Browsing by Author "Yuan, CJ"

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Caspase activation of mammalian Sterile 20-like kinase 3 (Mst3) - Nuclear translocation and induction of apoptosis
(2002-09-13)
Huang, CYF
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Wu, YM
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Hsu, CY
;
Lee, WS
;
Lai, MD
Mammalian Sterile 20-like kinase 3 (Mst3), the physiological functions of which are unknown, is a member of the germinal center kinase-III family. It contains a conserved kinase domain at its NH2 terminus, whereas there is a regulatory domain at its COOH terminus. In this study we demonstrate that endogenous Mst3 is specifically cleaved when Jurkat cells were treated with anti-Fas antibody or staurosporine and that this cleavage is inhibited by the caspase inhibitor, Ac-DEVD-CHO. Using apoptotic Jurkat cell extracts and recombinant caspases, we mapped the caspase cleavage site, AETD(313), which is at the junction of the NH2-terminal kinase domain and the COOH-terminal regulatory domain. Caspase-mediated cleavage of. Mst3 activates its intrinsic kinase activity, suggesting that the COOH-terminal domain of Mst3 negatively regulates the kinase domain. Furthermore, proteolytic removal of the Mst3 COOH-terminal domain by caspases promotes nuclear translocation. Ectopic expression of either wild-type or COOH-terminal truncated Mst3 in cells results in DNA fragmentation and morphological changes characteristic of apoptosis. By contrast, no such changes were exhibited for catalytically inactive Mst3, implicating the involvement of Mst3 kinase activity for mediation of these effects. Collectively, these results support the notion that caspase-mediated proteolytic activation of Mst3 contributes to apoptosis.
CMOS chip as luminescent sensor for biochemical reactions
(2003-06-01)
Lu, U
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Hu, BCP
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Shih, YC
;
Yang, YS
;
Wu, CY
We describe a novel biochemical sensing method and its potential new biosensing applications. A light-sensitive complementary metal oxide semiconductor (CMOS) chip prepared through a standard 0.5-mum CMOS process was developed for measuring biochemical reactions. A light producing enzymatic reaction catalyzed by horseradish peroxidase (HRP) was designed as a platform reaction to determine the concentration of hydrogen. peroxide (H2O2) by the CMOS chip with a standard semiconductor parameter analyzer (HP4145). The kinetics of enzymatic reaction were determined and compared with a standard and sophisticated fluorometer (Hitachi F-4500) in a biochemical laboratory. Similar results were obtained by both instruments. Using glucose oxidase as an example, we further demonstrated that the HRP platform can be used to determine other H2O2 producing reactions with the CMOS system. The result points to an important application of the CMOS chip in biological measurements and in diagnosis of various health factors.
Detection of serum uric acid using the optical polymeric enzyme biochip system
(2004-07-15)
Huang, SH
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Shih, YC
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Wu, CY
;
Yuan, CJ
;
Yang, YS
An optical polymeric biochip system based on the complementary metal oxide semiconductor (CMOS) photo array sensor and polymeric enzyme biochip for rapidly quantitating uric acid in a one-step procedure was developed. The CMOS sensor was designed with N+/P-well structure and manufactured using a standard 0.5 mum CMOS process. The polymeric enzyme biochip was immobilized with uricase-peroxidase and used to fill the reacting medium with the sample. This study encompasses the cloning of the Bacillus subtilis uricase gene and expression in Escherichia coli, as well as the purification of uricase and measurement of its activity. The cloned uricase gene included an open reading frame of 1491 nucleotides that encodes a protein of approximately 55 kDa. The expression of the putative MBP-fusion protein involved approximately 98 kDa of the protein. The CMOS sensor response was stronger at a higher temperature range of 20-40degreesC, with optimal pH at 8.5. The calibration curve of purified uric acid was linear in the concentration range from 2.5 to 12.5 mg/dL. The results obtained for serum uric acid correlated quite closely with those obtained using the Beckman Synchron method. (C) 2004 Elsevier B.V All rights reserved.
Eliminating the interference of ascorbic acid and uric acid to the amperometric glucose biosensor by cation exchangers membrane and size exclusion membrane
(2005-12-01)
Yuan, CJ
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Hsu, CL
;
Wang, SC
;
Chang, KS
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生物科技學系
The characteristics of a multiuse planar amperometric biosensor modified with Nafion and/or polyion membrane were investigated. A new enzyme immobilization process was proposed, in which the polyvinyl alcohol bearing a styrylpyridinium (SbQ)/glucose oxidase composite was treated with glutaraldehyde vapor prior to the photo-crosslinking reaction. The resulting planar enzyme electrode remains active for at least 150 days. Compared with poly-L-lysine/poly (4-stryenesulfonate) polyion complex membrane the Nafion membrane showed marked effect to reduce the electrochemical response of the modified planar enzyme electrode to the biological interferents, such as ascorbic acid and uric acid. Furthermore, Nafion membrane can effective restricting the oxidized anionic interferent to adhear on its surface, thereby the fouling of the electrode was avoided.
Identification and characterization of the nuclear import and export signals of the mammalian Ste20-like protein kinase 3
(2004-08-13)
Lee, WS
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Hsu, CY
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Wang, PL
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Huang, CYF
;
Chang, CH
Mst3, a human Ste20-like protein kinase, has been recently demonstrated to undergo a caspase-mediated cleavage during apoptosis. The proteolytic cleavage of the C-terminus of Mst3 caused nuclear translocation of its kinase domain. This work provides evidence that Mst3 may contain a bipartite-like nuclear localization sequence (NLS) at the C-terminus of its kinase domain (residues 278-292). The removal of NLS from the kinase domain of Mst3 led to the cytoplasmic accumulation of EGFP-Mst3 (Delta277). The presence of nuclear exporting signals in the Mst3 was also demonstrated by leptomycin B-treatment and serial deletion of the C-terminal regulatory domain of Mst3. A nuclear export signal was also postulated to be in the regions of amino acids 335-386. In conclusion, Mst3 contains both NLS and NES signals, which may cooperate to control the subcellular distribution of Mst3. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Identification of V23RalA-Ser(194) as a critical mediator for aurora-A-induced cellular motility and transformation by small pool expression screening
(2005-03-11)
Wu, JC
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Chen, TY
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Yu, CTR
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Tsai, SJ
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Hsu, JM
Human Aurora kinases have three gene family members: Aurora-A, Aurora-B, and Aurora-C. It is not yet established what the specificity of these kinases are and what signals relayed by their reactions. Therefore, we employed small pool expression screening to search for downstream substrates of Aurora-A. Interestingly, all of the identified Aurora-A substrates were resistant to serve as substrates for Aurora-B or Aurora-C, suggesting that these Aurora family members may have distinct substrate specificity for propagation of diverse signaling pathways, even though they share a conserved catalytic kinase domain. Of the candidate substrates, Aurora-A could increase the functional activity of RalA. Mutational analysis revealed that RalA-Ser(194) was the phosphorylation site for Aurora-A. Ectopic expression of V23RalA-WT could enhance collagen I-induced cell migration and anchorage-independent growth in Madin-Darby canine kidney (MDCK) Aurora-A stable cell lines. In contrast, overexpression of V23RalA-S194A in MDCK Aurora-Astable cell lines abolished the intrinsic migration and transformation abilities of Aurora-A. To our knowledge, this is the first systematic search for the downstream substrates of Aurora-A kinase. Moreover, these results support the notion that Aurora-A may act in concert with V23RalA through protein phosphorylation on Ser(194) to promote collagen I-induced cell motility and anchorage-independent growth in MDCK epithelial cells.
Induction of cyclooxygenase-2 by staurosporine through the activation of nuclear factor for IL-6 (NF-IL6) and activator protein 2 (AP2) in an osteoblast-like cell line
(2002-07-15)
Wang, CY
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Lei, HJ
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Huang, CYF
;
Zhang, ZJ
;
Mukherjee, AB
The induction of cyclooxygenase-2 (COX-2) plays a crucial role in many physiological and pathological processes. The expression of the COX-2 gene is regulated by many extracellular stimuli, including growth factors, cytokines, and tumor promoters. Staurosporine, a potential anti-tumor drug, was found recently to up-regulate the expression of the COX-2 gene in the mouse osteoblast-like cell line MC3T3-E1. The ability of staurosporine to induce the expression of the COX-2 gene was investigated using luciferase reporters controlled by various COX-2 core promoter regions. Two cis-acting sites for activator protein 2 (AP2) and nuclear factor for IL-6 (NF-IL6), respectively, were identified as responsible for the staurosporine-mediated COX-2 up-regulation. Mutational analysis further verified that both NF-IL6 and AP2 are involved in this process. Further studies showed the stimulatory effect of staurosporine on luciferase activity to be both time- and concentration-dependent. Luciferase activity could be induced at as low as 5 nM staurosporine and reached a maximum at around 20 nM. At 50 nM, the stimulatory effect of staurosporine on luciferase activity reached a maximum at about 8 hr and fell rapidly following 10 hr of incubation. Interestingly, a selective protein kinase C inhibitor, 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide (GF109203X), failed to stimulate luciferase activity under the same conditions. This finding implies that staurosporine-mediated COX-2 gene expression is specific and independent of protein kinase C activity. (C) 2002 Elsevier Science Inc. All rights reserved.
Mammalian Ste20-like protein kinase 3 (Mst3) plays an important role in spontaneous delivery
(2006-03-06)
Wu, HY
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Lin, CY
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Lin, TY
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Yuan, CJ
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生物科技學系
Mst3 plays an important role in the oxidative stress-induced apoptosis in human trophoblasts
(2006-03-06)
Lin, CY
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Wu, HY
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Yuan, CJ
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生物科技學系
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Department of Biological Science and Technology
Mst3, a novel mitochondrial protein kinase, regulates pro-apoptotic proteins in response to apoptotic stimuli
(2006-03-06)
Yuan, CJ
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Lin, CY
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Wu, HY
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生物科技學系
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Department of Biological Science and Technology
Series quartz crystal sensor for remote bacteria population monitoring in raw milk via the Internet
(2006-02-15)
Chang, KS
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Jang, HD
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Lee, CF
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Lee, YG
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Yuan, CJ
A remote monitoring system based on a piezoelectric quartz crystal (SPQC) sensor was developed for the determination of the bacteria population in raw milk. The system employs the Windows XP server operating system, and its programs for data acquisition, display and transmission were developed using the LabVIEW 7.1 programming language. The circuit design consists of a circuit with a piezoelectric quartz crystal (SPQC) and a pair of electrodes. This system can provide dynamic data monitoring on a web-page via the Internet. Immersion of the electrodes in a cell culture with bacteria inoculums resulted in a change of frequency caused by the impedance change due to microbial metabolism and the adherence of bacteria on the surface of the electrodes. The calibration curve of detection times against density of bacteria showed a linear correlation coefficient (R-2 = 0.9165) over the range of 70-10(6) CFU ml(-1). The sensor could acquire sufficient data rapidly (within 4 h) and thus enabled real-time monitoring of bacteria growth via the Internet. This system has potential application in the detection of bacteria concentration of milk at dairy farms. (c) 2005 Elsevier B.V. All rights reserved.
The role of mammalian Ste20-like kinase MST3 in apoptosis
(2001-03-07)
Yuan, CJ
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Hsu, CY
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Huang, CYF
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交大名義發表
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National Chiao Tung University
Zinc ion acts as a cofactor for serine/threonine kinase MST3 and has a distinct role in autophosphorylation of MST3
(2005-06-01)
Lu, TJ
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Huang, CYF
;
Yuan, CJ
;
Lee, YC
;
Leu, TH
We examined the metal ion cofactor preference for MST3 (mammalian Ste20-like kinase 3) of the Ste20 serine/threonine kinase family. Four metal ions (Mg+2, Mn+2, Zn2+, and Co2+) activate endogenous, exogenous, and baculovirus-expressed recombinant MST3 within the physiological concentration range. In contrast, Fe+2 and Ca+2 do not function as MST3 cofactors. Mn2+, Co2+, and Mg2+-dependent autophosphorylation of MST3 is mainly on threonine residue while Zn2+-stimulated MST3 autophosphorylation is on both serine and threonine residues. The distinct autophosphorylation pattern on MST3 suggests that MST3 may exert various types of kinase reactions depending on the type of metal ion cofactor used. To our knowledge, this is the first report showing Zn2+ as the metal ion cofactor of a recombinant serine/threonine kinase. (C) 2005 Elsevier Inc. All rights reserved.