A Circular Dichroism Spectroscopic Study Revealing the Cause of the Changes of Chlorophyll a Fluorescence Induction of Photosystem Ⅱ During Heat Treatment

Chlorophyll a fluorescence and circular dichroism (CD) spectra of photosystem Ⅱ (PSⅡ) membrane were measured after heat treatment. The chlorophyll fluorescence parameter Fo' remained stable after treatment at the temperatures from 30 ℃ to 40 ℃ and then reached a maximum after treatment at 55 ℃. In PSⅡ membranes and LHCⅡ (light-harvesting chlorophyll a/b binding complex)-enriched complexes, anomalous CD signals with extremely large amplitudes occurred during the heat treatment. The temperature corresponding to the maximum anomalous CD intensity peaking at 677 nm was 40 ℃. The results indicate that the aggregation state of the LHCⅡ in PSⅡ is related to the anomalous CD signal, and can be an important factor influencing Fo' in the heat treatment of PSⅡ membrane.     

MYCN-Related Suppression of Functional CD44 Expression Enhances Tumorigenic Properties of Human Neuroblastoma Cells

Highly malignant neuroblastoma tumors with MYCN amplification have been shown to downregulate the expression of the CD44 adhesion receptor. We have previously shown that MYCN amplified neuroblastoma cell lines either lack CD44 expression or express a nonfunctional, nonhyaluronic acid-binding CD44 receptor. By analysis of cells with manipulated expression of either CD44 or MYCN, we demonstrate that transfection of cells with a CD44 full-length cDNA construct produced a functional receptor in single copy MYCN cells and a nonfunctional CD44 receptor in MYCN amplified cells, similar to the CD44 receptor expressed by cells with enforced MYCN. Analysis of the in vivo growth properties of the transfectants revealed that the restoration of a functional CD44 receptor in nonamplified cells resulted in the suppression of in vivo cell growth, therefore linking the MYCN-related lack of hyaluronic acid-binding function of CD44 to the highly tumorigenic properties of a subset of neuroblastoma cells.     

Assembly of the CD8α/p56 protein complex in stably expressing rat epithelial cells

We have previously characterized the biogenesis of the human CD8α protein expressed in rat epithelial cells. We now describe the biosynthesis, post-translational maturation and hetero-oligomeric assembly of the human CD8α/p56^lck protein complex in stable transfectants obtained from the same cell line. There were no differences in the myristilation of p56^lck, or in the dimerization, O-glycosylation and transport to the plasma membrane of CD8α, between cells expressing either one or both proteins. In the doubly expressing cells, dimeric forms of CD8α established hetero-oligomeric complexes with p56^lck, as revealed by co-immunoprecipitation assays performed with anti-CD8α antibody. Moreover, p56^lck bound in these hetero-oligomeric complexes was endowed with auto- and hetero-phosphorylating activity. The present study shows that: (1) the newly synthesized p56^lck binds rapidly to CD8α and most of the p56^lck is bound to CD8α at steady state; (2) CD8α/p56^lck protein complexes are formed at internal membranes as well as at the plasma membrane; and (3) about 50% of complexed p56^lck reaches the cell surface.     

胶毒素与BSA的相互作用

应用荧光、圆二色和紫外—可见吸收等波谱法研究胶毒素与牛血清白蛋白(BSA)的相互作用。荧光光谱实验结果表明胶毒素主要靠疏水作用与BSA结合, 而对其内源荧光产生猝灭作用,其淬灭方式为静态猝灭, 胶毒素与BSA的结合常数为7.2×103 L/mol。圆二色光谱检测发现, 随着胶毒素浓度的增加, BSA的a-螺旋数量也增加, 当胶毒素浓度为BSA浓度的100倍时, BSA的a-螺旋增加40.1%, 表明胶毒素与BSA的结合改变了BSA的空间构象。     

假单胞菌株M18 gacA基因的克隆、表达及蛋白纯化

GacA是GacS/A双元调控系统的一个组分, 克隆假单胞菌株M18中的gacA基因。测序结果同Pseudomonas sp. PAO1比较, 该基因2个碱基的改变未引起氨基酸的改变。将该基因克隆到表达质粒pET28b (+)中, 将重组质粒经热激转化至大肠杆菌BL21 (DE3)中, IPTG诱导表达。表达产物经金属螯合层析柱纯化, 蛋白纯度约为98%, 质谱鉴定证明纯化的蛋白为GacA蛋白。CD谱分析GacA包含了4% α-helix, 48% β-sheet 和48%无规则卷曲。GacA蛋白的获得为进一步研究其晶体结构、生物学性能以及GacS/A双元调控系统奠定了基础。     

Oxidized high-density lipoprotein promotes CD36 palmitoylation and increases lipid uptake in macrophages

Oxidized high-density lipoprotein (oxHDL) reduces the ability of cells to mediate reverse cholesterol transport and also shows atherogenic properties. Palmitoylation of cluster of differentiation 36 (CD36), an important receptor mediating lipoprotein uptake, is required for fatty acid endocytosis. However, the relationship between oxHDL and CD36 has not been described in mechanistic detail. Here, we demonstrate using acyl-biotin exchange analysis that oxHDL activates CD36 by increasing CD36 palmitoylation, which promotes efficient uptake in macrophages. This modification increased CD36 incorporation into plasma lipid rafts and activated downstream signaling mediators, such as Lyn, Fyn, and c-Jun N-terminal kinase, which elicited enhanced oxHDL uptake and foam cell formation. Furthermore, blocking CD36 palmitoylation with the pharmacological inhibitor 2-bromopalmitate decreased cell surface translocation and lowered oxHDL uptake in oxHDL-treated macrophages. We verified these results by transfecting oxHDL-induced macrophages with vectors expressing wildtype or mutant CD36 (mCD36) in which the cytoplasmic palmitoylated cysteine residues were replaced. We show that cells containing mCD36 exhibited less palmitoylated CD36, disrupted plasma membrane trafficking, and reduced protein stability. Moreover, in ApoE^−/−CD36^−/− mice, lipid accumulation at the aortic root in mice receiving the mCD36 vector was decreased, suggesting that CD36 palmitoylation is responsible for lipid uptake in vivo. Finally, our data indicated that palmitoylation of CD36 was dependent on DHHC6 (Asp-His-His-Cys) acyltransferase and its cofactor selenoprotein K, which increased the CD36/caveolin-1 interaction and membrane targeting in cells exposed to oxHDL. Altogether, our study uncovers a causal link between oxHDL and CD36 palmitoylation and provides insight into foam cell formation and atherogenesis.     

Chronic cholesterol depletion increases F-actin levels and induces cytoskeletal reorganization via a dual mechanism

Previous work from us and others has suggested that cholesterol is an important lipid in the context of the organization of the actin cytoskeleton. However, reorganization of the actin cytoskeleton upon modulation of membrane cholesterol is rarely addressed in the literature. In this work, we explored the signaling crosstalk between cholesterol and the actin cytoskeleton by using a high-resolution confocal microscopic approach to quantitatively measure changes in F-actin content upon cholesterol depletion. Our results show that F-actin content significantly increases upon chronic cholesterol depletion, but not during acute cholesterol depletion. In addition, utilizing inhibitors targeting the cholesterol biosynthetic pathway at different steps, we show that reorganization of the actin cytoskeleton could occur due to the synergistic effect of multiple pathways, including prenylated Rho GTPases and availability of membrane phosphatidylinositol 4,5-bisphosphate. These results constitute one of the first comprehensive dissections of the mechanistic basis underlying the interplay between cellular actin levels and cholesterol biosynthesis. We envision these results will be relevant for future understating of the remodeling of the actin cytoskeleton in pathological conditions with altered cholesterol.     

人CD55基因的克隆及其转基因构件的组装

本实验采用人肝组织作为RNA的来源 ,经RT -PCR扩增得到CD55基因的cDNA片段。与人α -珠蛋白启动子及其polyA序列重组 ,插入质粒载体pGEM - 5zf,获得了可用于受精卵原核显微注射的基因构件 ,为建立人CD55转基因动物模型奠定了基础。