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81.
Background
Construction of plasmids is crucial in modern genetic manipulation. As of now, the common method for constructing plasmids is to digest specific DNA sequences with restriction enzymes and to ligate the resulting DNA fragments with DNA ligase. Another potent method to construct plasmids, known as gap-repair cloning (GRC), is commonly used in the budding yeast Saccharomyces cerevisiae. GRC makes use of the homologous recombination activity that occurs within the yeast cells. Due to its flexible design and efficiency, GRC has been frequently used for constructing plasmids with complex structures as well as genome-wide plasmid collections. Although there have been reports indicating GRC feasibility in the fission yeast Schizosaccharomyces pombe, this species is not commonly used for GRC as systematic studies of reporting GRC efficiency in S. pombe have not been performed till date.Methodology/Principal Findings
We investigated GRC efficiency in S. pombe in this study. We first showed that GRC was feasible in S. pombe by constructing a plasmid that contained the LEU2 auxotrophic marker gene in vivo and showed sufficient efficiency with short homology sequences (>25 bp). No preference was shown for the sequence length from the cut site in the vector plasmid. We next showed that plasmids could be constructed in a proper way using 3 DNA fragments with 70% efficiency without any specific selections being made. The GRC efficiency with 3 DNA fragments was dramatically increased >95% in lig4Δ mutant cell, where non-homologous end joining is deficient. Following this approach, we successfully constructed plasmid vectors with leu1+, ade6+, his5+, and lys1+ markers with the low-copy stable plasmid pDblet as a backbone by applying GRC in S. pombe.Conclusions/Significance
We concluded that GRC was sufficiently feasible in S. pombe for genome-wide gene functional analysis as well as for regular plasmid construction. Plasmids with different markers constructed in this research are available from NBRP-yeast (http://yeast.lab.nig.ac.jp/). 相似文献82.
Akio Watanabe Hitomi Ito Megumi Chiba Azumi Ito Hirono Shimizu Shin-ichi Fuji Shin-ichi Nakamura Hiroyuki Hattori Mitsuo Chino Namiko Satoh-Nagasawa Hidekazu Takahashi Kenji Sakurai Hiromori Akagi 《Planta》2010,232(4):825-836
We are interested in elucidating the molecular mechanisms underlying plant reactions to the toxic heavy metal cadmium (Cd). To this end, we devised a new screening strategy using agar plates with a gradient of Cd concentrations, termed Cd-gradient agar plates (CGAPs), to isolate Arabidopsis mutants that displayed altered reactions to the metal. Arabidopsis M2 seeds, derived from ethyl methanesulfonate (EMS) treated seeds, were germinated on the CGAPs such that the primary root of each seedling elongated against increasing concentrations of Cd on the surface of the plate. Under these conditions, the lengths of the primary roots reliably demonstrated the degree of Cd tolerance of individual seedlings. The use of CGAPs also allowed close observation of the root reaction of each seedling to Cd without causing lethal damage. The screen identified three mutant lines, MRC-32, MRC-22 and MRC-26, which showed distinctly different characteristics. MRC-32 plants exhibited enhanced tolerance to Cd and contained Cd at higher concentrations than wild-type (WT) plants treated with the heavy metal. The whole root system of MRC-22 plants showed a Cd-phobic response. MRC-26 plants accumulated less Cd in their aboveground tissues than WT plants, suggesting that they were defective in transporting the heavy metal from roots to aboveground tissues. We also determined the likely chromosomal location of each mutation. 相似文献
83.
Risa Nakamura Naoyoshi Maeda Kensuke Shibata Hisakata Yamada Tetsuo Kase Yasunobu Yoshikai 《Journal of virology》2010,84(11):5574-5582
Highly pathogenic influenza A viruses cause acute severe pneumonia to which the occurrence of “cytokine storm” has been proposed to contribute. Here we show that interleukin-15 (IL-15) knockout (KO) mice exhibited reduced mortality after infection with influenza virus A/FM/1/47 (H1N1, a mouse-adapted strain) albeit the viral titers of these mice showed no difference from those of control mice. There were significantly fewer antigen-specific CD44+ CD8+ T cells in the lungs of infected IL-15 KO mice, and adoptive transfer of the CD8+ T cells caused reduced survival of IL-15 KO mice following influenza virus infection. Mice deficient in β2-microglobulin by gene targeting and those depleted of CD8+ T cells by in vivo administration of anti-CD8 monoclonal antibody displayed a reduced mortality rate after infection. These results indicate that IL-15-dependent CD8+ T cells are at least partly responsible for the pathogenesis of acute pneumonia caused by influenza A virus.Highly pathogenic influenza A viruses cause acute severe pneumonia that results in high morbidity and significant mortality (11, 12, 24, 26). Elevated levels of serum cytokines and chemokines accompany these clinical manifestations, and the possibility that this “cytokine storm” contributes to increased severity of the disease caused by avian H5N1 virus and by other strains of influenza A virus has been proposed (10, 21, 33). In fact, CCR2-deficient mice [CCR2 is chemokine (C-C motif) receptor 2] were protected from early pathological manifestations despite higher pulmonary titers of the influenza virus A/PR/8/34 (H1N1) strain (7). Tumor necrosis factor receptor 1 (TNFR-1)-deficient mice exhibited significantly reduced morbidity following challenge with H5N1 virus (31). Other cytokines or chemokines have also been investigated (8, 28, 34, 35, 38). Thus, at least some of the elevated proinflammatory cytokines may contribute to the pathogenesis of influenza A virus.Interleukin-15 (IL-15) is a pleiotropic cytokine involved in both innate and adaptive immune responses (20, 36). IL-15 utilizes the β-chain of the IL-2 receptor (IL-2R) (CD122) and the common cytokine receptor γ-chain (CD132) for signal transduction in lymphocytes and therefore shares many biological properties with IL-2 (3). Memory CD8+ T cells, natural killer (NK) cells, NKT cells, and intraepithelial lymphocyte (IEL) T cells (15, 23, 42) decrease in mice with defective IL-15 signaling, indicating the importance of IL-15 in their development and/or maintenance. IL-15 regulates not only the number of memory CD8+ T cells but also activation of their functions, including gamma interferon (IFN-γ) production and cytotoxic activity (40), which are important to target the virus (9). Therefore, it is possible that we may be able to use IL-15 as an immune-enhancing molecular adjuvant in vaccines for protection against various pathogens, including influenza A virus (37).In the present study, we demonstrate that IL-15 knockout (KO) mice exhibited high resistance against infection with mouse-adapted influenza virus A/FM/1/47 (H1N1) strain. We show for the first time that IL-15-dependent CD8+ T cells are at least partly responsible for the pathogenesis of acute pneumonia caused by influenza A virus. In addition, our observations are important in the light of recent research into the use of IL-15 as an adjuvant for vaccination. 相似文献
84.
85.
So Nishimura Naoyoshi Nagata Takuro Shimbo Naoki Asayama Junichi Akiyama Norio Ohmagari Hirohisa Yazaki Shinichi Oka Naomi Uemura 《PloS one》2013,8(3)
Background
Candidia esophagitis (CE) is an AIDS-defining condition, usually occurring in individuals with low CD4 counts of <200 cells/µL. Endoscopy is a valuable definitive diagnostic method for CE but may not be indicated for asymptomatic patients or for those with high CD4 counts or without oral candidiasis. This study assessed such patients to clarify the factors associated with CE and its severity on endoscopy in the highly active antiretroviral therapy (HAART) era.Methodology/ Principal Findings
A total of 733 HIV-infected patients who underwent upper gastrointestinal (GI) endoscopy were analyzed. Sexual behavior, CD4+ count, HIV-RNA viral load (VL), history of HAART, GI symptoms, GI diseases, and oral candidiasis were assessed. Endoscopic severity of CE was classified as mild (Kodsi''s grade I/II) or severe (grade III/IV). Of the 733 subjects, 62 (8.46%) were diagnosed with CE (mild, n = 33; severe, n = 29). Of them, 56.5% (35/62) had no GI symptoms, 30.6% (19/62) had CD4 + ≥200 cells/μL, and 55.3% (21/38) had no oral candidiasis. Univariate analysis found lower CD4+ counts, higher HIV VL, and no history of HAART to be significantly associated with CE. With lower CD4+ counts and higher HIV VL, CE occurrence increased significantly (P<0.01 for trend in odds). Multivariate analysis showed low CD4+ counts and high HIV VL to be independently associated with CE. Of the severe CE patients, 55.2% (16/29) had no GI symptoms and 44.4% (8/18) had no oral candidiasis. Median CD4+ counts in severe cases were significantly lower than in mild cases (27 vs. 80; P = 0.04).Conclusions
Low CD4+ counts and high HIV VL were found to be factors associated with CE, and advanced immunosuppression was associated with the development of severity. Endoscopy is useful as it can detect CE, even severe CE, in patients without GI symptoms, those with high CD4 counts, and those without oral candidiasis. 相似文献86.
Naoyoshi Nagata Takuro Shimbo Hirohisa Yazaki Naoki Asayama Junichi Akiyama Katsuji Teruya Toru Igari Norio Ohmagari Shinichi Oka Naomi Uemura 《PloS one》2012,7(11)
Background
The diagnosis of gastrointestinal (GI) involvement in Kaposi''s sarcoma (KS) is important to make because the need for treatment depends on the extent of the disease. Moreover, severe GI lesions can cause serious complications. Endoscopy with biopsy is an extremely useful method to diagnose GI-KS. However, determining the indications for endoscopy is difficult because KS can occur without GI symptoms or cutaneous KS. This study sought to clarify predictive clinical factors for GI-KS and its severity on endoscopy.Methodology/Principal Findings
A total of 1,027 HIV-infected patients who underwent endoscopy were analyzed. Sexual behavior, CD4 count, HIV RNA, history of highly active antiretroviral therapy (HAART), GI symptoms, and cutaneous KS were assessed. Endoscopic severity including bulky tumor, ulceration, and number of lesions were evaluated. Thirty-three patients had GI-KS and 46 patients cutaneous KS. Among the GI-KS patients, 78.8% (26/33) had no GI symptoms and 24.2% (8/33) had no cutaneous KS. Univariate analysis identified men who have sex with men (MSM), CD4 <100 cells/µL, HIV RNA ≥10,000 copies/mL, no history of HAART, and cutaneous KS were significantly associated with GI-KS. Among these factors, cutaneous KS was closely related to GI-KS on multivariable analysis. Among patients without cutaneous KS, MSM and CD4 count <100 cells/µL were the only independent clinical factors related to GI-KS. Bulky tumor was significantly associated with CD4 <100 cells/µL and large number of lesions was significantly associated with HIV-RNA ≥10,000 copies/mL.Conclusions
To diagnose GI-KS, clinical factors need to be considered before endoscopy. The presence of GI symptoms is not useful in predicting GI-KS. MSM and CD4 count <100 cells/µL are predictive factors among patients without cutaneous KS. Caution should be exercised especially in patients with low CD4 counts or high HIV viral loads as they are more likely to develop severe GI-KS lesions. 相似文献87.
Naoyoshi Chino Shigeru Kubo Hideki Nishio Yuji Nishiuchi Masamitsu Nakazato Terutoshi Kimura 《International journal of peptide research and therapeutics》2006,12(3):203-209
Reduced human β-defensin (hBD)-1, -2, -3 and -4 synthesized by Boc chemistry were subjected to oxidative folding reaction under optimal conditions. Among the factors affecting the oxidative folding in the presence of reduced and oxidized glutathione (GSH/GSSG), the buffer concentration and reaction temperature were essential for the predominant formation of the native disulfide structure. The homogeneity of the four synthetic hBDs was confirmed by analytical procedures using RP-HPLC, IEX-HPLC, capillary zone electrophoresis (CZE) and MALDI-TOF MS as well as sequencing, although high temperature (70 °C) was used for the RP-HPLC analysis of hBD-3 and hBD-4 to exclude the influence of equilibrium with the respective conformers having native disulfide pairing. All synthetic hBDs were shown to possess the native disulfide structure by sequential analyses and mass measurements with cystine segments obtained by enzymatic digestion. Upon digestion of hBD-1 and hBD-4 with proline specific endopeptidase, the Cys-X bond was found to be reproducibly cleaved together with the Pro-X bond although the cleavage of Cys-X afforded the appropriate cystine segments for determining the disulfide structure of hBD-1 and hBD-4. With respect to antimicrobial activity against E. coli, the four synthetic hBDs of high homogeneity possessed the same potencies as those reported previously.Australian Peptide Conference Issue 相似文献
88.
Akira Otuka Tomonari Watanabe Yoshito Suzuki Masaya Matsumura Akiko Furuno Masamichi Chino 《Population Ecology》2005,47(2):143-150
Migrations of the rice planthopper Nilaparvata lugens (Stål) (Delphacidae) from the Philippines to Taiwan, southern China, and southern Japan were analyzed using three-dimensional migration simulations. The results strongly suggested that the Southeast Asian population of N. lugens mixes with the East Asian population. This highlighted the possibility that planthoppers from the Southeast Asian population, which have properties different from those in the East Asian population such as feeding of resistant rice varieties and wing polymorphism, could migrate to Japan via southern China and Taiwan. This study, therefore, emphasizes the special care that should be taken to monitor the properties of immigrants to Japan. 相似文献
89.
Kojima N Hayashi H Suzuki S Tominaga H Maezaki N Tanaka T Yamori T 《Bioorganic & medicinal chemistry letters》2008,18(24):6451-6453
C4-Fluorinated analogues of solamin, an antitumor acetogenin, were synthesized and investigated for their antitumor activities against 39 tumor cell lines. C4-Fluorinated solamins showed more potent growth inhibitory activity against cancer cell lines than solamin. 相似文献
90.
Mariko Oshiro Kenzo Ono Yasuhiko Suzuki Hiroyoshi Ota Tsutomu Katsuyama Naoyoshi Mori 《Histochemistry and cell biology》2001,115(5):373-380
Heparan sulfate proteoglycans (HS-PGs) are associated with important cell functions, for example, cell motility, cell adhesion, and oncogenesis. We examined the localization of HS-PGs in normal and carcinoma tissues of the gastrointestinal tract to help elucidate their roles in these organs. Fresh surgical materials from 134 patients with carcinoma of the stomach or large intestine and 26 patients with various diseases of the small intestine were immunostained after fixation with 10E4 (an antibody against the HS of HS-PG) as a primary antibody. Immunoelectron microscopy (immunogold method) was also performed. The basolateral surfaces of normal tissues of the large and small intestines were strongly stained with antibody confirmed by electron microscopy. In the stomach, lesions with intestinal metaplasia showed the same staining as the intestines, although normal gastric tissue showed staining only in some parts of the basal layer of fundic and pyloric glands. Carcinoma tissues in all cases examined showed staining with antibody. Better results were obtained after fixation in acetic alcohol or zinc-containing solutions than in ordinary formalin. These characteristic localizations of HS-PG in intestines and stomachs suggest that this kind of HS-PG staining could be a hallmark characteristic of the intestine. 相似文献