Biomedical subjects

Tetsuo Noda

Publications and source records attributed to Tetsuo Noda.

At least 19 recordsLinked to original sources

PubMed2005

Genetic analysis of Pten and Tsc2 functional interactions in the mouse reveals asymmetrical haploinsufficiency in tumor suppression.

The role of tumor suppressor haploinsufficiency in oncogenesis is still poorly understood. The PTEN and TSC2 tumor suppressors function to antagonize mTOR (mammalian target of rapamycin) activation by Akt; hence, compound heterozygous inactivation of Pten and Tsc2 in the mouse may in principle exacerbate the tumor phenotypes observed in the single mutants in a reciprocal manner. In contrast, we found that while Tsc2 heterozygosity unmasks Pten haploinsufficiency in growth and tumor suppression, tumorigenesis in Tsc2+/- mutants is surprisingly not accelerated by Pten heterozygosity, even though mTOR activation is cooperatively enhanced by compound Pten/Tsc2 heterozygosity. We show that the wild-type alleles of both Pten and Tsc2 are retained in prostate tumors from both Pten+/- and Pten+/-Tsc2+/- mice, whereas TSC-related tumor lesions are invariably associated with Tsc2 loss of heterozygosity (LOH) in both Tsc2+/- and Pten+/-Tsc2+/- mice. These findings demonstrate that inactivation of TSC2 is epistatic to PTEN in the control of tumor initiation and progression and, importantly, that both Pten and Tsc2 are haploinsufficient for suppression of tumorigenesis initiated by Pten heterozygosity, while neither Pten nor Tsc2 is haploinsufficient for repression of carcinogenesis arising from Tsc2 heterozygosity, providing a rationale for the differential cancer susceptibility of the two human conditions associated with PTEN or TSC2 heterozygous mutations.

Animals↗

PubMed2005

Achlorhydria by ezrin knockdown: defects in the formation/expansion of apical canaliculi in gastric parietal cells.

Loss of gastric acid secretion is pathologically known as achlorhydria. Acid-secreting parietal cells are characterized by abundant expression of ezrin (Vil2), one of ezrin/radixin/moesin proteins, which generally cross-link actin filaments with plasma membrane proteins. Here, we show the direct in vivo involvement of ezrin in gastric acid secretion. Ezrin knockout (Vil2(-/-)) mice did not survive >1.5 wk after birth, making difficult to examine gastric acid secretion. We then generated ezrin knockdown (Vil2(kd/kd)) mice by introducing a neomycin resistance cassette between exons 2 and 3. Vil2(kd/kd) mice born at the expected Mendelian ratio exhibited growth retardation and a high mortality. Approximately 7% of Vil2(kd/kd) mice survived to adulthood. Ezrin protein levels in Vil2(kd/kd) stomachs decreased to <5% of the wild-type levels without compensatory up-regulation of radixin or moesin. Adult Vil2(kd/kd) mice suffered from severe achlorhydria. Immunofluorescence and electron microscopy revealed that this achlorhydria was caused by defects in the formation/expansion of canalicular apical membranes in gastric parietal cells.

Achlorhydria↗

PubMed2005

The RNA-binding protein HuD regulates neuronal cell identity and maturation.

Neural Hu proteins (HuB/C/D) are RNA-binding proteins that have been shown to induce neuronal differentiation activity when overexpressed in immature neural progenitor cells or undifferentiated neuronal tumors. Newly generated HuD-deficient mice exhibited a transient impaired-cranial-nerve-development phenotype at an early embryonic stage. Adult HuD-deficient mice exhibited an abnormal hind-limb reflex and poor rotarod performance. Analysis of neurosphere formation revealed that the number and self-renewal capacity of the neural stem/progenitor cells were increased in HuD-deficient mice. HuD-deficient primary neurospheres also generated a smaller number of neurons. Cohort analysis of the cellular proliferative activity by using BrdUrd and iododeoxuridine labeling revealed that the number of differentiating quiescent cells in the embryonic cerebral wall was decreased. Long-term administration of BrdUrd revealed that the number of slowly dividing stem cells in the adult subventricular zone was increased in the HuD-deficient mice. Taken together, the results suggest that HuD is required at multiple points during neuronal development, including negative regulation of proliferative activity and neuronal cell-fate acquisition of neural stem/progenitor cells.

Animals↗

PubMed2005

Enamelin (Enam) is essential for amelogenesis: ENU-induced mouse mutants as models for different clinical subtypes of human amelogenesis imperfecta (AI).

Amelogenesis imperfecta (AI) is a group of commonly inherited defects of dental enamel formation, which exhibits marked genetic and clinical heterogeneity. The genetic basis of this heterogeneity is still poorly understood. Enamelin, the affected gene product in one form of AI (AIH2), is an extracellular matrix protein that is one of the components of enamel. We isolated three ENU-induced dominant mouse mutations, M100395, M100514 and M100521, which caused AI-like phenotypes in the incisors and molars of the affected individuals. Linkage analyses mapped each of the three mutations to a region of chromosome 5 that contained the genes encoding enamelin (Enam) and ameloblastin (Ambn). Sequence analysis revealed that each mutation was a single-base substitution in Enam. M100395 (Enam(Rgsc395)) and M100514 (Enam(Rgsc514)) were putative missense mutations that caused S to I and E to G substitutions at positions 55 and 57 of the translated protein, respectively. Enam(Rgsc395) and Enam(Rgsc514) heterozygotes showed severe breakage of the enamel surface, a phenotype that resembled local hypoplastic AI. The M100521 mutation (Enam(Rgsc521)) was a T to A substitution at the splicing donor site in intron 4. This mutation resulted in a frameshift that gave rise to a premature stop codon. The transcript of the Enam(Rgsc521) mutant allele was degraded, indicating that Enam(Rgsc521) is a loss-of-function mutation. Enam(Rgsc521) heterozygotes showed a hypomaturation-type AI phenotype in the incisors, possibly due to haploinsufficiency of Enam. Enam(Rgsc521) homozygotes showed complete loss of enamel on the incisors and the molars. Thus, we report here that the Enam gene is essential for amelogenesis, and that mice with different point mutations at Enam may provide good animal models to study the different clinical subtypes of AI.

Amelogenesis↗

PubMed2005

Targeted disruption of FSCN2 gene induces retinopathy in mice.

PURPOSE: To investigate the morphology and function of photoreceptors in mice with mutation of the FSCN2 gene. METHODS: A mouse line was generated carrying the 208delG mutation (point mutation, or p-type) and another with replacement of exon 1 by the cDNA of a green fluorescent protein (GFP knock-in, or g-type). The expression of retinal mRNA was determined by reverse transcription (RT)-polymerase chain reaction (PCR) and in situ hybridization performed on retinal sections. Morphologic analyses of the retinas were performed by light microscopy (LM) and transmission electron microscopy (TEM) and functional analyses by electroretinogram (ERG). RESULTS: mRNA of FSCN2 was not detected in the retinal mRNA extracted from FSCN2p/p and FSCN2g/g mice. Both FSCN2(+/p) and FSCN2(+/g) mice had progressive photoreceptor degeneration with increasing age detected by LM and structural abnormalities of the outer segment (OS) detected by TEM. Both FSCN2(+/p) and FSCN2(+/g) mice had depressed rod and cone ERGs that worsened with increasing age. CONCLUSIONS: These results indicate that haploinsufficiency of the FSCN2 gene may hamper maintenance and/or elongation of the OS disks and result in photoreceptor degeneration, as in human autosomal dominant retinitis pigmentosa.

Animals↗

PubMed2005

PKClambda regulates glucose-induced insulin secretion through modulation of gene expression in pancreatic beta cells.

Altered regulation of insulin secretion by glucose is characteristic of individuals with type 2 diabetes mellitus, although the mechanisms that underlie this change remain unclear. We have now generated mice that lack the lambda isoform of PKC in pancreatic beta cells (betaPKClambda(-/-) mice) and show that these animals manifest impaired glucose tolerance and hypoinsulinemia. Furthermore, insulin secretion in response to high concentrations of glucose was impaired, whereas the basal rate of insulin release was increased, in islets isolated from betaPKClambda(-/-) mice. Neither the beta cell mass nor the islet insulin content of betaPKClambda(-/-) mice differed from that of control mice, however. The abundance of mRNAs for Glut2 and HNF3beta was reduced in islets of betaPKClambda(-/-) mice, and the expression of genes regulated by HNF3beta was also affected (that of Sur1 and Kir6.2 genes was reduced, whereas that of hexokinase 1 and hexokinase 2 genes was increased). Normalization of HNF3beta expression by infection of islets from betaPKClambda(-/-) mice with an adenoviral vector significantly reversed the defect in glucose-stimulated insulin secretion. These results indicate that PKClambda plays a prominent role in regulation of glucose-induced insulin secretion by modulating the expression of genes important for beta cell function.

Animals↗

PubMed2004

Genomic organization of the mouse Msh4 gene producing bicistronic, chimeric and antisense mRNA.

By degenerate PCR and screening of mouse testis cDNA library, we have identified seven cDNAs from the meiotic recombination gene Msh4. Variant alpha and probably beta are likely involved in meiotic DNA recombination. Other variants have distinctive structures; variant epsilon, theta; and iota form a bicistronic operon, while variant delta contains antisense RNA for the endoplasmic reticulum (ER) chaperon Hspa5 gene and small open reading frame (ORF) identical to epsilon ORF2. Analysis of the exon-intron structures revealed unusual genomic organizations: the first three exons of delta and the first exon of epsilon are respectively mapped to the Hspa5 locus on chromosome 2 and the Pcbp3 locus on chromosome 10; the remaining exons of both variants are mapped to the Msh4 locus on chromosome 3. The first exon of variant beta is located on chromosome 16, while the others are located on chromosome 3. Synthesis of these mRNAs is assumed to require interchromosomal trans-splicing alone (beta and epsilon) or in combination with converse-splicing (delta). Most Msh4 variant mRNAs are mainly expressed in testis, but a small amount of each variant except for epsilon is also expressed in brain, heart, thymus, ovary and embryonic head. Enhanced green fluorescent protein (EGFP) fusion experiments showed that all the ORFs are translated, and most of those proteins are localized to a particular subcellular compartment. It also appeared that expression of variant delta induces cell death. This study suggests that the dynamic interchromosomal (intergenic) trans-splicing generates functional diversity of the mouse Msh4 gene.

Alternative Splicing↗

PubMed2004

Human parvovirus B19 transgenic mice become susceptible to polyarthritis.

Human parvovirus B19 (B19) often causes acute polyarthritis in adults. In this paper, we analyzed nucleotide sequences of the B19 genome of patients with rheumatoid arthritis (RA), and then introduced the nonstructural protein 1 (NS1) gene of B19 into C57BL/6 mice that had a genetic origin not susceptible to arthritis. The transgenic mice developed no lesions spontaneously, but were susceptible to type II collagen (CII)-induced arthritis. B19 NS1 was expressed in synovial cells on the articular lesions that were histologically characteristic of granulomatous synovitis and pannus formation in cartilage and bone. Serum levels of anti-CII Abs and TNF-alpha increased in NS1 transgenic mice to the same levels as those of DBA/1 mice, which were susceptible to polyarthritis. Stimulation with CII increased secretion of Th1-type- and Th2-type cytokines in NS1 transgenic mice, indicating that a nonpermissive H-2(b) haplotype in the wild type of C57BL/6 mice can be made susceptible to polyarthritis through the expression of NS1. This study is the first to show that a viral agent from the joints in humans can cause CII-induced arthritis resembling RA.

Animals↗

PubMed2004

MAGI-3 is involved in the regulation of the JNK signaling pathway as a scaffold protein for frizzled and Ltap.

A seven-transmembrane protein, frizzled, has been implicated in a planar cell polarity (PCP) pathway as well as the canonical Wnt signaling pathway. Although both pathways require a cytoplasmic protein, dishevelled, the molecular mechanism by which frizzled regulates intracellular signaling remains to be elucidated. In the mouse, nine frizzled family members have been identified and six of them contain a PDZ-binding motif at their carboxyl-termini. In this study, we show that a multi-PDZ containing protein, MAGI-3, specifically binds to frizzled-4 and -7. Furthermore, we also demonstrate that MAGI-3 interacts with Ltap, a mouse homolog of the Drosophila PCP protein, stbm, and that these three molecules can form a ternary complex. In epithelial cells, MAGI-3, frizzled-4, and Ltap colocalized at cell contact sites, indicating that these molecules form a physiologically significant complex. Finally, we found that MAGI-3 strongly activated JNK in conjunction with frizzled-4 and Ltap, and that this activation required the small GTPase, Rac. These results indicate that MAGI-3 functions as a scaffold protein for frizzled-4 and Ltap and regulates the JNK signaling cascade.

Animals↗

PubMed2004

Apollon ubiquitinates SMAC and caspase-9, and has an essential cytoprotection function.

Apollon (also known as BRUCE or BIRC6) is a large protein containing baculoviral-IAP-repeat (BIR) and ubiquitin-conjugating enzyme (UBC) domains at the amino- and carboxy termini, respectively. Apollon inhibits apoptosis, but its molecular and physiological function remains unclear. Here we report that Apollon binds to, ubiquitinates and facilitates proteasomal degradation of SMAC and caspase-9, which both contain IAP-binding motifs. Targeted disruption of Apollon in mice caused embryonic and neonatal lethality. Notably, SMAC induced apoptosis in Apollon-deficient cells, but not in Apollon-expressing cells. Furthermore, the IAP-binding motif of SMAC was required to induce apoptosis in Apollon-deficient cells. These results suggest that Apollon has an essential function in preventing SMAC-induced apoptosis.

Animals↗

PubMed2004

Oligo-astheno-teratozoospermia in mice lacking Cnot7, a regulator of retinoid X receptor beta.

Spermatogenesis is a complex process that involves cooperation of germ cells and testicular somatic cells. Various genetic disorders lead to impaired spermatogenesis, defective sperm function and male infertility. Here we show that Cnot7(-/-) males are sterile owing to oligo-astheno-teratozoospermia, suggesting that Cnot7, a CCR4-associated transcriptional cofactor, is essential for spermatogenesis. Maturation of spermatids is unsynchronized and impaired in seminiferous tubules of Cnot7(-/-) mice. Transplantation of spermatogonial stem cells from male Cnot7(-/-) mice to seminiferous tubules of Kit mutant mice (Kit(W/W-v)) restores spermatogenesis, suggesting that the function of testicular somatic cells is damaged in the Cnot7(-/-) condition. The testicular phenotypes of Cnot7(-/-) mice are similar to those of mice deficient in retinoid X receptor beta (Rxrb). We further show that Cnot7 binds the AF-1 domain of Rxrb and that Rxrb malfunctions in the absence of Cnot7. Therefore, Cnot7 seems to function as a coregulator of Rxrb in testicular somatic cells and is thus involved in spermatogenesis.

Animals↗

PubMed2004

A series of maturity onset diabetes of the young, type 2 (MODY2) mouse models generated by a large-scale ENU mutagenesis program.

Mutant mouse models are indispensable tools for clarifying the functions of genes and for elucidating the underlying pathogenic mechanisms of human diseases. Currently, several large-scale mutagenesis projects that employ the chemical mutagen N-ethyl-N-nitrosourea (ENU) are underway worldwide. One specific aim of our ENU mutagenesis project is to generate diabetic mouse models. We screened 9375 animals for dominant traits using a clinical biochemical test and thereby identified 11 mutations in the glucokinase (Gk) gene that were associated with hyperglycemia. GK is a key regulator of insulin secretion in the pancreatic beta-cell. Approximately 190 heterozygous mutations in the human GK gene have been reported to cause maturity onset diabetes of the young, type 2 (MODY2). In addition, five mutations have been reported to cause permanent neonatal diabetes mellitus (PNDM) when present on both alleles. The mutations in our 11 hyperglycemic mutants are located at different positions in Gk. Four have also been found in human MODY2 patients, and another mutant bears its mutation at the same location that is mutated in a PNDM patient. Thus, ENU mutagenesis is effective for developing mouse models for various human genetic diseases, including diabetes mellitus. Some of our Gk mutant lines displayed impaired glucose-responsive insulin secretion and the mutations had different effects on Gk mRNA levels and/or the stability of the GK protein. This collection of Gk mutants will be valuable for understanding GK gene function, for dissecting the function of the enzyme and as models of human MODY2 and PNDM.

Amino Acid Sequence↗

PubMed2004

Combined effects of cyclooxygenase-1 and cyclooxygenase-2 selective inhibitors on intestinal tumorigenesis in adenomatous polyposis coli gene knockout mice.

As with cyclooxygenase (COX)-2, genetic disruption of COX-1 gene or pharmacologic inhibition of its activity has been shown to decrease the number of intestinal polyps in Apc gene-deficient mice. The present study was designed to investigate the combined effects of COX-1 and COX-2 selective inhibitors on spontaneous polyp formation in APC1309 female mice. The animals were treated with 300 or 600 ppm mofezolac (a COX-1 selective inhibitor) alone, 200 or 400 ppm nimesulide (a COX-2 selective inhibitor) alone, 300 ppm mofezolac plus 200 ppm nimesulide, 600 ppm mofezolac plus 400 ppm nimesulide, or 10 ppm indomethacin (a dual-COX inhibitor) in the diet from 7 weeks of age for 4 weeks. Percentage inhibition of polyp area in the intestine was 17% with 600 ppm mofezolac alone and 25% with 400 ppm nimesulide alone, their sum of 42% being almost equal to the 37% observed for the combination treatment. Administration of 300 ppm mofezolac plus 200 ppm nimesulide also significantly decreased polyp area in the intestine by 30%. Moreover, the numbers of polyps more than 2.5 mm in diameter were markedly decreased by combined treatment of both COX inhibitors. With 10 ppm indomethacin, the dual inhibitor, polyp area was also clearly reduced by 46%. Our results indicate that COX-1 and COX-2 may to some extent contribute to polyp formation independently and inhibitor combination treatment thus has particular potential for chemoprevention of colon carcinogenesis.

Animals↗

PubMed2004

Development and implementation of a database system to manage a large-scale mouse ENU-mutagenesis program.

A mouse ENU-mutagenesis program at RIKEN GSC has been initiated to conduct a large-scale, genome-wide, early- and late-onset phenotypic screen of mutant mice. We screened about a hundred mice every week with a comprehensive set of phenotype assays including behavioral tests based on a modified SHIRPA protocol, blood tests (both clinical biochemical testing and hemogram), and measurement of locomotor activity in their home cages. To manage the entire program, we developed a client/server architecture database system and named it MUSDB (Mutagenesis Universal Support DataBase). It manages mouse husbandry, mating protocols, procedures for ENU injection and phenotypic screens, phenotype inheritance tests, preservation of sperm and organs, and other materials generated during the program. We have implemented MUSDB in quite a large-scale system that includes 150 client computers. It has, helped reduce typographical errors and provided simple and efficient operation via its front-end user interface. It significantly contributed to the communication within and between workgroups in the program and in the accumulation of various phenotypic and inheritance data.

Animals↗

PubMed2004

Phylogenetic conservation of a limb-specific, cis-acting regulator of Sonic hedgehog ( Shh).

Polarized expression of the Sonic hedgehog ( Shh) gene in the posterior mesenchyme is essential for pattern formation in the appendages of higher vertebrates, from teleost fins to tetrapod limb buds. We report on a sequence in intron 5 of the Lmbr1 gene, which resides approximately 1 Mb from the Shh coding region in the mouse genome and is highly conserved among teleost fishes and throughout the tetrapod lineage. Positional cloning revealed that two mouse mutations, Hx and M100081, characterized by mirror-image digit duplication and ectopic anterior Shh expression, have base substitutions in this sequence. Absence of the conserved sequence in limbless reptiles and amphibians and a cis- trans test using the Hx and Shh KO alleles suggest that the sequence is a cis-acting regulator that controls the polarized expression of Shh.

Animals↗

PubMed2004

Enhanced gastric emptying of a liquid gastric load in mice lacking cholecystokinin-B receptor: a study of CCK-A,B, and AB receptor gene knockout mice.

BACKGROUND: Although cholecystokinin (CCK) has been shown to inhibit gastric emptying via CCK-A receptors (CCK-ARs), the role of CCK-B receptors (CCK-BRs) has not been verified. We examined whether gastric emptying of a nonnutrient liquid load was modified in CCK-AR, BR, and ARBR gene knockout mice. METHODS: A liquid gastric load prepared with phenol red was administered via an orogastric tube (0.15 ml/mouse). The animals were killed by decapitation, and gastric emptying was estimated at 10 and 30 min after ingestion. The effects of the sulfated form of CCK-8 (CCK-8S) and of graded doses of atropine were examined. In addition, a proton pump inhibitor was administered to wild-type mice to examine the contribution of gastric acid to emptying. RESULTS: Gastric emptying was significantly enhanced in mice lacking CCK-BR, as compared with wild-type and CCK-AR(-/-) mice. CCK-8S inhibited gastric emptying in mice with CCK-AR, but not in mice without CCK-AR. A proton pump inhibitor did not affect gastric emptying. Atropine dose dependently inhibited gastric emptying in all genotypes. The thickness of smooth muscle was comparable for all genotypes. CONCLUSIONS: The gastric emptying of a nonnutrient liquid load was enhanced in mice without CCK-BR, although the precise mechanism is not known. Although cholecystokinin (CCK) has been shown to inhibit gastric emptying via CCK-A receptors (CCK-ARs), the role of CCK-B receptors (CCK-BRs) has not been verified. We examined whether gastric emptying of a nonnutrient liquid load was modified in CCK-AR, BR, and ARBR gene knockout mice.

Animals↗

PubMed2004

Putative gene loci associated with carcinogenesis and metastasis of endocervical adenocarcinomas of uterus determined by conventional and array-based CGH.

OBJECTIVES: This study aimed to estimate the gene loci associated with carcinogenesis of endocervical adenocarcinoma of uterus (EA) and metastasis. Study design Sixteen patients with EA were studied; 6 had nodal metastasis. DNA was extracted from EAs, and subjected to both conventional comparative genomic hybridization (CGH) and array-based CGH. Copy number abnormalities were compared between cases with and without nodal metastasis. RESULTS: In all EAs, high frequencies of copy number losses were detected in genes LRP1B (on 2q21.2), DAB2 (5p13), and DCC (18q21.3), as well as regions 3p, 16q, and 22q, and copy number amplifications in genes NRAS (1p13.2), TOP2A (17q21-q22), NCOA3(AIB1) (20q12), and ARSA (22q tel). Nodal metastasis was associated with high frequencies of copy number loss in genes PGRMC2 and LAMA3 and amplification in CDK6 and NCOA3(AIB1). CONCLUSION: This is the first report of gene copy number alterations spanning the whole genome in EA. These altered genes are speculated to be associated with EAs as a tumor suppressor and/or oncogene.

Acetyltransferases↗

PubMed2004

Synthesis of functional proteins by mixing peptide motifs.

Here, we describe a synthetic approach for generating artificial proteins by the assemblage of naturally occurring peptide motifs. Two motifs respectively related to apoptosis induction and protein transduction were encrypted into different reading frames of an artificial gene (microgene), which was then polymerized; random frame shifts at the junctions between the microgene units yielded combinatorial polymers of three reading frames. Among the proteins created, #284 was found to penetrate through cell membranes and exert a strong apoptotic effect on several cancer cell lines. Because a simple linkage of these motifs was not sufficient to construct a bifunctional peptide, and the successful reconstitution was dependent on how they were joined together, the combinatorial strategy is important for reconstituting functions from mixtures of motifs. This microgene-based approach represents a novel system for creating proteins with desired functions.

Amino Acid Motifs↗