耐甲氧西林金黄色葡萄球菌青霉素结合蛋白2a转肽酶区的原核表达及其纯化

目的原核表达耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)青霉素结合蛋白2a(penicillin binding protein 2a,PBP2a)转肽酶区,并进行纯化及鉴定。方法 PCR扩增编码PBP2a转肽酶区的mec Af基因片段,克隆至原核表达载体p GEX-6p-1中,构建重组表达质粒p GEX-6p-mec Af,经双酶切(Bam HⅠ和Eco RⅠ)及测序鉴定正确后,转化感受态大肠埃希菌(E.coli)BL21进行诱导表达;经蛋白亲和层析柱纯化后,进行SDS-PAGE和Western blot鉴定。结果重组表达质粒p GEX-6p-mec Af经双酶切(Bam HⅠ和Eco RⅠ)及测序鉴定证明构建正确;表达产物的相对分子质量约63 000,表达量约占菌体总蛋白的14.5%,纯化后纯度达90%。结论成功构建了PBP2a转肽酶区的原核表达质粒,目的蛋白在E.coli中获得高效表达,为制备单克隆抗体及建立MRSA快速检测方法奠定了基础。     

Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy

Live-cell microscopy is now routinely used to monitor the activities of the genetically encoded biosensor proteins that are designed to directly measure specific cell signaling events inside cells, tissues, or organisms. Most fluorescent biosensor proteins rely on Förster resonance energy transfer (FRET) to report conformational changes in the protein that occur in response to signaling events, and this is commonly measured with intensity-based ratiometric imaging methods. An alternative method for monitoring the activities of the FRET-based biosensor proteins is fluorescence lifetime imaging microscopy (FLIM). FLIM measurements are made in the time domain, and are not affected by factors that commonly limit intensity measurements. In this review, we describe the use of the digital frequency domain (FD) FLIM method for the analysis of FRET signals. We illustrate the methods necessary for the calibration of the FD FLIM system, and demonstrate the analysis of data obtained from cells expressing “FRET standard” fusion proteins. We then use the FLIM-FRET approach to monitor the changes in activities of two different biosensor proteins in specific regions of single living cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail.     

Effect of radiation on the friction-wear properties of polyetherketone with a cardo group

We studied the effect of radiation on the friction-wear properties of polyetherketone with a cardo group (PEK-C) with a MM-200 model friction and wear tester. We found that radiation could improve the friction-wear properties of PEK-C to a certain degree. The friction coefficient and wear rate of PEK-C decreased as the radiation dose increased from 5 × 10⁴ to 1 × 10⁷ Gy. Scanning electron microscopy results revealed that the size of wear debris of unirradiated PEK-C was larger than that of irradiated PEK-C. The worn surface of unirradiated PEK-C showed plough marks, whereas the worn surface of irradiated PEK-C did not show plough marks; its surface was quite smooth. With the frictional couple of a carbon steel ring and an irradiated PEK-C block, a relatively uniform and coherent transfer film was formed on the ring surface. It was inferred that the transfer film contributed largely to the decreased friction coefficient and wear rate of the irradiated PEK-C. An IR spectrum showed that no significant chemical change took place when the PEK-C sample was irradiated. Thermal analysis results showed that radiation changed the thermal properties of PEK-C, therefore, its friction-wear properties changed at the same time. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 962–967, 2001     

The Cross Talk between cGMP Signal Pathway and PKC in Pulmonary Endothelial Cell Angiogenesis

Angiogenic proliferation of vascular endothelial cells is believed to play an important role in pulmonary vascular remodeling in pulmonary arterial hypertension. In the present study, we found that c-GMP (cyclic guanosine monophosphate) inhibited the proliferation and tube formation of pulmonary vascular endothelial cells induced by TGF-β1, and that this process was reversed by PKG (protein kinase G) inhibitor and PKC (protein kinase C) inhibitor. In addition, small interfering RNA (siRNA) targeting ERK also reduced cellular proliferation. Furthermore, western blotting showed that cGMP down-regulated the phosphorylation level of ERK1/2, which was reversed not only by PKG inhibitor but also by PKC inhibitor. Silencing different PKC isoforms showed that PKCΔ, PKCγ and PKCα were involved in ERK phosphorylation, suggesting that PKC kinases have a permissive action. Three subtypes, PKCΔ, PKCγ and PKCα are likely to be involved the phosphorylation suppression of ERK included cGMP. Taken together, these data suggest that ERK phosphorylation mediates the proliferation of pulmonary vascular endothelial cells, and PKC kinases have a permissive action in this process.