• Title/Summary/Keyword: shRNA

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Isolation and Characterization of Marine Bacterial Strain SH-1 Producing Agar-Degrading Enzymes (한천 분해효소를 생산하는 해양 미생물 SH-1의 분리 및 특성 분석)

  • Lee, Jae-Hag;Lee, Soon-Youl
    • Microbiology and Biotechnology Letters
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    • v.42 no.4
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    • pp.324-330
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    • 2014
  • A marine bacterial strain producing agar-degrading enzymes was isolated from a mud flat in Jeboo-do (Korea) using a selective artificial sea water (ASW) agar plate containing agar as the sole carbon source. The isolate, designated as SH-1, was gram-negative, aerobic, and motile with single polar flagellum. 16S rRNA gene sequence similarity analysis showed the isolate SH-1 had the highest homology (96.5%) to marine bacterium Neiella marina J221. Cells could grow at $28-37^{\circ}C$ but not at $42^{\circ}C$, and the agarase activity of the cell culture supernatant was higher when grown at $28^{\circ}C$ than when grown at $37^{\circ}C$. Cells could grow when concentrations of 1-5% (w/v) NaCl were added to the growth media with the best growth observed at 3% NaCl, and the agardegrading enzyme activity of the cell culture supernatant was best when grown at 3% NaCl-containing growth media under the conditions we examined. The crude enzyme prepared from 48-h culture broth of strain SH-1 exhibited an optimum pH and temperature for agar-degrading activity at 7.0 and $40^{\circ}C$, respectively. Zymogram analysis of the crude supernatant and cell extract showed that strain SH-1 produced at least 3 agar-degrading enzymes with molecular weights of 15, 35, and 52 KD. Thinlayer chromatography (TLC) analysis also suggested that HS-1 produces ${\beta}$-agarase to degrade agarose to neoagarooligosaccharides.

Probiotic Properties of Pediococcus pentosaceus SH-10 Isolated from the Hard Clam Meretrix meretrix Shikhae (백합(Meretrix meretrix)식해에서 분리한 Pediococcus pentosaceus SH-10의 생균제적 특성)

  • Song, Hyun-Jung;Kim, Kang-Jin;Kim, Hee-Dai;Yoo, Jung-Hee;Koo, Jae-Geun;Park, Kwon-Sam
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.44 no.6
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    • pp.605-611
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    • 2011
  • This study examined the suitability of characteristics of potential strains of probiotic bacteria. Among 25 lactic acid bacteria isolated from Korean traditional fermented food, the Hard Clam Meretrix meretrix Shikhae, the SH-10 strain, which exhibited superior resistance to low pH and bile salts, was selected as a potential probiotic bacteria. By examining carbohydrate utilization, morphological properties, and the 16S rRNA gene sequence, the SH-10 strain was identified as Pediococcus pentosaceus (hereafter, P. pentosaceus SH-10). P. pentosaceus SH-10 was resistant to amikacin, cefotetan, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, and vancomycin. Tests of antimicrobial activities against pathogens such as Bacillus cereus, Listeria monocytogenes, Salmonella choleraesuis, and Staphylococcus aureus, indicated that P. pentosaceus SH-10 inhibited the growth of pathogenic bacteria. These results suggest that P. pentosaceus SH-10 can be developed as a probiotic bacteria.

Effects of Tissue Factor, PAR-2 and MMP-9 Expression on Human Breast Cancer Cell Line MCF-7 Invasion

  • Lin, Zeng-Mao;Zhao, Jian-Xin;Duan, Xue-Ning;Zhang, Lan-Bo;Ye, Jing-Ming;Xu, Ling;Liu, Yin-Hua
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.2
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    • pp.643-646
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    • 2014
  • Objective: This study aimed to explore the expression of tissue factor (TF), protease activated receptor-2 (PAR-2), and matrix metalloproteinase-9 (MMP-9) in the MCF-7 breast cancer cell line and influence on invasiveness. Methods: Stable MCF-7 cells transfected with TF cDNA and with TF ShRNA were established. TF, PAR-2, and MMP-9 protein expression was analyzed using indirect immunofluorescence and invasiveness was evaluated using a cell invasion test. Effects of an exogenous PAR-2 agonist were also examined. Results: TF protein expression significantly differed between the TF cDNA and TF ShRNA groups. MMP-9 protein expression was significantly correlated with TF protein expression, but PAR-2 protein expression was unaffected. The PAR-2 agonist significantly enhanced MMP-9 expression and slightly increased TF and PAR-2 expression in the TF ShRNA group, but did not significantly affect protein expression in MCF-7 cells transfected with TF cDNA. TF and MMP-9 expression was positively correlated with the invasiveness of tumor cells. Conclusion: TF, PAR-2, and MMP-9 affect invasiveness of MCF-7 cells. TF may increase MMP-9 expression by activating PAR-2.

Suppression of Ku80 Correlates with Radiosensitivity and Telomere Shortening in the U2OS Telomerase-negative Osteosarcoma Cell Line

  • Hu, Liu;Wu, Qin-Qin;Wang, Wen-Bo;Jiang, Huan-Gang;Yang, Lei;Liu, Yu;Yu, Hai-Jun;Xie, Cong-Hua;Zhou, Yun-Feng;Zhou, Fu-Xiang
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.2
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    • pp.795-799
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    • 2013
  • Ku70/80 heterodimer is a central element in the nonhomologous end joining (NHEJ) DNA repair pathway, Ku80 playing a key role in regulating the multiple functions of Ku proteins. It has been found that the Ku80 protein located at telomeres is a major contributor to radiosensitivity in some telomerase positive human cancer cells. However, in ALT human osteosarcoma cells, the precise function in radiosensitivity and telomere maintenance is still unknown. The aim of this study was to investigate the effects of Ku80 depletion in the U2OS ALT cell line cell line. Suppression of Ku80 expression was performed using a vector-based shRNA and stable Ku80 knockdown in cells was verified by Western blotting. U2OS cells treated with shRNA-Ku80 showed lower radiobiological parameters (D0, Dq and SF2) in clonogenic assays. Furthermore, shRNA-Ku80 vector transfected cells displayed shortening of the telomere length and showed less expression of TRF2 protein. These results demonstrated that down-regulation of Ku80 can sensitize ALT cells U2OS to radiation, and this radiosensitization is related to telomere length shortening.

The c-myc Expression on the Opioid Tolerance in Human Neuroblastoma SH-SY5Y Cells (사람 Neuroblastoma SH-SY5Y 세포주에서 Opiate 내성에 의한 c-myc 유전자 표현)

  • Park, Chang-Kyo;Kwon, Gee-Youn;Suh, Sung-Il;Kim, Soo-Kyung
    • The Korean Journal of Physiology and Pharmacology
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    • v.1 no.6
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    • pp.691-697
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    • 1997
  • The mechanisms underlying opiate tolerance and dependence are not fully understood. We used human neuroblastoma SH-SY5Y cells as a model system for studying effects of morphine tolerance and withdrawal on c-myc induction and cAMP levels. It has been reported that regulation of c-fos by acute and chronic morphine withdrawal is mediated through alterations in CREB transcription factor. In this study, we examined the effects of morphine tolerance on c-myc expression and cAMP concentrations. The activation of opiate receptors by an acute morphine administration resulted in an increase in c-myc mRNA and a decrease in cAMP concentrations in a dose-dependent manner $(5,\;10,\;15,\;and\;20\;{\mu}M)$. On the other hand, the chronic treatment of morphine $(10\;{\mu}M\;for\;six\;days)$ did not induce the elevated expression of c-myc mRNA. The c-myc expression was slightly inhibited in comparison with that of the acute morphine response. However, cAMP concentrations were increased with regard to morphine withdrawal response. These results suggest that the alterations in c-myc expression might imply a significant opiate regulation relating to morphine tolerance. This observation differs from increased expression of c-fos via regulation of cAMP pathway.

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High Density Cell Culture of Bifidobacterium by Optimization of Medium Composition and Culture Conditions. (배지조성 및 배양환경 최적화에 의한 Bifidobacterium의 고농도 배양)

  • 송수한;김택범;지근억;오훈일;오덕근
    • Microbiology and Biotechnology Letters
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    • v.30 no.1
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    • pp.63-67
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    • 2002
  • Bifidobacterium strain was isolated from the feces of brast fed infants. The isolated strain was identified as Bifidobacterium longum by 16S rRNA sequence analysis and named as Bifidobacterium SH2. The MRS medium was modified to obtain high density cells of Bifidobacterium SH2. The optimal medium was determined to be 50 g/L lactose, 10 g/L beef extract, 10 g/L peptone, 5 g/L yeast extract, 7 g/L sodium acetate, 2 g/L ammonium citrate, 2 g/L disodium phosphate,1 g/L tween 80, 0.2 g/L MnSO$_4$ and 0.5 g/L L-cysteine. The pH and temperature were optimized as 5.0 and $37^{\circ}C$, respectively. Through out the optimization of medium composition and culture conditions, the dry cell weight and viable cell count were 2.5 times and 1.8 times higer than those in MRS medium, respectively.

Isolation of Simiduia sp. SH-2 and Characterization of Its β-Agarase (한천분해세균 Simiduia sp. SH-2 균주의 분리 및 β-agarase의 특성조사)

  • Lee, Dong-Geun;Kim, Geun-Dae;Lee, Sang-Hyeon
    • Journal of Life Science
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    • v.32 no.10
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    • pp.778-783
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    • 2022
  • This study isolated a new agarase-producing bacterium and characterized its agarase. A new agar-degrading strain was isolated from the seashore of Namhae in Gyeongnam province, Korea, and was purely cultured using the Marine Agar 2216 media. The isolated bacterium was identified as Simiduia sp. SH-2 after 16S rRNA gene sequencing. The crude agarase was obtained from the culture medium of the Simiduia sp. SH-2 strain, and the agar-degrading activity was measured. The highest level of activity of the Simiduia sp. SH-2-derived agar-degrading enzyme was 625 U/l. Agar degradation activity was most significant at 40℃ and pH 7.0. Compared to the activity at 40℃, the relative activity was 31% at 20℃ and 71% at 30℃. Compared to the activity at pH 7.0, the relative activity was 94% and 89% at pH 6.0 and pH 8.0, respectively. Residual activity was greater than 96% after exposure to 20℃ and 30℃ for 2 hr and more than 49% after exposure to 40℃ for 2 hr. Simiduia sp. SH-2 was identified as a strain producing β-agarase that creates neoagarooligosaccharides, such as neoagarotetraose and neoagarohexaose. Therefore, the Simiduia sp. SH-2 strain and its β-agarase are expected to be useful functional material producers in the food, cosmetic, and pharmaceutical industries.

Biochemical Characteristics of a Killer Toxin Produced by Ustilago maydis Virus SH14 Isolated in Korea

  • Ha, Eun-Soo;Yie, Se-Won;Choi, Hyoung-Tae
    • Journal of Microbiology
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    • v.35 no.4
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    • pp.323-326
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    • 1997
  • Toxin protein from Ustilago maydis virus SH14 isolated in Korea was purified using ethanol precipitation, cation exchange, gel filtration and anion exchange chromatography. The molecular weight of the purified protein was estimated to be 8.3 kDa by SDS-PAGE analysis. The Nterminal sequence of the protein is L-G-I-N-C(K)-R-G-S-S-Q--C(K)-G-L-S-G which is highly homologous with that of P4 toxin, but the amino acid composition and electrophoretic mobility in a native PAGE of the toxin protein were totally different from those of P4 toxin respectively. The SH14 toxin was shown to have immunological cross-reactivity about 50% with P4 toxin when examined by Western hybridization.

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SR proteins regulate V6 exon splicing of CD44 pre-mRNA

  • Loh, Tiing Jen;Moon, Heegyum;Jang, Ha Na;Liu, Yongchao;Choi, Namjeong;Shen, Shengfu;Williams, Darren Reece;Jung, Da-Woon;Zheng, Xuexiu;Shen, Haihong
    • BMB Reports
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    • v.49 no.11
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    • pp.612-616
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    • 2016
  • CD44 pre-mRNA includes 20 exons, of which exons 1-5 ($C_1-C_5$) and exons 16-20 ($C_6-C_{10}$) are constant exons, whereas exons 6-15 ($V_1-V_{10}$) are variant exons. $V_6$-exon-containing isoforms have been known to be implicated in tumor cell invasion and metastasis. In the present study, we performed a SR protein screen for CD44 $V_6$ splicing using overexpression and lentivirus-mediated shRNA treatment. Using a CD44 $V_6$ minigene, we demonstrate that increased SRSF3 and SRSF4 expression do not affect $V_6$ splicing, but increased expression of SRSF1, SRSF6 and SRSF9 significantly inhibit $V_6$ splicing. In addition, using a constitutive exon-specific primer set, we could not detect alterations of CD44 splicing after SR protein-targeting shRNA treatment. However, using a $V_6$ specific primer, we identified that reduced SRSF2 expression significantly reduced the $V_6$ isoform, but increased $V_{6-10}$ and $V_{6,8-10}$ isoforms. Our results indicate that SR proteins are important regulatory proteins for CD44 $V_6$ splicing.