| 1. |
- Chen, Qing-wen, et al.
(författare)
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Role of ERK/MAPK in endothelin receptor signaling in human aortic smooth muscle cells.
- 2009
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Ingår i: BMC Cell Biology. - : Springer Science and Business Media LLC. - 1471-2121. ; 10:Jul 3
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Tidskriftsartikel (refereegranskat)abstract
- ABSTRACT: muscle cells (VSMCs) through activation of endothelin type A (ETA) and type B (ETB) receptors. The extracellular signal-regulated kinase 1 and 2 (ERK1/2) mitogen-activated protein kinases (MAPK) are involved in ET-1-induced VSMC contraction and proliferation. This study was designed to investigate the ETA and ETB receptor intracellular signaling in human VSMCs and used phosphorylation (activation) of ERK1/2 as a functional signal molecule for endothelin receptor activity. RESULTS: Subconfluent human VSMCs were stimulated by ET-1 at different concentrations (1 nM-1 microM). The activation of ERK1/2 was examined by immunofluorescence, Western blot and phosphoELISA using specific antibody against phosphorylated ERK1/2 protein. ET-1 induced a concentration- and time- dependent activation of ERK1/2 with a maximal effect at 10 min. It declined to baseline level at 30 min. The ET-1-induced activation of ERK1/2 was completely abolished by MEK1/2 inhibitors U0126 and SL327, and partially inhibited by the MEK1 inhibitor PD98059. A dual endothelin receptor antagonist bosentan or the ETA antagonist BQ123 blocked the ET-1 effect, while the ETB antagonist BQ788 had no significant effect. However, a selective ETB receptor agonist, Sarafotoxin 6c (S6c) caused a time-dependent ERK1/2 activation with a maximal effect by less than 20% of the ET-1-induced activation of ERK1/2. Increase in bosentan concentration up to 10 microM further inhibited ET-1-induced activation of ERK1/2 and had a stronger inhibitory effect than BQ123 or the combined use of BQ123 and BQ788. To further explore ET-1 intracellular signaling, PKC inhibitors (staurosporin and GF109203X), PKC-delta inhibitor (rottlerin), PKA inhibitor (H-89), and phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin) were applied. The inhibitors showed significant inhibitory effects on ET-1-induced activation of ERK1/2. However, blockage of L-type Ca2+ channels or calcium/calmodulin-dependent protein kinase II, chelating extracellular Ca2+ or emptying internal Ca2+ stores, did not affect ET-1-induced activation of ERK1/2. CONCLUSION: The ETA receptors predominate in the ET-1-induced activation of ERK1/2 in human VSMCs, which associates with increments in intracellular PKC, PKA and PI3K activities, but not Ca2+ signalling.
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| 2. |
- Li, Jing-Jing, et al.
(författare)
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Immobilized Triton X-100-assisted refolding of Green Fluorescent Protein-Tobacco Etch Virus protease fusion protein using β-cyclodextrin as the eluent
- 2009
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Ingår i: Process Biochemistry. - : Elsevier BV. - 1359-5113 .- 1873-3298. ; 44:3, s. 277-282
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Tidskriftsartikel (refereegranskat)abstract
- A new protein refolding technique based on the use of the non-charged detergent Triton X-100 immobilized to the cross-linked agarose gel Sepharose High Performance has been developed. The new solid phase was used in combination with soluble β-cyclodextrin (β-CD) to refold recombinant Green Fluorescent Protein fused to Tobacco Etch Virus protease (GFPTEVP) expressed as inclusion bodies in E. coli. Previous attempts to refold recombinant GFPTEVP by dilution had failed. In the new procedure a column packed with Triton X-100-coupled Sepharose High Performance was used to capture unfolded GFPTEVP followed by elution using an increasing β-CD concentration gradient. The yield of properly refolded GFPTEVP was 46% at a protein concentration of 380 μg/ml. In contrast, dilution refolding of GFPTEVP at 200 μg/ml refolding buffer resulted in only 4.7% of native protein.
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| 3. |
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| 4. |
- Yu, Ling-Zhu, et al.
(författare)
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MEK1/2 regulates microtubule organization, spindle pole tethering and asymmetric division during mouse oocyte meiotic maturation.
- 2007
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Ingår i: Cell cycle (Georgetown, Tex.). - 1551-4005. ; 6:3, s. 330-8
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Tidskriftsartikel (refereegranskat)abstract
- It is well known that MAPK plays pivotal roles in oocyte maturation, but the function of MEK (MAPK kinase) remains unknown. We have studied the expression, subcellular localization and functional roles of MEK during meiotic maturation of mouse oocytes. Firstly, we found that MEK1/2 phoshorylation (p-MEK1/2, indicative of MEK activation) was low in GV (germinal vesicle) stage, increased 2h after GVBD (germinal vesicle breakdown), and reached the maximum at metaphase II. Secondly, we found that P-MEK1/2 was restricted in the GV prior to GVBD. In prometaphase I and metaphase I, P-MEK1/2 was mainly associated with the spindle, especially with the spindle poles. At anaphase I and telophase I, p-MEK1/2 became diffusely distributed in the region between the separating chromosomes, and then became associated with the midbody. The association of p-MEK1/2 with spindle poles was further confirmed by its colocalization with the centrosomal proteins, gamma-tubulin and NuMA. Thirdly, we have investigated the possible functional role of MEK1/2 activation by intravenous administration and intrabursal injection of a specific MEK inhibitor, U0126, and by microinjection of MEK siRNA into oocytes. All these manipulations cause disorganized spindle poles and spindle structure, misaligned chromosomes and larger than normal polar bodies. Our results suggest that MEK1/2 may function as a centrosomal protein and may have roles in microtubule organization, spindle pole tethering and asymmetric division during mouse oocyte maturation.
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| 5. |
- Zhong, Hongmei, et al.
(författare)
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A novel photoluminescence transition influenced by O implantation in ZnO bulk
- 2006
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Ingår i: Chemical Physics Letters. - : Elsevier BV. - 0009-2614 .- 1873-4448. ; 421:1-3, s. 309-311
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Tidskriftsartikel (refereegranskat)abstract
- The bulk zinc oxide (ZnO) sample implanted O with implantation concentration of 5 × 1019/cm3 was investigated by photoluminescence. A novel transition at emission energy of 3.08 eV at 77 K appears in the O-implanted sample. In order to find the origin of the novel transition, the O-implanted effects on the luminescence of ZnO bulk have been studied by first principle calculations based on the local density approximation. The theoretical results show that the novel transition at emission energy of 3.08 eV is attributed to O-antisite (Ozn) produced by O-implanted procedure.
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