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Controlled Expression and Secretion of Aspergillus oryzae Alkaline Protease in Aspergillus nidulans
Kim, Eun-Ah,Lee, Jeong-Goo,Whang, Mi-Kyung,Park, Hee-Moon,Kim, Jeong-Yoon,Chae, Suhn-Kee,Maeng, Pil-Jae The Microbiological Society of Korea 2001 The journal of microbiology Vol.39 No.2
In an effort to develop an efficient expression and secretion system for heterologous proteins in Aspergilius nidulans, the PCR-amplified coding sequence for alkaline pretense (AlpA) of A. oryzae was cloned into a fungal expression vector downstream of A. nidulans aicA (alcohol dehydrogenase) promoter to yield pRAAlp. Transformation of A. nidulans with pRAAlp gave stable transformants harboring various copy numbers (3 to 10) of integrated alpA gene, from among which 6 representatives were selected. On a medium containing 0.8% ammonium sulfate that represses the expression of the host's own pretense, the alcA prumoter-controlled AlpA expression was strongly induced by threonine but repressed by glucose. The level of AlpA secretion was highest (approximately 666 mU/ml) in transformant ALP6 containing the largest copy number integrated alpA. However, the level of AlpA secretion was not necessarily proportional to the copy numbers of the integrated alpA genes. The N-terminal sequence or the secreted mature AlpA was determined to be Gly-Leu-Thr-Thr-Gln-Lys-Ser and its molecular mass to be approximately 34 kDa, indicating that AlpA is properly processed by the removal of 121 N-terminal amino acids.
Shin, Jae-Sik,Maeng, Hyung-Gun,Hong, Seung-Woo,Moon, Jai-Hee,Kim, Jin-Sun,Suh, Young-Ah,Kim, Eun-Sung,Lee, Young-Min,Kim, Ye-Seul,Choi, Eun-Kyung,Kim, Inki,Lee, Sok-Young,Cho, Dong-Hyung,Hong, Nam-Joo Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.12
Recently, we reported that an ethanol extract of Iris nertschinskia induces p53-dependent apoptosis in the MCF7 human breast cancer cell line. However, the detailed mechanisms were not fully explored. Here, we demonstrate another aspect of the activity of I. nertschinskia in breast cancer cells. We compared the response to an ethanol extract of I. nertschinskia in two different human breast cancer cell lines, Hs578Tand MDA-MB231, respectively with relatively low and high AKT1/2 activity by trypan blue exclusion assay and FACS analysis. Knockdown of endogenous AKT1 or AKT2 in breast cancer cells by RNA interference determined the sensitivity to I. nertschinskia ethanol extract compared to control cells. The I. nertschinskia ethanol extract induced cell death in a manner that depended on the level of phosphorylated AKT1/2 protein and was associated with a significant increase in the sub-G1 cell population, indicative of apoptosis. Our results indicate that an ethanol extract of I. nertschinskia differentially induces cell death in breast cancer cells depending on their level of phosphorylated AKT1/2.
Kiyong Jeong,정태영,Kyung-ah Maeng,Jihyun Song,Sungyoul Hong,권무식 한국생물공학회 2008 Biotechnology and Bioprocess Engineering Vol.13 No.1
Fowl cholera, caused by Pasteurella multocida (A:3), is a fearsome disease leading to a nonproductive influence upon poultry industry. It has been known that outer membrane protein H (OmpH) in the bacterium is a strong candidate to bring on the notorious ailment. Genetically modified (GM) tobacco (Nicotiana tabacum cv. Petit Havana) harboring ompH(A:3) was constructed to develop a plant expression system for the protein, OmpH(A:3). Some 987 bp-long (ORF with the stop codon, TAA) of the ompH(A:3) excluding the nucleotide for signal peptide, was amplified by RT-PCR with the gene specific primers and pGEM-T-ompH(A:3) as template DNA. The PCR-amplified DNA was ligated into BamHI/SacI-cut pBI121 to obtain a recombinant plasmid, pBI121-ompH(A:3). It was then transformed into Agrobacterium tumefaciens (LBA 4404) by liquid nitrogen method to generate a recombinant clone of Agrobacterium LBA4404/pBI121-ompH(A:3). The Agrobacterium LBA4404/pBI121-ompH(A:3) was inoculated into leaf discs of tobacco (2 day old). The gene-transfected leaves were cultured on Murashige-Skoog basal medium containing kanamycin (50 mg/mL) to generate numerous calli, from which some GM tobacco plants were obtained. Transgenicity of the tobacco plant was confirmed by PCR screening along with the DNA sequencing. Also, its expression in the GM-tobacco was examined qualitatively as well as quantitatively by ELISA/Western blot. These results suggest that the genetically modified tobacco plant can be potentially used as a model system to develop plant-based vaccine against the fowl cholera.
조성윤,김기용,김수진,손영배,맹세현,김치화,고아라,송정한,여성희,김경효,진동규,Cho, Sung Yoon,Kim, Ki-Yong,Kim, Su Jin,Sohn, Young Bae,Maeng, Se Hyun,Kim, Chi Hwa,Ko, Ah-Ra,Song, Junghan,Yeau, Sung-Hee,Kim, Kyung-Hyo,Jin, Dong-Kyu The Korean Society of Inherited Metabolic Disease 2012 대한유전성대사질환학회지 Vol.12 No.1
I 세포 질환(뮤코지방증 2형; MIM 252500)과 pseudo-Hurler polydystrophy (뮤코지방증 3형; MIM 252600)는 세포내 비정상적인 리소좀 관련 운송으로 인해 발병한다. 특징적인 소견으로는 섬유아 세포의 세포질에 다수의 봉입체, 뮤코다당뇨의 부재, 혈청 내 리소좀 효소 활성도의 증가, GlcNAc-phosphotransferase 활성도의 감소를 보인다. 이 연구에서는 GlcNAc-phosphotransferase 알파/베타 아형에 대한 knockout 마우스의 표현형과 생화학적 특징을 조사하였다. 또한, 태반으로부터 추출한 리소좀 농축 분획을 knockout 마우스에 투여하였을 때 체중 증가에 대한 효과를 볼 수 있는지에 대해 알아보고자 하였다. knockout 마우스는 뮤코지방증 2형 환자에서 그렇듯이 정상적인 체중 증가를 보이지 않았고 낮은 골밀도를 보였다. 게다가 knockout 마우스의 피부 섬유 아세포의 배양액에서는 리소좀 효소 활성도가 증가한 반면, 세포 내에서는 리소좀 효소 활성도가 감소되어 있는 것을 확인할 수 있었으며 이러한 특징은 뮤코지방증 2형 환자에서 볼 수 있는 특징과 일치한다. knockout 마우스의 꼬리 정맥내로 태반에서 추출한 리소좀 농축 분획을 투여한 결과, 체중이 증가하는 것을 확인할 수 있었고, 반면 생리식염수를 투여한 knockout 마우스의 경우는 체중이 증가하지 않았다. 결론적으로, knockout 마우스의 표현형과 생화학적 특징이 뮤코지방증 2형 환자와 유사하다는 것을 확인하였으며, 리소좀 농축 분획의 치료적 가능성을 증명하였다. 더 큰 범위의 동물 실험을 진행할 필요가 있으나, 이 연구는 질병에 대한 동물 모델을 개발하고 리조솜 농축 분획의 치료적 가능성을 제시하는 것을 통해 현재까지 치료가 불가능한 뮤코지방증 2형의 새로운 치료 방법의 가능성을 열었다고 볼 수 있다. I-cell disease (mucolipidosis type II; MIM 252500) and pseudo-Hurler polydystrophy (mucolipidosis type III; MIM 252600) are disorders caused by abnormal lysosomal transport in cells. The presence of numerous inclusion bodies in the cytoplasm of fibroblasts, a lack of mucopolysacchariduria, increased lysosomal enzyme activity in serum, and decreased GlcNAc-phosphotransferase activity are hallmark. Here, we attempted to investigate phenotypical and biochemical characteristics of the knockoutmouse of GlcNAc-phosphotransferase ${\alpha}/{\beta}$ subunits; in addition, we also attempted to determine whether the lysosome enriched fraction derived from placenta can be beneficial to phenotype and biochemistry of the knockout mouse.We found that the knockout mouse failed to thrive and had low bone density, as is the case in human. In addition, skin fibroblasts from the animal had the same biochemical characteristics, including increased lysosomal enzyme activity in the culture media, in contrast to the relatively low enzyme activity within the cells. Intravenous injection of the lysosome rich fraction derived from placenta into the tail vein of the animal resulted in a gain of weight, while saline injected animals didn't.In conclusion, our study demonstrated the phenotypical and biochemical similarities of the knockout mouse to a mucolipidosis type II patient and showed the therapeutic potential of the lysosome enriched fraction. We admit that a larger scale animal study will be needed; however, the disease model and the therapeutic potential of the lysosome enriched fraction will highlight the hope for a novel treatment approach to mucopolipidosis type II, for which no therapeutic modality is available.