Browsing by Author "Chiu, Yi-Rong"

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Development of Single-Vector Tet-on Inducible Systems with High Sensitivity to Doxycycline
(2012-07-01)
Chao, Jiun-Shuan
;
Chao, Chun-Chieh
;
Chang, Chu-Li
;
Chiu, Yi-Rong
;
Yuan, Chiun-Jye
Single-vector Tet-on systems were developed to enable the tight regulation of transgenes in mammalian cells with a low dosage of doxycycline. Both the regulatory and the responsive units were integrated in a single vector and separated by a short DNA segment (214 bp). In the developed single-vector Tet-on systems, a high level of expression of the transgene can be induced by doxycycline at a concentration of as low as 1 ng/ml, which is 500-1,000 times lower than that usually utilized in other Tet-on systems. The single-vector Tet-on system developed here exhibited 3.5-10.8 times greater inducibility of the transgene in response to doxycycline than did a dual-vector system from a commercial source. Further studies indicate that the basal activity of Tet-on systems depends greatly on the strength of the promoter that controls the transactivator. The basal activity of Tet-on systems was high when the transactivator that was directed by the human cytomegalovirus promoter, and it was almost undetectable when the transactivator was placed under the control of a moderate strength mouse mammary tumor virus promoter. Moreover, the introduction of selectable markers allows the developed single-vector Tet-on systems to facilitate the generation of conditional transgenic cells and animals with high inducibility, low basal activity and detrimental effects of the long-term administration of doxycycline.
Enzyme-encapsulated silica nanoparticle for cancer chemotherapy
(2012-04-01)
Chiu, Yi-Rong
;
Ho, Wei-Jen
;
Chao, Jiun-Shuan
;
Yuan, Chiun-Jye
;
生物科技學系
A novel horseradish peroxidase-encapsulated silica nanoparticle (SNP) was generated in this study under relatively mild conditions. The generated enzyme-encapsulated SNP were relatively uniform in size (average 70 +/- 14.3 nm), monodispersed, and spherical, as characterized by transmission electron microscopy and scanning electron microscopy. The horseradish peroxidase encapsulated in silica nanoparticle exhibits biological properties, such as a pH-dependent activity profile and k(m) value, similar to that of free enzymes. Furthermore, enzyme-encapsulated SNP exhibited good operational stability for the repetitive usage with a relative standard deviation of 5.1 % ( n = 10) and a high stability for long term storage (>60 days) at 4 degrees C. The feasibility of using enzyme-encapsulated SNP in prodrug cancer therapy was also demonstrated by its capability to convert the prodrug indole-3-acetic acid into cytotoxic peroxyl radicals and trigger the death of tumor cells. These results indicate that the developed enzyme-encapsulated SNP has potential in the applications of prodrug cancer therapy.
Monitoring the cellular dynamic distribution of mammalian Ste20-like protein kinase 3 by peptide aptamer from phage display
(2010-04-01)
Chiu, Yi-Rong
;
Yuan, Chiun-Jye
;
生物科技學系
;
Department of Biological Science and Technology
Site-Directed Mutations of Thermostable Direct Hemolysin from Grimontia hollisae Alter Its Arrhenius Effect and Biophysical Properties
(2011)
Wang, Yu-Kuo
;
Huang, Sheng-Cih
;
Wu, Yi-Fang
;
Chen, Yu-Ching
;
Lin, Yen-Ling
Recombinant thermostable direct hemolysin from Grimontia hollisae (Gh-rTDH) exhibits paradoxical Arrhenius effect, where the hemolytic activity is inactivated by heating at 60 degrees C but is reactivated by additional heating above 80 degrees C. This study investigated individual or collective mutational effect of Tyr53, Thr59, and Ser63 positions of Gh-rTDH on hemolytic activity, Arrhenius effect, and biophysical properties. In contrast to the Gh-rTDH wild-type (Gh-rTDH(WT)) protein, a 2-fold decrease of hemolytic activity and alteration of Arrhenius effect could be detected from the Gh-rTDH(Y53H/T59I) and Gh-rTDH(T59I/S63T) double-mutants and the Gh-rTDH(Y53H/T59I/S63T) triple-mutant. Differential scanning calorimetry results showed that the Arrhenius effect-loss and -retaining mutants consistently exhibited higher and lower endothermic transition temperatures, respectively, than that of the Gh-rTDHWT. Circular dichroism measurements of Gh-rTDH(WT) and Gh-rTDH(mut) showed a conspicuous change from a beta-sheet to alpha-helix structure around the endothermic transition temperature. Consistent with the observation is the conformational change of the proteins from native globular form into fibrillar form, as determined by Congo red experiments and transmission electron microscopy.