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Biomedical subjects

Keiko Ikeda

Publications and source records attributed to Keiko Ikeda.

13 recordsLinked to original sources
PubMed2006

Six1 and Six4 promote survival of sensory neurons during early trigeminal gangliogenesis.

Survival of sensory neurons is tightly regulated in cell-type and developmental-stage-specific manners. The transcriptional regulatory mechanisms underlying this regulation remain to be elucidated. In the present study, we investigated the role of Six1 and Six4 in the development of trigeminal ganglia. Abundant expression of Six1 and Six4 was noted in sensory neurons during early trigeminal gangliogenesis. Loss of both Six1 and Six4 in mice caused severe defects in the trigeminal ganglia, wherein massive apoptosis accompanied by activation of caspase-3 was observed at early but not late stages of gangliogenesis. In Six1(-/-)Six4(-/-) mice, trigeminal sensory neurons were generated, but showed reduced expression of Bcl-x compared with the wild-type mice. Accordingly, neurons from the deficient mice could not survive in culture even in the presence of neurotrophins. Our results suggest a cell-intrinsic role of Six1 and Six4 in the survival of early-generated trigeminal sensory neurons.

Animals↗
PubMed2006

Immobilization of diverse foreign proteins in viral polyhedra and potential application for protein microarrays.

Cypoviruses are insect viruses that produce a cytoplasmic crystalline particle called the polyhedron in which progeny virions are occluded. The virion structural protein, VP3, is implicated in the occlusion of viral particles into polyhedra. In this study, we determined the amino acid sequence of VP3 required for occlusion of viral particles into polyhedra and proposed that this sequence could be used as an immobilization signal to direct the stable incorporation of foreign proteins into polyhedra. A large-scale survey revealed that the immobilization signal could, in fact, direct the incorporation of a variety of human proteins into polyhedra. Immune reactivity and protein-protein interactions were detected on the surface of polyhedra containing immobilized foreign proteins, and these particles were shown to be highly stabilized against dehydration. We showed that these particles could be arrayed onto a glass slide by standard spotting and laser manipulation methods. Thus, this approach is well suited for protein expression, purification, and the development of protein microarrays.

Animals↗
PubMed2005

Hyperphagia and obesity in Na,K-ATPase alpha2 subunit-defective mice.

OBJECTIVE: The Na,K-ATPase alpha2 subunit gene (Atp1a2) is expressed in the brain, skeletal muscles, heart, and adipocytes. Specific function of the alpha2 subunit, such as involvement in differentiation and function of adipocytes, has not been addressed. The aim of this study was to examine whether Atp1a2-defective heterozygous mice show obesity and reveal the mechanisms underlying the obesity. RESEARCH METHODS AND PROCEDURES: We measured the differentiation and glucose uptake function of in vitro-differentiated adipocytes derived from embryonic fibroblasts of Atp1a2-defective mice. Food intake, body temperature, metabolic rate, and spontaneous activity and mRNA levels of neuropeptide genes were compared between the heterozygous and wild-type adult mice. RESULTS: Atp1a2 heterozygous female mice developed obesity after middle age. The time course of in vitro adipocyte differentiation of embryonic fibroblasts isolated from wild type, heterozygous, and homozygous mice was not different, glucose and Rb uptake activities of the in vitro-differentiated adipocytes were not altered, and the effects of insulin on glucose uptake and those of monensin and ouabain on Rb uptake were similar among the genotypes. However, food intake in the light phase was significantly greater in the heterozygous mice than the wild type in the 24-hour dark-light cycle, whereas it was similar under constant-light condition. Body temperature, metabolic rate at rest, and spontaneous motor activity of the heterozygous mice were similar to those of the wild type. Orexin mRNA level was lower in heterozygous than wild-type mice. DISCUSSION: The Na,K-ATPase alpha2 subunit is not involved in the differentiation or in glucose and Rb uptake function of in vitro-differentiated adipocytes. Hyperphagia is the likely primary cause of obesity in Atp1a2 heterozygous mice.

Adipocytes↗
PubMed2005

Slc12a2 is a direct target of two closely related homeobox proteins, Six1 and Six4.

Six genes are homologs of Drosophila sine oculis and encode transcription factors that are characterized by a conserved Six domain and homeodomain. Of the six family members (Six1-Six6) in mice, Six1 and Six4 show similar expression patterns during embryogenesis. Six1-/- mice show defective formation of various organs such as inner ear, nose, skeletal muscle, kidney and thymus, whereas Six4-/- mice show little anomaly in organogenesis. To understand the molecular basis for the differential function of Six1 and Six4 in vivo, we screened target genes of Six1 and Six4 and found that Six1 and Six4 differentially regulated a set of target genes. Gel-retardation assays indicated that the promoter region of one of the targets, sodium-potassium-chloride cotransporter 1 (Slc12a2), contains multiple Six1-binding sites and one common binding site of Six1 and Six4, suggesting that the DNA-binding specificity of Six1 is distinct from that of Six4. This underlies the differential regulation of common target genes by Six1 and Six4. Furthermore, in situ hybridization demonstrated that the expression of Slc12a2 was reduced in the developing dorsal root ganglia of Six1-/-/Six4-/- mice, suggesting that Six1 and Six4 regulate Slc12a2 in vivo.

Animals↗
PubMed2004

Malfunction of respiratory-related neuronal activity in Na+, K+-ATPase alpha2 subunit-deficient mice is attributable to abnormal Cl- homeostasis in brainstem neurons.

Na+, K+-ATPase 2 subunit gene (Atp1a2) knock-out homozygous mice (Atp1a2-/-) died immediately after birth resulting from lack of breathing. The respiratory-related neuron activity in Atp1a2-/- was investigated using a brainstem-spinal cord en bloc preparation. The respiratory motoneuron activity recorded from the fourth cervical ventral root (C4) was defective in Atp1a2-/- fetuses of embryonic day 18.5. The C4 response to electrical stimulation of the ventrolateral medulla (VLM) recovered more slowly in Atp1a2-/- than in wild type during superfusion with Krebs' solution, consistent with the high extracellular GABA in brain of Atp1a2-/-. Lack of inhibitory neural activities in VLM of Atp1a2-/- was observed by optical recordings. High intracellular Cl- concentrations in neurons of the VLM of Atp1a2-/- were detected in gramicidin-perforated patch-clamp recordings. The alpha2 subunit and a neuron-specific K-Cl cotransporter KCC2 were coimmunoprecipitated in a purified synaptic membrane fraction of wild-type fetuses. Based on these results, we propose a model for functional coupling between the Na+, K+-ATPase alpha2 subunit and KCC2, which excludes Cl- from the cytosol in respiratory center neurons.

Amino Acid Sequence↗
PubMed2003

Six1 controls patterning of the mouse otic vesicle.

Six1 is a member of the Six family homeobox genes, which function as components of the Pax-Six-Eya-Dach gene network to control organ development. Six1 is expressed in otic vesicles, nasal epithelia, branchial arches/pouches, nephrogenic cords, somites and a limited set of ganglia. In this study, we established Six1-deficient mice and found that development of the inner ear, nose, thymus, kidney and skeletal muscle was severely affected. Six1-deficient embryos were devoid of inner ear structures, including cochlea and vestibule, while their endolymphatic sac was enlarged. The inner ear anomaly began at around E10.5 and Six1 was expressed in the ventral region of the otic vesicle in the wild-type embryos at this stage. In the otic vesicle of Six1-deficient embryos, expressions of Otx1, Otx2, Lfng and Fgf3, which were expressed ventrally in the wild-type otic vesicles, were abolished, while the expression domains of Dlx5, Hmx3, Dach1 and Dach2, which were expressed dorsally in the wild-type otic vesicles, expanded ventrally. Our results indicate that Six1 functions as a key regulator of otic vesicle patterning at early embryogenesis and controls the expression domains of downstream otic genes responsible for respective inner ear structures. In addition, cell proliferation was reduced and apoptotic cell death was enhanced in the ventral region of the otic vesicle, suggesting the involvement of Six1 in cell proliferation and survival. In spite of the similarity of otic phenotypes of Six1- and Shh-deficient mice, expressions of Six1 and Shh were mutually independent.

Animals↗
PubMed2003

Degeneration of the amygdala/piriform cortex and enhanced fear/anxiety behaviors in sodium pump alpha2 subunit (Atp1a2)-deficient mice.

The sodium pump is the enzyme responsible for the maintenance of Na+ and K+ gradients across the cell membrane. Four isoforms of the catalytic alpha subunit have been identified, but their individual roles remain essentially unknown. To investigate the necessary functions of the alpha2 subunit in vivo, we generated and analyzed mice defective in the alpha2 subunit gene. Mice homozygous for the alpha2 mutation died just after birth and displayed selective neuronal apoptosis in the amygdala and piriform cortex. In these regions, high expression of c-Fos before apoptosis indicated neural hyperactivity, and re-uptake of glutamic acid and GABA into P2 fraction containing crude synaptosome was impaired. These results indicate that the alpha2 subunit plays a critical role regulating neural activity in the developing amygdala and piriform cortex. Further supporting a role of the alpha2 subunit in the function of the amygdala, heterozygous adult mice showed augmented fear/anxiety behaviors and enhanced neuronal activity in the amygdala and piriform cortex after conditioned fear stimuli.

Amygdala↗
PubMed2003

Facilitative role of endogenous oxytocin in noradrenaline release in the rat supraoptic nucleus.

Oxytocin is released not only from the axon terminals in the neurothypophysis but also from the dendrites in the hypothalamus. In the present study, we examined the role of dendritic oxytocin release in regulating presynaptic noradrenaline release within the hypothalamus. In vivo microdialysis experiments showed that local application of oxytocin augmented high-K+-induced noradrenaline release in the hypothalamic supraoptic nucleus. Oxytocin application to the hypothalamic synaptosomal preparation in vitro also potentiated high-K+-induced noradrenaline release. The effect of oxytocin was dose-dependent and was blocked by an oxytocin receptor antagonist. We then examined roles of oxytocin released from the dendrites using in vivo microdialysis. Local application of an oxytocin receptor antagonist impaired noradrenaline release in the supraoptic nucleus in response to high-K+ solution or noxious stimuli. An i.c.v. injection of an oxytocin receptor antagonist also impaired oxytocin release from the pituitary after noxious stimuli. These data suggest that dendritic oxytocin facilitates activation of oxytocin neurons, at least in part by augmentation of noradrenaline release via a presynaptic action.

Analysis of Variance↗
PubMed2003

Associations of blood glucose control with self-efficacy and rated anxiety/depression in type II diabetes mellitus patients.

We investigated the possible relations of hemoglobin A1c (HbA1c) level, a parameter of blood glucose control with scores on anxiety, depression, and self-efficacy for 113 outpatients (55 men and 58 women) with type II diabetes mellitus. The relation between the HbA1c level and the self-efficacy scores as well as with the ratings of anxiety and depression was evaluated. A significant relation was found for the HbA1c level with the scores; however, self-efficacy and anxiety and depression showed no association. These results suggest that HbA1c can be maintained at better levels by increasing self-efficacy of diabetic patients.

Adult↗
PubMed2002

Impaired interactions between mouse Eyal harboring mutations found in patients with branchio-oto-renal syndrome and Six, Dach, and G proteins.

Mutations in the EYA1 gene are responsible for branchio-oto-renal (BOR) syndrome as well as for other ocular defects. Most of the mutations are located within or in the vicinity of the EYA domain, which is highly conserved in the EYA protein family. The EYA domain is required for protein-protein interactions, which are important to the biological function of EYA proteins. To determine how EYA1 mutations cause BOR syndrome and/or ocular defects, we tested the effects of Eya1 mutations on interactions with Six. Dach, and G proteins by mammalian two-hybrid and GST-pulldown assays. Defective interactions were noted between BOR-type mutations S486P and L504R of Eya1 and Dach1, G proteins, and some Six proteins. These mutations impaired the activation of transcription from a Six-responsive gene, myogenin, with Six5. S486P and L504R showed an altered digestion pattern with trypsin, and L504R also decreased the sensitivity to V8 protease digestion and produced a peptide fragment with a different M(r). Our results suggest that defective protein-protein interactions of the mutations in the EYA domain underlie BOR syndrome and that SIX, DACH, and/or G proteins are possibly involved in the pathogenic processes.

Amino Acid Substitution↗
PubMed2002

The H1 and H2 regions of the activation domain of herpes simplex virion protein 16 stimulate transcription through distinct molecular mechanisms.

BACKGROUND: The Herpes Simplex Virion Protein 16 (VP16) contains a strong activation domain which can be subdivided into two regions, H1 and H2, both of which independently activate transcription in vivo. Several components of the basal transcription machinery have been shown to interact with the activation domain of VP16, mostly through the H1 region. RESULTS: We show that the H2 region binds directly to histone acetyltransferase, CBP (CREB (cAMP Responsive Element Binding Protein) Binding Protein) both in vivo and in vitro. The sites of interaction with the H2 region were mapped to both the amino- and carboxy-terminal segments of CBP. A mutation in the H2 region disrupts the interaction with CBP and abolishes the ability of VP16 to mediate in vitro transactivation from chromatin templates in an acetyl-CoA dependent manner. In contrast, human Mediator, another co-activator complex, binds specifically to both the H1 and H2 regions. CONCLUSION: The H1 and H2 regions of the VP16 activation domain activate transcription via distinct pathways. The H2 requires CBP for activation, whereas the H1 may function through Mediator and general transcription factors.

Binding Sites↗
PubMed2002

Molecular interaction and synergistic activation of a promoter by Six, Eya, and Dach proteins mediated through CREB binding protein.

Drosophila sine oculis, eyes absent, and dachshund are essential for compound eye formation and form a gene network with direct protein interaction and genetic regulation. The vertebrate homologues of these genes, Six, Eya, and Dach, also form a similar genetic network during muscle formation. To elucidate the molecular mechanism underlying the network among Six, Eya, and Dach, we examined the molecular interactions among the encoded proteins. Eya interacted directly with Six but never with Dach. Dach transactivated a multimerized GAL4 reporter gene by coproduction of GAL4-Eya fusion proteins. Transactivation by Eya and Dach was repressed by overexpression of VP16 or E1A but not by E1A mutation, which is defective for CREB binding protein (CBP) binding. Recruitment of CBP to the immobilized chromatin DNA template was dependent on FLAG-Dach and GAL4-Eya3. These results indicate that CBP is a mediator of the interaction between Eya and Dach. Contrary to our expectations, Dach binds to chromatin DNA by itself, not being tethered by GAL4-Eya3. Dach also binds to naked DNA with lower affinity. The conserved DD1 domain is responsible for binding to DNA. Transactivation was also observed by coproduction of GAL4-Six, Eya, and Dach, indicating that Eya and Dach synergy is relevant when Eya is tethered to DNA through Six protein. Our results demonstrated that synergy is mediated through direct interaction of Six-Eya and through the interaction of Eya-Dach with CBP and explain the molecular basis for the genetic interactions among Six, Eya, and Dach. This work provides fundamental information on the role and the mechanism of action of this gene cassette in tissue differentiation and organogenesis.

3T3 Cells↗