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51.
Yoneda T Kumagai T Nagatomo I Furukawa M Yamane H Hoshino S Mori M Takeda Y Horai T Nishida S Watanabe D Kijima T Yoshida M Osaki T Tachibana I Greene MI Kawase I 《DNA and cell biology》2006,25(9):530-540
EGFR is involved in the density-dependent inhibition of cell growth, while coexpression of EGFR with erbB2 can render normal cells transformed. In this study, we have examined the effect of a species of p185 that contains the transmembrane domain and the extracellular domain of p185(c-neu), on growth properties of a human malignant mesothelioma cell line that coexpresses EGFR and erbB2. The ectodomain form of p185(c-neu) enhanced density-dependent inhibition of cell growth and we found that p21 induction appeared to be responsible for this inhibitory effect. Previously, the extracellular domain species was shown to suppress the transforming abilities of EGFR and p185(c-neu/erbB2) in a dominant-negative manner. The ability of this subdomain to affect tumor growth is significant, as it reduced in vivo tumor growth. Unexpectedly, we found that the domain did not abrogate all of EGFR functions. We noted that EGFR-induced density-dependent inhibition of cell growth was retained. Tyrosine kinase inhibitors of EGFR did not cause density-dependent inhibition of cell growth of malignant mesothelioma cells. Therefore, simultaneously inhibiting the malignant phenotype and inducing density-dependent inhibition of cell growth in malignant mesothelioma cells by the extracellular domain of p185(c-neu) may represent an important therapeutic advance. 相似文献
52.
Reduced Contamination by the Fusarium Mycotoxin Zearalenone in Maize Kernels through Genetic Modification with a Detoxification Gene 总被引:4,自引:0,他引:4
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Tomoko Igawa Naoko Takahashi-Ando Noriyuki Ochiai Shuichi Ohsato Tsutomu Shimizu Toshiaki Kudo Isamu Yamaguchi Makoto Kimura 《Applied microbiology》2007,73(5):1622-1629
Maize is subject to ear rot caused by toxigenic Aspergillus and Fusarium species, resulting in contamination with aflatoxins, fumonisins, trichothecenes, and zearalenone (ZEN). The trichothecene group and ZEN mycotoxins are produced by the cereal pathogen Fusarium graminearum. A transgenic detoxification system for the elimination of ZEN was previously developed using an egfp::zhd101 gene (gfzhd101), encoding an enhanced green fluorescent protein fused to a ZEN-degrading enzyme. In this study, we produced a transgenic maize line expressing an intact copy of gfzhd101 and examined the feasibility of transgene-mediated detoxification in the kernels. ZEN-degrading activity has been detected in transgenic kernels during seed maturation (for a period of 6 weeks after pollination). The level of detoxification activity was unaltered after an additional storage period of 16 weeks at 6°C. When the seeds were artificially contaminated by immersion in a ZEN solution for 48 h at 28°C, the total amount of the mycotoxin in the transgenic seeds was uniformly reduced to less than 1/10 of that in the wild type. The ZEN in the transgenic maize kernels was also efficiently decontaminated under conditions of lower water activity (aw) and temperature; e.g., 16.9 μg of ZEN was removed per gram of seed within 48 h at an aw of 0.90 at 20°C. F. graminearum infection assays demonstrated an absence of ZEN in the transgenic maize seeds, while the mycotoxin accumulated in wild-type kernels under the same conditions. Transgene-mediated detoxification may offer simple solutions to the problems of mycotoxin contamination in maize. 相似文献
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55.
Eiji Takita Katsunori Kohda Hajime Tomatsu Shigeru Hanano Kanami Moriya Tsutomu Hosouchi Nozomu Sakurai Hideyuki Suzuki Atsuhiko Shinmyo Daisuke Shibata 《DNA research》2013,20(6):583-592
Ligation, the joining of DNA fragments, is a fundamental procedure in molecular cloning and is indispensable to the production of genetically modified organisms that can be used for basic research, the applied biosciences, or both. Given that many genes cooperate in various pathways, incorporating multiple gene cassettes in tandem in a transgenic DNA construct for the purpose of genetic modification is often necessary when generating organisms that produce multiple foreign gene products. Here, we describe a novel method, designated PRESSO (precise sequential DNA ligation on a solid substrate), for the tandem ligation of multiple DNA fragments. We amplified donor DNA fragments with non-palindromic ends, and ligated the fragment to acceptor DNA fragments on solid beads. After the final donor DNA fragments, which included vector sequences, were joined to the construct that contained the array of fragments, the ligation product (the construct) was thereby released from the beads via digestion with a rare-cut meganuclease; the freed linear construct was circularized via an intra-molecular ligation. PRESSO allowed us to rapidly and efficiently join multiple genes in an optimized order and orientation. This method can overcome many technical challenges in functional genomics during the post-sequencing generation. 相似文献
56.
A preponderance of small, dense (sd) LDL is strongly associated with the development of coronary heart disease, but the method for the measurement of sd LDL is too laborious for clinical use. We report a simple method for the quantification of sd LDL that is applicable to an autoanalyzer. This method consists of two steps: first, to precipitate the lipoprotein of density (d) <1.044 g/ml using heparin-magnesium; and second, to measure LDL-cholesterol in the supernatant by the homogeneous method or apolipoprotein B (apoB) by an immunoturbidometric assay. The cholesterol and apoB values obtained by the precipitation method (45 +/- 26 and 33 +/- 20 mg/dl, respectively) were similar to those obtained in the lipoprotein (d = 1.044-1.063) separated by ultracentrifugation (42 +/- 22 and 31 +/- 17 mg/dl, respectively), and there was an excellent correlation between the two methods for sd LDL-cholesterol (y = 1.05X + 1, r = 0.88, n = 69) and apoB (y = 1.07X, r = 0.90). Sd LDL values had a significant inverse correlation with LDL size. A high correlation was found between sd LDL-cholesterol and apoB values (r = 0.94). Sd LDL value was related to triglyceride, apoB, and LDL-cholesterol, but not to the buoyant LDL level. These results suggest that this precipitation method is a simple and rapid method for the measurement of sd LDL concentration. 相似文献
57.
Nishi M Yasue A Nishimatu S Nohno T Yamaoka T Itakura M Moriyama K Ohuchi H Noji S 《Biochemical and biophysical research communications》2002,293(1):247-251
Myostatin, which is a member of the TGF-beta superfamily, is a negative regulator of skeletal muscle formation. Double-muscled Piedmontese cattle have a C313Y mutation in myostatin and show increased skeletal muscle mass which resulted from an increase of myofiber number (hyperplasia) without that of myofiber size (hypertrophy). To examine whether this mutation in myostatin gene affects muscle development in a dominant negative manner, we generated transgenic mice overexpressing the mutated gene. The transgenic mice exhibited dramatic increases in the skeletal muscle mass resulting from hyperplasia without hypertrophy. In contrast, it has been reported that a myostatin mutated at its cleavage site produces hypertrophy without hyperplasia in the muscle. Thus, these results suggest that (1) the myostatin containing the missense mutation exhibits a dominant negative activity and that (2) there are two types in the dominant negative form of myostatin, causing either hypertrophy or hyperplasia. 相似文献
58.
Tsutomu Ishikawa Tetsuya Tosa Ichiro Chibata 《Bioscience, biotechnology, and biochemistry》2013,77(7):412-416
?-Lysine acylase of Achromobacter pestifer EA was purified by fractionations with ammonium sulfate and acetone, and by vertical zone electrophoresis. As a result, this bacterial ?-lysine acylase was obtained as an electrophoretically homogeneous protein, specific activity of which is the highest among ?-lysine acylases ever reported. 相似文献
59.
Takashima N Odaka YS Sakoori K Akagi T Hashikawa T Morimura N Yamada K Aruga J 《PloS one》2011,6(7):e22716
Recent genetic linkage analysis has shown that LRRTM1 (Leucine rich repeat transmembrane neuronal 1) is associated with schizophrenia. Here, we characterized Lrrtm1 knockout mice behaviorally and morphologically. Systematic behavioral analysis revealed reduced locomotor activity in the early dark phase, altered behavioral responses to novel environments (open-field box, light-dark box, elevated plus maze, and hole board), avoidance of approach to large inanimate objects, social discrimination deficit, and spatial memory deficit. Upon administration of the NMDA receptor antagonist MK-801, Lrrtm1 knockout mice showed both locomotive activities in the open-field box and responses to the inanimate object that were distinct from those of wild-type mice, suggesting that altered glutamatergic transmission underlay the behavioral abnormalities. Furthermore, administration of a selective serotonin reuptake inhibitor (fluoxetine) rescued the abnormality in the elevated plus maze. Morphologically, the brains of Lrrtm1 knockout mice showed reduction in total hippocampus size and reduced synaptic density. The hippocampal synapses were characterized by elongated spines and diffusely distributed synaptic vesicles, indicating the role of Lrrtm1 in maintaining synaptic integrity. Although the pharmacobehavioral phenotype was not entirely characteristic of those of schizophrenia model animals, the impaired cognitive function may warrant the further study of LRRTM1 in relevance to schizophrenia. 相似文献
60.
Umeda N Suzuki T Yukawa M Ohya Y Shindo H Watanabe K Suzuki T 《The Journal of biological chemistry》2005,280(2):1613-1624
Human mitochondrial (mt) tRNA(Lys) has a taurine-containing modified uridine, 5-taurinomethyl-2-thiouridine (taum5s2U), at its anticodon wobble position. We previously found that the mt tRNA(Lys), carrying the A8344G mutation from cells of patients with myoclonus epilepsy associated with ragged-red fibers (MERRF), lacks the taum5s2U modification. Here we describe the identification and characterization of a tRNA-modifying enzyme MTU1 (mitochondrial tRNA-specific 2-thiouridylase 1) that is responsible for the 2-thiolation of the wobble position in human and yeast mt tRNAs. Disruption of the yeast MTU1 gene eliminated the 2-thio modification of mt tRNAs and impaired mitochondrial protein synthesis, which led to reduced respiratory activity. Furthermore, when MTO1 or MSS1, which are responsible for the C5 substituent of the modified uridine, was disrupted along with MTU1, a much more severe reduction in mitochondrial activity was observed. Thus, the C5 and 2-thio modifications act synergistically in promoting efficient cognate codon decoding. Partial inactivation of MTU1 in HeLa cells by small interference RNA also reduced their oxygen consumption and resulted in mitochondria with defective membrane potentials, which are similar phenotypic features observed in MERRF. 相似文献