• Title/Summary/Keyword: amoeba

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Development of a Plasmid Vector for Overproduction of $\beta$-Galactosidase in Escherichia coli by Using Genetic Components of groEx from Symbiotic Bacteria in Amoeba proteus

  • Lee, Jung-Eun;Ahn, Eun-Young;Ahn, Tae-In
    • Journal of Microbiology and Biotechnology
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    • v.8 no.5
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    • pp.509-516
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    • 1998
  • A plasmid vector, pXGPRMATG-lac-Tgx, was developed for overproduction of $\beta$-galactosidase in Escherichia coli using the genetic components of groEx, a heat-shock gene cloned from symbiotic X-bacteria in Amoeba proteus. The vector is composed of intragenic promoters P3 and P4 of groEx, the structural gene of lac operon, transcription tenninator signals of lac and groEx, and ColEl and amp'of pBluescript SKII. The optimized host, E. coli DH5$\alpha$, transfonned with the vector constitutively produced 117,310-171,961 Miller units of $\beta$-galactosidase per mg protein in crude extract. The amount of enzyme in crude extract was 53% of total water-soluble proteins. About 43% of the enzyme could be purified to a specific activity of 322,249 Miller units/mg protein after two-fold purification, using two cycles of precipitation with ammonium sulfate and one step of gel filtration. Thus, the expression system developed in this study presents a low-cost and simple method for purifying overproduced $\beta$-galactosidase in E. coli.

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Down-Regulation of Cellulose Synthase Inhibits the Formation of Endocysts in Acanthamoeba

  • Moon, Eun-Kyung;Hong, Yeonchul;Chung, Dong-Il;Goo, Youn-Kyoung;Kong, Hyun-Hee
    • Parasites, Hosts and Diseases
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    • v.52 no.2
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    • pp.131-135
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    • 2014
  • Acanthamoeba cysts are resistant to unfavorable physiological conditions and various disinfectants. Acanthamoeba cysts have 2 walls containing various sugar moieties, and in particular, one third of the inner wall is composed of cellulose. In this study, it has been shown that down-regulation of cellulose synthase by small interfering RNA (siRNA) significantly inhibits the formation of mature Acanthamoeba castellanii cysts. Calcofluor white staining and transmission electron microscopy revealed that siRNA transfected amoeba failed to form an inner wall during encystation and thus are likely to be more vulnerable. In addition, the expression of xylose isomerase, which is involved in cyst wall formation, was not altered in cellulose synthase down-regulated amoeba, indicating that cellulose synthase is a crucial factor for inner wall formation by Acanthamoeba during encystation.

The UV Images and Aging Judgement with Behaviors Caused by Surface Contamination on 22.9[kV] Class Insulators (22.9[kV]급 애자의 표면오염에 의한 자외선 영상과 열화판정)

  • Shong, Kil-Mok;Kim, Young-Seok
    • Journal of the Korean Institute of Illuminating and Electrical Installation Engineers
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    • v.24 no.1
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    • pp.167-174
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    • 2010
  • In this paper, the ultraviolet rays images and aging judgement caused by surface contamination on 22.9[kV] class insulators was defined. UV images represented by the corona discharge are divided 3 types such as sunflower, jellyfish, amoeba and in detail, there are classified the progress mechanism of eight. In the field which is installed the power facilities, immediately, this judgement method can be found out the deteriorated parts of the power facilities. These steps are possible to judge the deterioration of power facilities through reliable data. Hereafter, this study as the diagnostic technology suitable for the sites is used.

The Fate of Strain-Specific Protein in xD Strain of Amoeba proteus (Amoeba proteus xD Strain의 변이주 특이성 단백질의 운영)

  • 안태인
    • The Korean Journal of Zoology
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    • v.26 no.3
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    • pp.181-192
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    • 1983
  • Cytosol protein patterns of two strains of A. proteus, tD and xD strain, were compared by two dimensional gel electrophoresis. Among the 200 major polypeptides that could be stained by silver stain method, tD strain contained a cell specific protein whose molecular weight was 45,000 dalton, pI 5.9. On the other hand, the cytosol and the symbiotic vesicles of xD strain contained a symbiosis specific protein (M.W. 29,000; pI 5.5). The fate of the symbiosis specific protein depended on the presence of symbiotic bacteria in the experiment of high temperature effect and of experimental infection. The significance of these results is discussed in relation to their function in organismic association on the basis of the previous findings.

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Effects of Mannose on Pathogenesis of Acanthamoeba castellanii

  • Yoo, Kyung-Tae;Jung, Suk-Yul
    • Parasites, Hosts and Diseases
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    • v.50 no.4
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    • pp.365-369
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    • 2012
  • Acanthamoeba spp. are single-celled protozoan organisms that are widely distributed in the environment. In this study, to understand functional roles of a mannose-binding protein (MBP), Acanthamoeba castellanii was treated with methyl-alpha-D-mannopyranoside (mannose), and adhesion and cytotoxicity of the amoeba were analyzed. In addition, to understand the association of MBP for amoeba phagocytosis, phagocytosis assay was analyzed using non-pathogenic bacterium, Escherichia coli K12. Amoebae treated with mannose for 20 cycles exhibited larger vacuoles occupying the most area of the amoebic cytoplasm in comparison with the control group amoebae and glucose-treated amoebae. Mannose-selected amoebae exhibited lower levels of binding to Chinese hamster ovary (CHO) cells. Exogenous mannose inhibited >50% inhibition of amoebae (control group) binding to CHO cells. Moreover, exogenous mannose inhibited amoebae (i.e., man-treated) binding to CHO cells by <15%. Mannose-selected amoebae exhibited significantly decreased cytotoxicity to CHO cells compared with the control group amoebae, 25.1% vs 92.1%. In phagocytic assay, mannose-selected amoebae exhibited significant decreases in bacterial uptake in comparison with the control group, 0.019% vs 0.03% (P<0.05). Taken together, it is suggested that mannose-selected A. castellanii trophozoites should be severely damaged and do not well interact with a target cell via a lectin of MBP.

The role of domestic tap water in Acanthamoeba contamination in contact lens storage cases in Korea

  • JEONG Hae Jin;YU Hak Sun
    • Parasites, Hosts and Diseases
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    • v.43 no.2 s.134
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    • pp.47-50
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    • 2005
  • A survey was carried out from August to December 2004 in Pusan, Korea to document the presence of free-living amoeba (FLA), including the genus Acanthamoeba, in both contact lens storage cases and domestic tap water. Acanthamoeba was isolated from $5(4.2\%)$ in 120 contact lens storage cases. Four house tap water samples from residents, whose contact lens storage cases had been contaminated by Acanthamoeba, were also found to be contaminated with Acanthamoeba. Therefore, the contamination rate of FLA and Acanthamoeba in domestic tap water was investigated in order to examine the role of domestic tap water in Acanthamoeba contamination of contact lens storage cases. FLA and Acanthamoeba were identified in $97(46.8\%)\;and\;16(7.7\%)$ of the 207 domestic tap water samples, respectively. There were no significant differences between the contamination rates of FLA in tap water according to the filtration plant of origin. No FLA was detected in the tap water directly supplied by the water purification plants. Water storage tanks appear to promote FLA colonization, including Acanthamoeba, in domestic tap water. This increases the risk of Acanthamoeba contamination in contact lens storage cases as well as increasing the risk of Acanthamoeba keratitis.

Characterization of Ubiquitinated Lysosomal Membrane Proteins in Acanthamieba castellanii

  • Oh, Sekyung;Ahn, Tae-In
    • Animal cells and systems
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    • v.4 no.2
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    • pp.165-171
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    • 2000
  • Ubiquitinated proteins in lysosomes were characterized by using two monoclonal antibodies (mAbs): LYS8-1, a mAb to lysosomal proteins, and NYA124, a mAb to ubiquitin. LYS8-1 stained lysosome-like vesicles in immunofluorescence microscopy of Amoeba proteus and Acanthamoeba castellanii. In immunoblotting, LYS8-1's antigens (LYS proteins) were detected as 68-kDa and 77-kDa proteins in A. proteus, and as 30-kDa and 39-kDa proteins in A. castellanii. In immunoprecipitation of A. castellanii, at least four distinct LYS proteins, LVS35p, LyS39p, LyS42p, and LYS46p, were detected and accumulated upon inhibition of lysosome functions but not upon that of 26S proteasome functions. They were all found to be ubiquitinated, and were recovered in the lysosome fractions in subcellular fractionation experiments. In chemical fractionation analyses, LYS35p and LYS39p were demonstrated to be peripherally associated with lysosome membrane, while LYS42p and LYS46p tightly bound to the membrane. These results suggest that the LYS proteins become associated to lysosomal membrane upon ubiquitination.

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