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Yuko Hayashi

Publications and source records attributed to Yuko Hayashi.

17 recordsLinked to original sources
PubMed2007

Complex interference in the eye developmental pathway by Drosophila NF-YA.

The CCAAT motif-binding factor NF-Y consists of three different subunits, NF-YA, NF-YB, and NF-YC, all of which are required for formation of the NF-Y complex and DNA-binding. NF-YA contains a DNA binding domain in its C-terminal region. We established transgenic fly lines carrying the UAS-HA-dNF-YA or UAS-dNF-YAIR and showed over-expression or knockdown with various GAL4 drivers to be lethal at various developmental stages, suggesting that dNF-YA participate in various gene regulatory pathways during Drosophila development. Expression of dNF-YA with eyeless-GAL4 mainly resulted in lethality with a headless phenotype in pharate-adults. Reduction of the eyeless gene dose enhanced the dNF-YA-induced phenotype, while reduction of the Distal-less gene dose suppressed the phenotype. On the other hand, crossing the dNF-YA over-expressing flies with Notch mutant resulted in no apparent effect on the phenotype. These results suggest that dNF-YA can disturb eye disc specification, but not eye disc growth.

Amino Acid Sequence↗
PubMed2006

Enalapril improves impairment of SERCA-derived relaxation and enhancement of tyrosine nitration in diabetic rat aorta.

We investigated the involvement of angiotensin II and vascular smooth muscle sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) function in the impaired NO-induced relaxation seen in established streptozotocin-induced diabetes. Plasma angiotensin II levels, which were elevated in untreated diabetic rats (vs age-matched controls), were improved by treatment with the angiotensin-converting enzyme inhibitor enalapril. Systolic blood pressure was significantly decreased in chronic enalapril-treated diabetics (vs the other two groups). Intact aortae from diabetic rats and chronic angiotensin II-infused control rats, but not those from diabetic rats treated with enalapril, showed impaired endothelium-dependent relaxations to acetylcholine (vs controls). The relaxation induced by Angeli's Salt (a NO donor) was significantly impaired in endothelium-denuded aortae from diabetic rats (vs controls) but it was normalised by enalapril treatment. After preincubation with the irreversible SERCA inhibitor, thapsigargin, the relaxation induced by Angeli's Salt was significantly impaired in endothelium-denuded aortae from the controls, but not from the diabetics, and there was no significant difference between the thapsigargin-treated groups. Nitrotyrosine, an indirect marker of peroxynitrite, was markedly increased in aortic smooth muscle from diabetic rats, while chronic enalapril administration reduced this increase. These results suggest that in streptozotocin-induced diabetic rats, excessive angiotensin II production may lead to the generation of peroxynitrite and that this may in turn trigger a dysfunction of vascular smooth muscle SERCA. Enalapril improved the diabetes-related impairments.

Acetylcholine↗
PubMed2006

Drosophila distal-less negatively regulates dDREF by inhibiting its DNA binding activity.

The Drosophila DNA replication-related element binding factor (dDREF) is required for expression of many proliferation-related genes carrying the DRE sequence, 5'-TATCGATA. Over-expression of dDREF in the eye imaginal disc induces ectopic DNA synthesis, apoptosis and inhibition of photoreceptor cell specification, and results in rough eye phenotype in adults. In the present study, half dose reduction of the Distal-less (Dll) gene enhanced the dDREF-induced rough eye phenotype, suggesting that Dll negatively regulates dDREF activity in eye imaginal disc cells. Biochemical analyses revealed the N-terminal (30aa to 124aa) and C-terminal (190aa to 327aa) regions of Dll to interact with the DNA binding domain (16aa to 125aa) of dDREF, although it is not clear yet whether the interaction is direct or indirect. Electrophoretic mobility shift assays showed that Dll thereby inhibits DNA binding. The repression of this dDREF-function by a homeodomain protein like Dll may contribute to the differentiation-coupled repression of cell proliferation during development.

Animals↗
PubMed2006

ANG II enhances contractile responses via PI3-kinase p110 delta pathway in aortas from diabetic rats with systemic hyperinsulinemia.

We investigated the involvement of ANG II and phosphatidylinositol 3-kinase (PI3-K) in the enhanced aortic contractile responses induced by hyperinsulinemia in chronic insulin-treated Type 1 diabetic rats. Plasma ANG II levels were elevated in untreated compared with control diabetic rats and further increased in insulin-treated diabetic rats. Aortic contractile responses and systolic blood pressure were significantly enhanced in chronic insulin-treated diabetic rats compared with the other groups. These insulin-induced increases were largely prevented by cotreatment with losartan (an ANG II type 1 receptor antagonist) or enalapril (an angiotensin-converting enzyme inhibitor). LY-294002 (a PI3-K inhibitor) diminished the increases in contractile responses in ANG II-incubated aortas and aortas from chronic insulin-treated diabetic rats. The norepinephrine (NE)-stimulated levels of p110 delta-associated PI3-K activity and p110 delta protein expression were increased in aortas from insulin-treated diabetic compared with control and untreated diabetic rats, and chronic administration of losartan blunted these increases. Contractions were significantly larger in aortas from diabetic rats incubated with a low concentration (inducing approximately 10% of the maximum contraction) of ANG II or with NE or isotonic K+ than in aortas from nonincubated diabetic rats. NE-stimulated p110 PI3-K activity was elevated in aortas from diabetic rats coincubated with a noncontractile dose of ANG II. These results suggest that, in insulin-treated Type 1 diabetic rats with hyperinsulinemia, chronic ANG II type 1 receptor blockade blunts the increases in vascular contractility and blood pressure via a decrease in p110 delta-associated PI3-K activity.

Angiotensin II↗
PubMed2006

Antagonistic regulation of the Drosophila PCNA gene promoter by DREF and Cut.

The gene promoter of Drosophila proliferating cell nuclear antigen (dPCNA) contains several transcriptional regulatory elements, such as upstream regulatory element (URE), DNA replication-related element (DRE, 5'-TATCGATA), and E2F recognition sites. In the present study, a yeast one-hybrid screen using three tandem repeats of DRE in dPCNA promoter as the bait allowed isolation of a cDNA encoding Cut, a Drosophila homolog of mammalian CCAAT-displacement protein (CDP)/Cux. Electrophoretic mobility shift assays showed that Cut bound to both DRE and the sequence 5'-AATCAAAC in URE, with much higher affinity to the former. Measurement of dPCNA promoter activity by transient luciferase expression assays in Drosophila S2 cells after an RNA interference for Cut or DREF showed DREF activates the dPCNA promoter while Cut functions as a repressor. Chromatin immunoprecipitation assays in the presence or absence of 20-hydroxyecdysone further showed both DREF and Cut proteins to be localized in the genomic region containing the dPCNA promoter in S2 cells, especially in the Cut case upon induction of differentiation. These results indicate that Cut functions as a transcriptional repressor of dPCNA gene by binding to the promoter region in the differentiated state, while DREF binds to DRE to promote expression of dPCNA during cell proliferation.

Animals↗
PubMed2005

Changes in aortic endothelial gene expressions and relaxation responses following chronic short-term insulin treatment in diabetic rats.

The purpose of the present study was to examine the relationship between the changes in the expressions of several mRNAs and changes in endothelial function in streptozotocin-induced diabetic and chronic short-term insulin-treated rats. Aortas from later-stage (10 week) diabetics, but not those from their insulin-treated counterparts, showed an impaired endothelial function. We found that the mRNA expressions for 30 genes were significantly upregulated, while those for 13 other genes were downregulated in aortic endothelial cells from diabetes. In later-stage diabetes, chronic insulin treatment ameliorated the endothelial dysfunction and normalized the expressions for 20 out of the 43 genes altered in diabetes. Further, 12 of the remaining 23 genes were altered by high-dose insulin treatment in the controls. In early-stage (1 week) diabetic aortas, which did not show impaired endothelial function, expression changes were shown by only 12/30 and 5/13 of the genes up- or downregulated, respectively, in later-stage diabetes. Thus, in the diabetic aortas endothelial gene expressions and function exhibited time-related changes, and several gene expressions and endothelial function were normalized by insulin treatment. The hyperinsulinemia caused by this treatment may oppose the alterations in some gene expressions and the endothelial proliferation (cell growth-related gene expressions) that occur in established diabetes.

Animals↗
PubMed2005

Identification of trichoplein, a novel keratin filament-binding protein.

Keratins 8 and 18 (K8/18) are major components of the intermediate filaments (IFs) of simple epithelia. We report here the identification of a novel protein termed trichoplein. This protein shows a low degree of sequence similarity to trichohyalin, plectin and myosin heavy chain, and is a K8/18-binding protein. Among interactions between trichoplein and various IF proteins that we tested using two-hybrid methods, trichoplein interacted significantly with K16 and K18, and to some extent with K5, K6a, K8 and K14. In in vitro co-sedimentation assays, trichoplein directly binds to K8/18, but not with vimentin, desmin, actin filaments or microtubules. An antibody raised against trichoplein specifically recognized a polypeptide with a relative molecular mass of 61 kDa in cell lysates. Trichoplein was immunoprecipitated using this antibody in a complex with K8/18 and immunostaining revealed that trichoplein colocalized with K8/18 filaments in HeLa cells. In polarized Caco-2 cells, trichoplein colocalized not only with K8/18 filaments in the apical region but also with desmoplakin, a constituent of desmosomes. In the absorptive cells of the small intestine, trichoplein colocalized with K8/18 filaments at the apical cortical region, and was also concentrated at desmosomes. Taken together, these results suggest that trichoplein is a keratin-binding protein that may be involved in the organization of the apical network of keratin filaments and desmosomes in simple epithelial cells.

Amino Acid Sequence↗
PubMed2005

Cytotoxic and mutagenic effects of chronic low-dose-rate irradiation on TERT-immortalized human cells.

To analyze the genetic effects of low-dose-rate radiation on human cells, we used human telomere reverse transcriptase (TERT)-immortalized fibroblast cells obtained from normal individuals. We studied the effect of low-dose-rate (0.3 mGy/ min) and high-dose-rate (2 Gy/min) radiation on cells in a confluent state. Survival and micronucleus induction frequency showed higher resistance after irradiation at low dose rate than at high dose rate. The survival after 5 Gy of high-dose-rate radiation was 0.01 compared to 0.3 after low-dose-rate irradiation at the same dose. In accordance with this, the level of HPRT mutation induction by low-dose-rate radiation decreased to approximately one-eighth that for high-dose-rate radiation. We then characterized the mutants by multiplex PCR analysis, which showed that the fraction of deletion mutations was lower in the mutant cells induced at low dose rate than at high dose rate. Furthermore, the size of the deletions in mutant cells induced by low-dose-rate radiation appeared to be smaller than those in mutant cells irradiated at high dose rate. Only a few exons were deleted in the former mutants while all exons were deleted in most of the latter mutants. The present study indicates that the genetic effects of low-dose-rate radiation on nonproliferating normal human cells are quantitatively and qualitatively less severe than the effect of high-dose-rate radiation.

Cell Survival↗
PubMed2004

Characterization of dRFX2, a novel RFX family protein in Drosophila.

A transcriptional regulatory element was identified in the region between URE (upstream regulatory element) and DRE (DNA replication-related element) in the Drosophila PCNA gene promoter. This element plays an important role in promoter activity in living flies. A yeast one-hybrid screening using this element as a bait allowed isolation of a cDNA encoding a protein which binds to the element in vitro. Nucleotide sequence analyses revealed that the cDNA encodes a novel protein containing a characteristic DNA-binding domain conserved among the regulatory factor X (RFX) family proteins. We termed this protein Drosophila RFX2 (dRFX2) and this element dRFX2 site. To investigate the function of dRFX2 in vivo, we took the strategy of analyzing the dominant negative effects against the endogenous dRFX2. Transgenic flies were established in which expression of HA-dRFX(202-480) carrying the amino acid sequences from 202 to 480 containing the RFX domain (DNA-binding domain) of dRFX2 was targeted to the cells in the eye imaginal discs. In the eye imaginal disc expressing the HA-dRFX(202-480), the G1-S transition and/or the progression of S phase were/was interrupted, and the ectopic apoptosis was induced, though photoreceptor cells differentiated normally. These results indicate that dRFX2 plays a role in G1-S transition and/or in progression of S phase.

Animals↗
PubMed2004

Armadillo/Pangolin regulates PCNA and DREF promoter activities.

Here we show that Armadillo and Pangolin (dTCF), downstream effectors of the Wingless (Wg) signal transduction pathway, activate transcription of the important DNA replication-related genes encoding Drosophila proliferating cell nuclear antigen (PCNA) and DNA replication-related element-binding factor (DREF). By transient luciferase expression assays and band mobility shift assays, we demonstrated the PCNA gene to be a direct target gene for the Armadillo/Pangolin complex. Using a GAL4-UAS system, stimulation of the PCNA gene by Armadillo/Pangolin was confirmed in adult females. From the published reports of an inhibitory role, we expected that Drosophila CREB-binding protein (dCBP) would interfere with activation. However, effects were only observed with the DREF but not the PCNA gene. In the latter case, as in mammals, dCBP could potentiate Armadillo-mediated activation. These results suggest that first, PCNA and DREF genes are targets of the Armadillo/Pangolin complex and second, dCBP modulates Wg signaling in a gene-specific manner.

Animals↗
PubMed2004

Genetic link between p53 and genes required for formation of the zonula adherens junction.

Ectopic expression of human p53 in Drosophila eye imaginal disc cells induces apoptosis and results in a rough eye phenotype in the adult flies. We have screened Drosophila stocks to identify mutations that enhance or suppress the p53-induced rough eye phenotype. One of the dominant enhancers of the p53-induced rough eye phenotype corresponds to a loss-of-function mutation of the crumbs gene, which is essential for the biogenesis of the zonula adherens junction and the establishment of apical polarity in epithelial cells. Enhancement of p53-induced apoptosis in the eye imaginal discs by a half-reduction of the crumbs gene dose was confirmed by a TUNEL method. Furthermore, mutations of genes for Shotgun (Drosophila E-cadherin) and Armadillo (Drosophila beta-catenin), the two main components of the adherens junction, also strongly enhanced the p53-induced rough eye phenotype. These results suggest that human p53 senses subtle abnormality at the adherens junction or in signals derived from the junction, and consequently induces apoptosis to remove abnormal cells from tissue. Thus p53 likely plays a role as a guardian of the tissue not only by sensing the damaged DNA, but also by sensing signals from the adherens junction.

Adherens Junctions↗
PubMed2004

No induction of p53 phosphorylation and few focus formation of phosphorylated H2AX suggest efficient repair of DNA damage during chronic low-dose-rate irradiation in human cells.

Human fibroblast cells obtained from a normal individual and immortalized by introduction of the hTERT gene were irradiated with 0 to 5 Gy of acute high-dose-rate radiation (1.8 Gy/min) or chronic low-dose-rate radiation (0.3 mGy/min) in the G0 phase, and p53 activation was studied. After high-dose-rate irradiation, a dose-dependent induction of Ser15 phosphorylation was observed, whereas after low-dose-rate irradiation almost none was observed. Then we analyzed the focus formation of phosphorylated histone H2AX protein, which is closely correlated with the induction of double-strand breaks. High-dose-rate radiation induced a significant number of foci in a dose-dependent manner, whereas, low-dose-rate radiation could induce only a few foci even at the highest dose. These results strongly suggest that DNA damage induced by low-dose-rate radiation such as a double-strand break is efficiently repaired during chronic irradiation.

Blotting, Western↗
PubMed2002

Fluorescent whitening agents in Tokyo Bay and adjacent rivers: their application as anthropogenic molecular markers in coastal environments.

Two kinds of stilbene-type fluorescent whitening agents (i.e., DSBP and DAS1), minor components of laundry detergents, were analyzed in surface waters of Tokyo Bay and adjacent rivers and in sewage effluents to examine their usefulness as molecular markers in the marine environment. Sensitive determination using HPLC (high performance liquid chromatography) with fluorescence detection with postcolumn UV radiator was employed. DSBP and DAS1 were found in Tokyo rivers at concentrations of a few microg/L and approximately 1 microg/L, respectively. DSBP and DAS1 were widely distributed in Tokyo Bay waters at concentrations in the range of 0.019-0.264 microg/L and 0.021-0.127 microg/L, respectively. Comparison of these concentrations with those in sewage effluents (DSBP: 8 microg/L and DAS1: 2.5 microg/L on average) yielded sewage dilutions in Tokyo Bay on the order of 10(2). FWAs-salinity diagram in the Tamagawa Estuary showed fairly conservative behaviors of the FWAs with approximately 20% and approximately 10% removal of DSBP and DAS1, respectively. This is thought to be caused by photodegradation. The persistent nature of FWAs and their widespread distribution in coastal environments demonstrates the utility of FWAs in tracing the behavior of water from rivers and sewage outfalls. The DSBP/DAS1 ratio showed a decreasing trend from sewage effluents, to rivers, to Tokyo Bay, indicating selective photodegradation of DSBP. The DSBP/DAS1 ratio is proposed as an index of the degree of photodegradation and residence time and freshness of water mass in coastal environments.

Biphenyl Compounds↗
PubMed2002

Multiple retinal holes in the macular region: a case report.

PURPOSE: To describe the first case of multiple retinal holes in the macular region successfully treated with vitrectomy. METHOD: A single case report. A 44-year-old man was treated for impaired vision caused by multiple macular holes in the right eye. RESULTS: Fundus examinations detected six retinal holes in the right macula and a giant macular hole in the left eye. Optical coherence tomography disclosed multiple vitreoretinal adhesions in the right macula. We vitrectomized the right eye to produce posterior vitreous detachment and then tamponaded the vitreous cavity with 20% SF(6) gas, resulting in closure of holes and improvement of the vision. CONCLUSION: Multiple macular holes may be treated by surgical posterior detachment combined with gas tamponade. Vitreoretinal adhesions are a possible cause of these holes. Similar multiple holes may have coalesced into one giant hole in this patient's left eye.

Adult↗
PubMed2002

Establishment of immortal normal and ataxia telangiectasia fibroblast cell lines by introduction of the hTERT gene.

To establish immortal human cells, we introduced the human catalytic subunit of telomerase (hTERT) gene into skin fibroblast cells obtained from normal and ataxia telangiectasia (AT) individuals of Japanese origin. After hTERT introduction, these cells continue to grow beyond a population doubling number of 200 while maintaining their original radiosensitivity. Inductions of p53, phosphorylation of Ser15 in p53, and induction of p21 by X-ray irradiation in immortal cells derived from normal individual were not affected by the hTERT introduction. Both normal and AT immortal cells exhibited an apparent inhibition of growth as original primary cells when they reached confluence. Karyotype analysis has revealed that they are in a diploid range. These results suggest that cells immortalized by hTERT introduction retain their original characteristics except for immortalization, and that they may be useful for analyzing various effects of radiation on human cells.

Ataxia Telangiectasia↗
PubMedDate not supplied

Cornea with Peters' anomaly: perturbed differentiation of corneal cells and abnormal extracellular matrix in the corneal stroma.

PURPOSE: We examined histopathologically the anterior ocular segment including the cornea and lens of an eye which had been enucleated in a patient with Peters' anomaly because of untreatable corneal perforation. Special effort was made to differentiate the corneal stromal and endothelial cells, and the stromal extracellular matrix. METHODS: Light microscopy, with hematoxylin and eosin staining, and transmission electron microscopy were employed. RESULTS: Corneal endothelial cells and Descemet's membrane were not detected in the central cornea, where there were immature cells with a fibroblastic configuration. The inner surface of the peripheral cornea was covered with cells containing pigment granules in the cytoplasm. Cell density in the central corneal stroma was relatively high. The diameter of the stromal collagen fibrils was not uniform. A mature collagen fibril-free area was also seen in the central corneal stroma. CONCLUSIONS: Differentiation of neural crest-derived cells in corneal stroma and endothelium might have been perturbed in the cornea of this patient with Peters' anomaly, inducing the defect in the corneal endothelium and the qualitative and quantitative abnormalities of the extracellular matrix.

Cell Count↗