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271.
Fumio Kasai Noriko Hirayama Midori Ozawa Motonobu Satoh Arihiro Kohara 《Human cell》2018,31(3):261-267
Human cell lines represent a valuable resource as in vitro experimental models. A hepatoma cell line, HuH-7 (JCRB0403), has been used extensively in various research fields and a number of studies using this line have been published continuously since it was established in 1982. However, an accurate genome profile, which can be served as a reliable reference, has not been available. In this study, we performed M-FISH, SNP microarray and amplicon sequencing to characterize the cell line. Single cell analysis of metaphases revealed a high level of heterogeneity with a mode of 60 chromosomes. Cytogenetic results demonstrated chromosome abnormalities involving every chromosome in addition to a massive loss of heterozygosity, which accounts for 55.3% of the genome, consistent with the homozygous variants seen in the sequence analysis. We provide empirical data that the HuH-7 cell line is composed of highly heterogeneous cell populations, suggesting that besides cell line authentication, the quality of cell lines needs to be taken into consideration in the future use of tumor cell lines. 相似文献
272.
Hidekazu Tokuhara Yasuhiro Imaeda Yoshiyuki Fukase Koichi Iwanaga Naohiro Taya Koji Watanabe Ray Kanagawa Keisuke Matsuda Yumiko Kajimoto Keiji Kusumoto Mitsuyo Kondo Gyorgy Snell Craig A. Behnke Takanobu Kuroita 《Bioorganic & medicinal chemistry》2018,26(12):3261-3286
We previously identified 2-tert-butyl-4-[(3-methoxypropyl)amino]-N-(2-methylpropyl)-N-[(3S,5R)-5-(morpholin-4-ylcarbonyl)piperidin-3-yl]pyrimidine-5-carboxamide 3 as a potent renin inhibitor. Since 3 showed unacceptably low bioavailability (BA) in rats, structural modification, using SBDD and focused on physicochemical properties was conducted to improve its PK profile while maintaining renin inhibitory activity. Conversion of the amino group attached at the 4-position of pyrimidine to methylene group improved PK profile and decreased renin inhibitory activity. New central cores with carbon side chains were explored to improve potency. We had designed a series of 5-membered azoles and fused heterocycles that interacted with the lipophilic S3 pocket. In the course of modification, renin inhibitory activity was enhanced by the formation of an additional hydrogen bonding with the hydroxyl group of Thr77. Consequently, a series of novel benzimidazole derivatives were discovered as potent and orally bioavailable renin inhibitors. Among those, compound 13 exhibited more than five-fold of plasma renin inhibition than aliskiren in cynomolgus monkeys at dose ratio. 相似文献
273.
Machida M Kosako H Shirakabe K Kobayashi M Ushiyama M Inagawa J Hirano J Nakano T Bando Y Nishida E Hattori S 《The FEBS journal》2007,274(6):1576-1587
Prefractionation procedures facilitate the identification of lower-abundance proteins in proteome analysis. Here we have optimized the conditions for immobilized metal affinity chromatography (IMAC) to enrich for phosphoproteins. The metal ions, Ga(III), Fe(III), Zn(II), and Al(III), were compared for their abilities to trap phosphoproteins; Ga(III) was the best. Detailed analyses of the pH and ionic strength for IMAC enabled us to determine the optimal conditions (pH 5.5 and 0.5 m NaCl). When whole cell lysates were fractionated in this way, about one-tenth of the total protein was recovered in the eluate, and the recovery of phosphorylated extracellular signal-regulated kinase (ERK) was more than 90%. Phosphorylated forms of ribosomal S6 kinase (RSK) and Akt were also enriched efficiently under the same conditions. Our Ga(III) IMAC and a commercially available purification kit for phosphoproteins performed similarly, with a slight difference in the spectrum of phosphoproteins. When phosphoproteins enriched from NIH3T3 cells in which ERK was either activated or suppressed were analyzed by two-dimensional fluorescence difference gel electrophoresis, phosphorylated ERK was detected as discrete spots unique to ERK-activated cells, which overlapped with surrounding spots in the absence of prefractionation. We applied the same technique to search for Akt substrates and identified Abelson interactor 1 as a novel potential target. These results demonstrate the efficacy of phosphoprotein enrichment by IMAC and suggest that this procedure will be of general use in phosphoproteome research. 相似文献
274.
Hatano Jiro Kusama Shoko Tanaka Kenya Kohara Ayaka Miyake Chikahiro Nakanishi Shuji Shimakawa Ginga 《Photosynthesis research》2022,153(1-2):113-120
Photosynthesis Research - Live cyanobacteria and algae integrated onto an extracellular electrode can generate a light-induced current (i.e., a photocurrent). Although the photocurrent is expected... 相似文献
275.
Mashiba T Udaka K Hirachi Y Hiasa Y Miyakawa T Satta Y Osoda T Kataoka S Kohara M Onji M 《Immunogenetics》2007,59(3):197-209
Developing a peptide-based vaccine for the highly variable hepatitis C virus (HCV) remains a challenging task. Variant viruses
not only escape antigen presentation but also persist in a patient as quasi-species. Such variants are often antagonistic
to the responding T cell repertoire. To overcome these problems, we herein propose a cocktail vaccine consisting of a few
epitope peptides, which make it possible to outpace the emergence of variant viruses. To design such a vaccine, we developed
a way to identify HLA-A*2402-binding peptides efficiently by means of the computational scanning of the whole genome of the
pathogen. Most of the predicted peptides exhibited strong binding to the HLA-A*2402 molecule, while also inducing CD8 T cell
responses from the patients’ peripheral blood mononuclear cells (PBMCs). Peptide-induced T cells were capable of lysing HCV-expressing
HepG2 cells which process antigens endogenously. The amount of HCV core antigen in the patients’ livers suggested that the
lytic activity of the peptide-induced T cells was clearly in a range suitable for therapeutic use. If T cells were activated
under optimal conditions by high density peptides, then they tended to be relatively tolerant of single amino acid variations
for cytolysis. Finally, an analysis of the viral population isolated in Japan suggested no obvious changes due to immune evasion
in the viral genome even in a host population highly biased toward HLA-A*2402.
Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. 相似文献
276.
277.
278.
Ono K Satoh M Yoshida T Ozawa Y Kohara A Takeuchi M Mizusawa H Sawada H 《In vitro cellular & developmental biology. Animal》2007,43(5-6):168-175
We developed a highly sensitive and convenient method of nested polymerase chain reaction (PCR) targeted to mitochondrial
deoxyribonucleic acid (DNA) to identify animal species quickly in cultured cells. Fourteen vertebrate species, including human,
cynomolgus monkey, African green monkey, mouse, rat, Syrian hamster, Chinese hamster, guinea pig, rabbit, dog, cat, cow, pig,
and chicken, could be distinguished from each other by nested PCR. The first PCR amplifies mitochondrial DNA fragments with
a universal primer pair complementary to the conserved regions of 14 species, and the second PCR amplifies the DNA fragments
with species-specific primer pairs from the first products. The species-specific primer pairs were designed to easily distinguish
14 species from each other under standard agarose gel electrophoresis. We further developed the multiplex PCR using a mixture
of seven species-specific primer pairs for two groups of animals. One was comprised of human, mouse, rat, cat, pig, cow, and
rabbit, and the other was comprised of African green monkey, cynomolgus monkey, Syrian hamster, Chinese hamster, guinea pig,
dog, and chicken. The sensitivity of the PCR assay was at least 100 pg DNA/reaction, which was sufficient for the detection
of each species of DNA. Furthermore, the nested PCR method was able to identify the species in the interspecies mixture of
DNA. Thus, the method developed in this study will provide a useful tool for the authentication of animal species. 相似文献
279.
The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells 总被引:124,自引:0,他引:124
Mitsui K Tokuzawa Y Itoh H Segawa K Murakami M Takahashi K Maruyama M Maeda M Yamanaka S 《Cell》2003,113(5):631-642
Embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocysts grow infinitely while maintaining pluripotency. Leukemia inhibitory factor (LIF) can maintain self-renewal of mouse ES cells through activation of Stat3. However, LIF/Stat3 is dispensable for maintenance of ICM and human ES cells, suggesting that the pathway is not fundamental for pluripotency. In search of a critical factor(s) that underlies pluripotency in both ICM and ES cells, we performed in silico differential display and identified several genes specifically expressed in mouse ES cells and preimplantation embryos. We found that one of them, encoding the homeoprotein Nanog, was capable of maintaining ES cell self-renewal independently of LIF/Stat3. nanog-deficient ICM failed to generate epiblast and only produced parietal endoderm-like cells. nanog-deficient ES cells lost pluripotency and differentiated into extraembryonic endoderm lineage. These data demonstrate that Nanog is a critical factor underlying pluripotency in both ICM and ES cells. 相似文献
280.