcl-6 immunoreactivity suggestive of overexpression was seen, respectively, in 5 (19%) and 6 (22%) cases; 2 cases were positive for both immunoreactivities. Interestingly, overexpression of Bcl-2 or Bcl-6 was not seen in the cases which showed p53 overexpression (P < 0.03; chi-square test). EBER-1 expression was not detected in any of the 27 immunocompetent cases, but was found in the AIDS-related CNS lymphoma, which also showed an overexpression of Bcl-6, but not Bcl-2. None of the cases showed c-Myc or Mdm2 overexpression. Taken together, it is suggested that CNS lymphoma in immunocompetent hosts is a distinct disease that has a different molecular profile from those of systemic lymphoma and/or AIDS-related CNS lymphoma.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Epigenotype switching of imprintable loci in embryonic germ cells.

Expression of imprinted genes is dependent on their parental origin. This is reflected in the heritable differential methylation of parental alleles. The gametic imprints are however reversible as they do not endure for more than one generation. To investigate if the epigenetic changes in male and female germ line are similar or not, we derived embryonic germ (EG) cells from primordial germ cells (PGCs) of day 11.5 and 12.5 male and female embryos. The results demonstrate that they have an equivalent epigenotype. First, chimeras made with EG cells derived from both male and female embryos showed comparable fetal overgrowth and skeletal abnormalities, which are similar to but less severe than those induced by androgenetic embryonic stem (ES) cells. Thus, EG cells derived from female embryos resemble androgenetic ES cells more than parthenogenetic cells. Furthermore, the methylation status of both alleles of a number of loci in EG cells was similar to that of the paternal allele in normal somatic cells. Hence, both alleles of Igf2r region 2, Peg1/Mest, Peg3, Nnat were consistently unmethylated in EG cells as well as in the primary embryonic fibroblasts (PEFs) rescued from chimeras. More strikingly, both alleles of p57kip2 that were also unmethylated in EG cells, underwent de novo methylation in PEFs to resemble a paternal allele in somatic cells. The exceptions were the H19 and Igf2 genes that retained the methylation pattern in PEFs as seen in normal somatic tissues. These studies suggest that the initial epigenetic changes in germ cells of male and female embryos are similar.

Animals↗
PubMed1998

Pyloric stenosis due to Crohn's disease.

Gastroduodenal involvement by Crohn's disease is relatively rare and occasionally causes pyloric stenosis, for which medical therapy may be ineffective and surgery may be required. We report on a 44-year-old man patient in whom corticosteroids had a marked effect in reducing pyloric stenosis caused by Crohn's disease. A proton-pump inhibitor was not effective, and Helicobacter pylori was negative.

Adult↗