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221.
222.
Watanabe T Saito D Tanabe K Suetsugu R Nakaya Y Nakagawa S Takahashi Y 《Developmental biology》2007,305(2):625-636
The in ovo electroporation technique in chicken embryos has enabled investigators to uncover the functions of numerous developmental genes. In this technique, the ubiquitous promoter, CAGGS (CMV base), has often been used for overexpression experiments. However, if a given gene plays a role in multiple steps of development and if overexpression of this gene causes fatal consequences at the time of electroporation, its roles in later steps of development would be overlooked. Thus, a technique with which expression of an electroporated DNA can be controlled in a stage-specific manner needs to be formulated. Here we show for the first time that the tetracycline-controlled expression method, "tet-on" and "tet-off", works efficiently to regulate gene expression in electroporated chicken embryos. We demonstrate that the onset or termination of expression of an electroporated DNA can be precisely controlled by timing the administration of tetracycline into an egg. Furthermore, with this technique we have revealed previously unknown roles of RhoA, cMeso-1 and Pax2 in early somitogenesis. In particular, cMeso-1 appears to be involved in cell condensation of a newly forming somite by regulating Pax2 and NCAM expression. Thus, the novel molecular technique in chickens proposed in this study provides a useful tool to investigate stage-specific roles of developmental genes. 相似文献
223.
Stable integration and conditional expression of electroporated transgenes in chicken embryos 总被引:2,自引:0,他引:2
Sato Y Kasai T Nakagawa S Tanabe K Watanabe T Kawakami K Takahashi Y 《Developmental biology》2007,305(2):616-624
The in ovo electroporation in chicken embryos has widely been used as a powerful tool to study roles of genes during embryogenesis. However, the conventional electroporation technique fails to retain the expression of transgenes for more than several days because transgenes are not integrated into the genome. To overcome this shortcoming, we have developed a transposon-mediated gene transfer, a novel technique in chicken manipulations. It was previously reported that the transposon Tol2, originally found in medaka fish, facilitates an integration of a transgene into the genome when co-acting with Tol2 transposase. In this study, we co-electroporated a plasmid containing a CAGGS-EGFP cassette cloned in the Tol2 construct along with a transposase-encoding plasmid into early presomitic mesoderm or optic vesicles of chicken embryos. This resulted in persistent expression of EGFP at least until embryonic day 8 (E8) and E12 in somite-derived tissues and developing retina, respectively. The integration of the transgene was confirmed by genomic Southern blotting using chicken cultured cells. We further combined this transposon-mediated gene transfer with the tetracycline-dependent conditional expression system that we also developed recently. With this combined method, expression of a stably integrated transgene could be experimentally induced upon tetracycline administration at relatively late stages such as E6, where a variety of organogenesis are underway. Thus, the techniques proposed in this study provide a novel approach to study the mechanisms of late organogenesis, for which chickens are most suitable model animals. 相似文献
224.
Lysosomal cathepsins in embryonic programmed cell death 总被引:1,自引:0,他引:1
Zuzarte-Luis V Montero JA Kawakami Y Izpisua-Belmonte JC Hurle JM 《Developmental biology》2007,301(1):205-217
During limb development, expression of cathepsin D and B genes prefigure the pattern of interdigital apoptosis including the differences between the chick and the webbed digits of the duck. Expression of cathepsin L is associated with advanced stages of degeneration. Analysis of Gremlin-/- and Dkk-/- mouse mutants and local treatments with BMP proteins reveal that the expression of cathepsin B and D genes is regulated by BMP signaling, a pathway responsible for triggering cell death. Further cathepsin D protein is upregulated in the preapoptotic mesenchyme before being released into the cytosol, and overexpression of cathepsin D induces cell death in embryonic tissues by a mechanism including mitochondrial permeabilization and nuclear translocation of AIF. Combined inhibition of cathepsin and caspases suggests a redundancy in the apoptotic molecular machinery, providing evidence for compensatory activation mechanisms in the cathepsin pathway when caspases are blocked. It is concluded that lysosomal enzymes are functionally implicated in embryonic programmed cell death. 相似文献
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Yoko Nakagawa Naofumi Nakayama Hitoshi Goto Ikuhide Fujisawa Soda Chanthamath Kazutaka Shibatomi Seiji Iwasa 《Chirality》2019,31(1):52-61
Computational chemical analysis of Ru(II)‐Pheox–catalyzed highly enantioselective intramolecular cyclopropanation reactions was performed using density functional theory (DFT). In this study, cyclopropane ring–fused γ‐lactones, which are 5.8 kcal/mol more stable than the corresponding minor enantiomer, are obtained as the major product. The results of the calculations suggest that the enantioselectivity of the Ru(II)‐Pheox–catalyzed intramolecular cyclopropanation reaction is affected by the energy differences between the starting structures 5l and 5i . The reaction pathway was found to be a stepwise mechanism that proceeds through the formation of a metallacyclobutane intermediate. This is the first example of a computational chemical analysis of enantioselective control in an intramolecular carbene‐transfer reaction using C1‐symmetric catalysts. 相似文献
228.
Tetsuya Hirata Atsushi Kobayashi Tamio Furuse Ikuko Yamada Masaru Tamura Hiroyuki Tomita Yuko Tokoro Akinori Ninomiya Yoshitaka Fujihara Masahito Ikawa Yusuke Maeda Yoshiko Murakami Yasuhiko Kizuka Taroh Kinoshita 《The Journal of biological chemistry》2022,298(3)
Glycosylphosphatidylinositol (GPI) is a posttranslational glycolipid modification of proteins that anchors proteins in lipid rafts on the cell surface. Although some GPI-anchored proteins (GPI-APs), including the prion protein PrPC, have a glycan side chain composed of N-acetylgalactosamine (GalNAc)−galactose−sialic acid on the core structure of GPI glycolipid, in vivo functions of this GPI-GalNAc side chain are largely unresolved. Here, we investigated the physiological and pathological roles of the GPI-GalNAc side chain in vivo by knocking out its initiation enzyme, PGAP4, in mice. We show that Pgap4 mRNA is highly expressed in the brain, particularly in neurons, and mass spectrometry analysis confirmed the loss of the GalNAc side chain in PrPC GPI in PGAP4-KO mouse brains. Furthermore, PGAP4-KO mice exhibited various phenotypes, including an elevated blood alkaline phosphatase level, impaired bone formation, decreased locomotor activity, and impaired memory, despite normal expression levels and lipid raft association of various GPI-APs. Thus, we conclude that the GPI-GalNAc side chain is required for in vivo functions of GPI-APs in mammals, especially in bone and the brain. Moreover, PGAP4-KO mice were more vulnerable to prion diseases and died earlier after intracerebral inoculation of the pathogenic prion strains than wildtype mice, highlighting the protective roles of the GalNAc side chain against prion diseases. 相似文献
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230.
Tanaka AR Ikeda Y Abe-Dohmae S Arakawa R Sadanami K Kidera A Nakagawa S Nagase T Aoki R Kioka N Amachi T Yokoyama S Ueda K 《Biochemical and biophysical research communications》2001,283(5):1019-1025
ABCA1 has been suggested to play a key role in cellular lipid release from peripheral cells. In order to study structure-function relationship of this protein, the protein product of a full-length human ABCA1 cDNA was examined for its functions and topological orientation. The electrophoretic mobilities of human ABCA1 expressed in transfected cells increased when treated with N-glycosidase F, suggesting that ABCA1 is highly glycosylated. The ABCA1 was photoaffinity-labeled with ATP and mediated the apoA-I-dependent-release of cholesterol and phospholipid. The influenza hemagglutinin (HA) epitope was introduced into the amino-terminus (N-HA) or between the residues 207 and 208 (207-HA) of the protein. While an antibody against the C-terminus peptide of ABCA1 detected both fusion proteins, an anti-HA antibody did not react with the N-HA fusion protein. Confocal microscopy demonstrated strong cell surface signal with the anti-HA antibody of nonpermeabilized HEK293 cells expressing the 207-HA fusion protein. The results suggested that the signal peptide in the amino-terminal region is cleaved off in its mature form and that the following large hydrophilic region is exposed to outside of cells unlike previously proposed models. We found that this amino-terminal extracellular domain contains a segment homologous to the autoantigen SS-N, an epitope of Sj?gren's syndrome, and further identified that ABCA7 codes for the autoantigen SS-N. 相似文献