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Biomedical subjects

H Kondoh

Publications and source records attributed to H Kondoh.

At least 55 records · Page 3Linked to original sources
PubMed1999

Transcription factors for lens development assessed in vivo.

Lens-cell differentiation occurs at a fairly early stage of embryogenesis and results in very simple tissue architecture. These features allow the embryonic lens to provide a paradigm of tissue development starting from tissue induction to tissue maturation. Not only have a number of transcription factors participating in lens development been identified but their actual functions are now assessed by modern approaches utilizing genetic and tissue manipulations of embryos.

Animals↗
PubMed1999

Mouse Lefty2 and zebrafish antivin are feedback inhibitors of nodal signaling during vertebrate gastrulation.

Mammalian lefty and zebrafish antivin form a subgroup of the TGF beta superfamily. We report that mouse mutants for lefty2 have an expanded primitive streak and form excess mesoderm, a phenotype opposite to that of mutants for the TGF beta gene nodal. Analogously, overexpression of Antivin or Lefty2 in zebrafish embryos blocks head and trunk mesoderm formation, a phenotype identical to that of mutants caused by loss of Nodal signaling. The lefty2 mutant phenotype is partially suppressed by heterozygosity for nodal. Similarly, the effects of Antivin and Lefty2 can be suppressed by overexpression of the nodal-related genes cyclops and squint or the extracellular domain of ActRIIB. Expression of antivin is dependent on Nodal signaling, revealing a feedback loop wherein Nodal signals induce their antagonists Lefty2 and Antivin to restrict Nodal signaling during gastrulation.

Activin Receptors, Type II↗
PubMed1999

Expression of tyrosinase, TRP-1 and TRP-2 in ultraviolet-irradiated human melanomas and melanocytes: TRP-2 protects melanoma cells from ultraviolet B induced apoptosis.

Tyrosinase related protein (TRP)-1 and TRP-2 are known to regulate the quality of melanin, and recently their potential role in inhibiting apoptosis have also been reported. To study the role of tyrosinase, TRP-1 and TRP-2 in the growth, differentiation and cell death of ultraviolet B (UVB) irradiated melanocytes, the expression of these proteins in amelanotic and melanotic cells was examined. Expression of tyrosinase and TRP-1 correlated with melanin content, which was upregulated after repeated irradiation of melanotic cells by low doses of UVB. In contrast, the expression and activity of TRP-2 correlated with cell proliferation, but not with pigmentation. In one melanotic melanoma cell line, significant suppression of cell proliferation was observed after low or high doses of UVB irradiation, possibly due to apoptotic changes. TRP-2 expression was remarkably reduced in UVB-irradiated cells, and transfection with TRP-2 expression vector rescued these cells from UVB-induced apoptosis. These results indicate that TRP-2 expression is closely associated with the regulation of cell growth/survival of melanocytes exposed to UVB and that TRP-2 plays a role in protecting melanoma cells from UVB-induced apoptosis.

Adult↗
PubMed1999

Mechanism of regulatory target selection by the SOX high-mobility-group domain proteins as revealed by comparison of SOX1/2/3 and SOX9.

SOX proteins bind similar DNA motifs through their high-mobility-group (HMG) domains, but their action is highly specific with respect to target genes and cell type. We investigated the mechanism of target selection by comparing SOX1/2/3, which activate delta-crystallin minimal enhancer DC5, with SOX9, which activates Col2a1 minimal enhancer COL2C2. These enhancers depend on both the SOX binding site and the binding site of a putative partner factor. The DC5 site was equally bound and bent by the HMG domains of SOX1/2 and SOX9. The activation domains of these SOX proteins mapped at the distal portions of the C-terminal domains were not cell specific and were independent of the partner factor. Chimeric proteins produced between SOX1 and SOX9 showed that to activate the DC5 enhancer, the C-terminal domain must be that of SOX1, although the HMG domains were replaceable. The SOX2-VP16 fusion protein, in which the activation domain of SOX2 was replaced by that of VP16, activated the DC5 enhancer still in a partner factor-dependent manner. The results argue that the proximal portion of the C-terminal domain of SOX1/2 specifically interacts with the partner factor, and this interaction determines the specificity of the SOX1/2 action. Essentially the same results were obtained in the converse experiments in which COL2C2 activation by SOX9 was analyzed, except that specificity of SOX9-partner factor interaction also involved the SOX9 HMG domain. The highly selective SOX-partner factor interactions presumably stabilize the DNA binding of the SOX proteins and provide the mechanism for regulatory target selection.

Animals↗
PubMed1999

Identification of CtBP1 and CtBP2 as corepressors of zinc finger-homeodomain factor deltaEF1.

deltaEF1, a representative of the zinc finger-homeodomain protein family, is a transcriptional repressor which binds E2-box (CACCTG) and related sequences and counteracts the activators through transrepression mechanisms. It has been shown that the N-proximal region of the protein is involved in the transrepression. Here we demonstrate that deltaEF1 has a second mechanism of transrepression recruiting CtBP1 or CtBP2 as its corepressor. A two-hybrid screen of mouse cDNAs with various portions of deltaEF1 identified these proteins, which bind to deltaEF1 in a manner dependent on the PLDLSL sequence located in the short medial (MS) portion of deltaEF1. CtBP1 is the mouse orthologue of human CtBP, known as the C-terminal binding protein of adenovirus E1A, while CtBP2 is the second homologue. Fusion of mouse CtBP1 or CtBP2 to Gal4DBD (Gal4 DNA binding domain) made them Gal4 binding site-dependent transcriptional repressors in transfected 10T1/2 cells, indicating their involvement in a transcriptional repression mechanism. When the MS portion of deltaEF1 was used to Gal4DBD and used to transfect cells, a strong transrepression activity was generated, but this activity was totally dependent on the PLDLSL sequence which served as the site for interaction with endogenous CtBP proteins, indicating that CtBP1 and -2 can act as corepressors. Exogenous CtBP1/2 significantly enhanced transcriptional repression by deltaEF1, and this enhancement was lost if the PLDLSL sequence was altered, demonstrating that CtBP1 and -2 act as corepressors of deltaEF1. In the mouse, CtBP1 is expressed from embryo to adult, but CtBP2 is mainly expressed during embryogenesis. In developing embryos, CtBP1 and CtBP2 are expressed broadly with different tissue preferences. Remarkably, their high expression occurs in subsets of deltaEF1-expressing tissues, e.g., cephalic and dorsal root ganglia, spinal cord, posterior-distal halves of the limb bud mesenchyme, and perichondrium of forming digits, supporting the conclusion that CtBP1 and -2 play crucial roles in the repressor action of deltaEF1 in these tissues.

Alcohol Oxidoreductases↗
PubMed1999

Regulation of Gli2 and Gli3 activities by an amino-terminal repression domain: implication of Gli2 and Gli3 as primary mediators of Shh signaling.

Gli family zinc finger proteins are mediators of Sonic hedgehog (Shh) signaling in vertebrates. The question remains unanswered, however, as to how these Gli proteins participate in the Shh signaling pathway. In this study, regulatory activities associated with the Gli2 protein were investigated in relation to the Shh signaling. Although Gli2 acts as a weak transcriptional activator, it is in fact a composite of positive and negative regulatory domains. In cultured cells, truncation of the activation domain in the C-terminal half results in a protein with repressor activity, while removal of the repression domain at the N terminus converts Gli2 into a strong activator. In transgenic mouse embryos, N-terminally truncated Gli2, unlike the full length protein, activates a Shh target gene, HNF3beta, in the dorsal neural tube, thus mimicking the effect of Shh signal. This suggests that unmasking of the strong activation potential of Gli2 through modulation of the N-terminal repression domain is one of the key mechanisms of the Shh signaling. A similar regulatory mechanism involving the N-terminal region was also found for Gli3, but not for Gli1. When the Shh signal derived from the notochord is received by the neural plate, the widely expressed Gli2 and Gli3 proteins are presumably converted to their active forms in the ventral cells, leading to activation of transcription of their target genes, including Gli1.

Amino Acid Sequence↗
PubMed1999

Isolation and characterization of a novel TP53-inducible gene, TP53TG3.

We applied the differential mRNA display method to isolate genes regulated by wild-type TP53 in cells of a colon-cancer line (SW480) in which we had established an inducible TP53 expression system under the control of the lactose operon. Here we report isolation and characterization of a novel TP53-inducible gene, termed TP53TG3 (TP53 target gene 3). Its DNA sequence was identical to sequences present in two BAC clones that had been mapped to chromosome band 16p13. The gene expressed several transcripts by alternative splicing; the two major transcripts, TP53TG3a and TP53TG3b, encoded 124- and 132-amino-acid peptides that were expressed predominantly in testis. Immunohistochemical analysis using cancer cells (HeLa or H1299) that had been transfected with plasmid DNA designed to express the MYC-fused TP53TG3 proteins indicated that these products were present mainly in the cytoplasm 20 hr after transfection. However, 40 hr after transfection, the recombinant proteins had accumulated in the nuclei of some cells. Because no known nuclear localization domain was present in the amino acid sequence, we suspect that this protein plays an important role in the TP53-mediated signaling pathway, when it forms complexes with other protein(s) and is transferred by them into the nucleus. Genes Chromosomes Cancer 26:329-335, 1999.

Amino Acid Sequence↗
PubMed1998

lefty-1 is required for left-right determination as a regulator of lefty-2 and nodal.

lefty-1, lefty-2, and nodal are expressed on the left side of developing mouse embryos and are implicated in left-right (L-R) determination. The role of lefty-1 was examined by analyzing mutant mice lacking this gene. The lefty-1-deficient mice showed a variety of L-R positional defects in visceral organs. Unexpectedly, however, the most common feature of lefty-1-/- mice was thoracic left isomerism (rather than right isomerism). The lack of lefty-1 resulted in bilateral expression of nodal, lefty-2, and Pitx2 (a homeobox gene normally expressed on the left side). These observations suggest that the role of lefty-1 is to restrict the expression of lefty-2 and nodal to the left side, and that lefty-2 or nodal encodes a signal for "leftness."

Animals↗
PubMed1998

Identification and functional analysis of the mouse lens filensin gene promoter.

Filensin (also called CP94; CP95; CP97; 115kDa protein) is a component of the lens-specific beaded filament which is believed to be functionally important in lens fiber cell differentiation and in maintaining lens fiber cell conformation and transparency. A 17.2kb fragment containing the 5'-upstream sequence of the filensin gene was isolated. S1-mapping analysis determined the transcription start point (tsp; +1) which locates at 94base pairs upstream from the initiating ATG on the filensin gene. In addition to a major tsp, a minor tsp (-136) was observed. DNA sequence of the fragment around the tsp (-2144 to +155) was identified. Analysis of the DNA sequence of the promoter region around tsp revealed two motifs with sequence homology to Sox2 and Maf recognition sequences in addition to one GATA-1 site, two Sp1 binding sites, and three AP-2 binding motifs. No TATA-box or CCAAT-motif was found around the tsp region. A series of sequentially deleted fragments of (-2144 to +40) were fused to firefly luciferase reporter plasmid pGL2 and tested for activity in chicken embryonic lens explants. A minimal promoter region for mouse filensin of (-70 to +40) was identified. The lens-specific promoter activity was detected using lens explants cultured within 12h after dissection. The activity was remarkably enhanced by culture in the presence of 5ng/ml of basic fibroblast growth factor. Each one of the Sp1 and AP-2 binding motifs was localized to the fragment of (-27 to +40) using electrophoretic mobility shift assays. These are the first data to identify the basic elements to the 5'-upstream sequences of the filensin gene, namely the tsp and the minimal filensin promoter.

Animals↗
PubMed1998

Sox1 directly regulates the gamma-crystallin genes and is essential for lens development in mice.

gamma-Crystallins are major structural components of the lens fiber cells in amphibians and mammals. Many dominant inherited cataracts in humans and mice have been shown to map within the gamma-crystallin gene cluster. Several transcription factors, including PAX6 and SOX proteins, have been suggested as candidates for crystallin gene regulation. Here we show that the targeted deletion of Sox1 in mice causes microphthalmia and cataract. Mutant lens fiber cells fail to elongate, probably as a result of an almost complete absence of gamma-crystallins. It appears that the direct interaction of the SOX1 protein with a promoter element conserved in all gamma-crystallin genes is responsible for their expression.

Animals↗
PubMed1998

Expression of the glutathione peroxidase gene lacking its 3' untranslated region.

In order to investigate the expression of human cellular glutathione peroxidase (GPx), we mutated the gene encoding GPx by deleting either the 5' or 3' untranslated region (utr), subcloned the deleted fragments into plasmid pSVL followed by transfection into COS-7 cells and measured the amount of GPx expressed. When the 5' utr of the gene was deleted, GPx was not expressed. However, the deletion of the 3' utr resulted in some expression of GPx. Deletion of the poly A region of the GPx gene resulted in the expression of GPx but the level was lower than that of the full-length cGPx. The complete deletion of the 3' utr resulted in a half of the expression of the poly A deletion mutant. Thus, the expression of GPx increased according to the length of the 3' utr. These results suggest that the GPx gene carrying one SECIS on 5' utr (FEBS Lett. 312(1992)10-14) is essential for GPx expression. SECIS on 3' utr might not play a key role of GPx expression. Expression of GPx by COS-7 cells was not observed when a plasmid harboring an antisense gene was transfected.

Animals↗
PubMed1998

Site-specific confocal fluorescence imaging of biological microstructures in a turbid medium.

Normally transparent biological structures in a turbid medium are imaged using a laser confocal microscope and multiwavelength site-specific fluorescence labelling. The spatial filtering capability of the detector pinhole in the confocal microscope limits the number of scattered fluorescent photons that reach the photodetector. Simultaneous application of different fluorescent markers on the same sample site minimizes photobleaching by reducing the excitation time for each marker. A high-contrast grey-level image is also produced by summing confocal images of the same site taken at different fluorescence wavelengths. Monte Carlo simulations are performed to obtain the quantitative behaviour of confocal fluorescence imaging in turbid media. Confocal images of the following samples were also obtained: (i) 15 microm diameter fluorescent spheres placed 1.16 mm deep beneath an aqueous suspension of 0.0823 microm diameter polystyrene latex spheres, and (ii) hindbrain of a whole-mount mouse embryo (age 10 days) that was stained to fluoresce at 515 nm and 580 nm peak wavelengths. Expression of RNA transcripts of a gene within the embryo hindbrain was detected by a fluorescence-based whole-mount in situ hybridization procedure that we recently tested.

Animals↗
PubMed1998

Enhanced melanogenesis induced by tyrosinase gene-transfer increases boron-uptake and killing effect of boron neutron capture therapy for amelanotic melanoma.

Specific and powerful cancer killing effect for melanoma by boron neutron capture therapy (BNCT) using DOPA analogue, 10B-p-boronophenylalanine (10B-BPA), has been established, but amelanotic melanoma is insufficiently responsive to 10B-BPA BNCT in comparison with actively melanin-producing melanoma. Although the accumulation mechanism of 10B-BPA within melanoma was not established, we have recently obtained findings suggesting that melanin monomers, key intermediates for melanin polymer formation, play a critical role in 10B-BPA accumulation. In addition, there are some kinds of human amelanotic melanomas, such as MEL2A, in which expression of tyrosinase is repressed or lacking though tyrosinase-related protein (TRP)-1 and TRP-2 are well expressed. Thus, by using a similarly tyrosinase-lacking mouse amelanotic melanoma cell line, A1059, we constructed TA1059 cells by transfecting human tyrosinase-cDNA into these cells. TA1059 cells acquired higher DOPA-oxidase and DOPAchrome tautomerase activity as well as eumelanin content at even higher levels than those of B16F10 cells. TA1059 cells showed about 2.5 times higher P-boronophenylalanine (BPA) uptake than A1059 cells in culture. In animal experiments, by using these cell lines, tumor growth of TA1059 was significantly suppressed by 10B-BPA BNCT as compared with A1059. These findings indicate that the induction of active melanin biosynthesis by melanogenic gene-transfer effectively improves the treatment of amelanotic melanoma by BNCT.

Animals↗
PubMed1998

DeltaEF1, a zinc finger and homeodomain transcription factor, is required for skeleton patterning in multiple lineages.

DeltaEF1 is a DNA binding protein containing a homeodomain and two zinc finger clusters, and is regarded as a vertebrate homologue of zfh-1 (zinc finger homeodomain-containing factor-1) in Drosophila. In the developing embryo, deltaEF1 is expressed in the notochord, somites, limb, neural crest derivatives and a few restricted sites of the brain and spinal cord. To elucidate the regulatory function of deltaEF1 in mouse embryogenesis, we generated deltaEF1 null mutant (deltaEF1null(lacZ)) mice. The deltaEF1null(lacZ) homozygotes developed to term, but never survived postnatally. In addition to severe T cell deficiency of the thymus, the deltaEF1null(lacZ) homozygotes exhibited skeletal defects of various lineages. (1) Craniofacial abnormalities of neural crest origin: cleft palate, hyperplasia of Meckel's cartilage, dysplasia of nasal septum and shortened mandible. (2) Limb defects: shortening and broadening of long bones, fusion of carpal/tarsal bone and fusion of joints. (3) Fusion of ribs. (4) Sternum defects: split and asymmetric ossification pattern of the sternebrae associated with irregular sternocostal junctions. (5) Hypoplasia of intervertebral discs. These results indicate that deltaEF1 has an essential role in regulating development of these skeletal structures. Since the skeletal defects were not observed in deltaEF1deltaC727 mice, deltaEF1 bears distinct regulatory activities which are dependent on different domains of the molecule.

Abnormalities, Multiple↗
PubMed1998

Involvement of Sox1, 2 and 3 in the early and subsequent molecular events of lens induction.

Activation of the first lens-specific gene of the chicken, delta 1-crystallin, is dependent on a group of lens nuclear factors, deltaEF2, interacting with the delta1-crystallin minimal enhancer, DC5. One of the deltaEF2 factors was previously identified as SOX2. We show that two related SOX proteins, SOX1 and SOX3, account for the remaining members of deltaEF2. Activation of the DC5 enhancer is dependent on their C-terminal domains. Expression of Sox1-3 in the eye region during lens induction was studied in comparison with Pax6 and delta1-crystallin. Pax6, known to be required for the inductive response of the ectoderm, is broadly expressed in the lateral head ectoderm from before lens induction. After tight association of the optic vesicle (around stage 10-11, 40 hours after egg incubation), expression of Sox2 and Sox3 is activated in the vesicle-facing ectoderm at stage 12 (44 hours). These cells, expressing together Pax6 and Sox2/3, subsequently give rise to the lens, beginning with formation of the lens placode and expression of delta-crystallin at stage 13 (48 hours). Sox1 then starts to be expessed in the lens-forming cells at stage 14. When the prospective retina area of the neural plate was unilaterally ablated at stage 7, expression of Sox2/3 was lost in the side of lateral head ectoderm lacking the optic cup, implying that an inductive signal from the optic cup activates Sox2/3 expression. In the mouse embryonic lens, this subfamily of Sox genes is expressed in an analogous fashion, although Sox3 transcripts have not been detected and Sox2 expression is down-regulated when Sox1 is activated. In ectodermal tissues of the chicken embryo, delta -crystallin expression occurs in a few ectopic sites. These are always characterized by overlapping expression of Sox2/3 and Pax6. Thus, an essential molecular event in lens induction is the 'turning on' of the transcriptional regulators SOX2/3 in the Pax6-expressing ectoderm and these SOX proteins activate crystallin gene expression. Continued activity, especially of SOX1, is then essential for further development of the lens.

Amino Acid Sequence↗
PubMed1997

Impairment of T cell development in deltaEF1 mutant mice.

Using the method of gene targeting in mouse embryonic stem cells, regulatory function of deltaEF1, a zinc finger and homeodomain-containing transcription factor, was investigated in vivo by generating the deltaEF1 mutant mice. The mutated allele of deltaEF1 produced a truncated form of the deltaEF1 protein lacking a zinc finger cluster proximal to COOH terminus. The homozygous deltaEF1 mutant mice had poorly developed thymi with no distinction of cortex and medulla. Analysis of the mutant thymocyte showed reduction of the total cell number by two orders of magnitude accompanying the impaired thymocyte development. The early stage intrathymic c-kit+ T precursor cells were largely depleted. The following thymocyte development also seemed to be affected as assessed by the distorted composition of CD4- or CD8-expressing cells. The mutant thymocyte showed elevated alpha4 integrin expression, which might be related to the T cell defect in the mutant mice. In the peripheral lymph node tissue of the mutant mice, the CD4-CD8+ single positive cells were significantly reduced relative to CD4+CD8-single positive cells. In contrast to T cells, other hematopoietic lineages appeared to be normal. The data indicated that deltaEF1 is involved in regulation of T cell development at multiple stages.

Animals↗
PubMed1997

Time and flow study results before and after installation of a hospital information system and radiology information system and before clinical use of a picture archiving and communication system.

The effectiveness of a hospital information system (HIS) and a radiological information system (RIS) was evaluated to optimize preparation for the planned full clinical operation of a picture archiving and communication system (PACS), which is now linked experimentally to the HIS and the RIS. One thousand IC (integrated circuit) cards were used for time studies and flow studies in the hospital. Measurements were performed on image examination order entry, image examination, reporting, and image delivery times. Even though after the HIS and the RIS operation only a small amount of time savings were realized in each time fraction component, such as in the patient movement time, examination time, and film delivery time, the total turn-around time was shortened markedly, by more than 23 hours on average. It was verified that the HIS and the RIS was beneficial in the outpatient clinics of the orthopedic department. Our method of measurement employing IC cards before and after HIS and RIS operations can be applied in other hospitals.

Computers↗