钙库调控的钙通道Orai1体外研究方法的建立

目的:建立钙通道Orai1的体外研究方法。方法:利用脂质体重组技术,将体外纯化的Orai1蛋白重组到脂质体膜上,利用蔗糖密度梯度离心来检测其重组效率及Orai1蛋白在脂质体膜上的结构,并利用钙染料Fura-2检测脂质体内钙离子的释放。结果:成功制备了脂质体及体外纯化了GST-Orai1融合蛋白,蔗糖密度梯度离心结果证明GST-Orai1蛋白成功重组到脂质体上,以及Orai1蛋白以多聚体的形式定位在脂质体膜上。钙离子释放实验证明脂质体内钙离子包装完好,可用于后续Orai1钙通道的功能研究。结论:利用脂质体重组技术建立了一种新的Orai1的研究方法,能够更直接有效地研究其功能及其活化机制。     

20(S)-Protopanaxadiol Inhibition of Progression and Growth of Castration-Resistant Prostate Cancer

Castration-resistant progression of prostate cancer after androgen deprivation therapies remains the most critical challenge in the clinical management of prostate cancer. Resurgent androgen receptor (AR) activity is an established driver of castration-resistant progression, and upregulation of the full-length AR (AR-FL) and constitutively-active AR splice variants (AR-Vs) has been implicated to contribute to the resurgent AR activity. We reported previously that ginsenoside 20(S)-protopanaxadiol-aglycone (PPD) can reduce the abundance of both AR-FL and AR-Vs. In the present study, we further showed that the effect of PPD on AR expression and target genes was independent of androgen. PPD treatment resulted in a suppression of ligand-independent AR transactivation. Moreover, PPD delayed castration-resistant regrowth of LNCaP xenograft tumors after androgen deprivation and inhibited the growth of castration-resistant 22Rv1 xenograft tumors with endogenous expression of AR-FL and AR-Vs. This was accompanied by a decline in serum prostate-specific antigen levels as well as a decrease in AR levels and mitoses in the tumors. Notably, the 22Rv1 xenograft tumors were resistant to growth inhibition by the next-generation anti-androgen enzalutamide. The present study represents the first to show the preclinical efficacy of PPD in inhibiting castration-resistant progression and growth of prostate cancer. The findings provide a rationale for further developing PPD or its analogues for prostate cancer therapy.     

AMPK regulates homeostasis of invasion and viability in trophoblasts by redirecting glucose metabolism: Implications for pre‐eclampsia

Pre‐eclampsia (PE) is deemed an ischemia‐induced metabolic disorder of the placenta due to defective invasion of trophoblasts during placentation; thus, the driving role of metabolism in PE pathogenesis is largely ignored. Since trophoblasts undergo substantial glycolysis, this study aimed to investigate its function and regulatory mechanism by AMPK in PE development. Metabolomics analysis of PE placentas was performed by gas chromatography–mass spectrometry (GC–MS). Trophoblast‐specific AMPKα1‐deficient mouse placentas were generated to assess morphology. A mouse PE model was established by Reduced Uterine Perfusion Pressure, and placental AMPK was modulated by nanoparticle‐delivered A769662. Trophoblast glucose uptake was measured by 2‐NBDG and 2‐deoxy‐d‐[³H] glucose uptake assays. Cellular metabolism was investigated by the Seahorse assay and GC–MS.PE complicated trophoblasts are associated with AMPK hyperactivation due not to energy deficiency. Thereafter, AMPK activation during placentation exacerbated PE manifestations but alleviated cell death in the placenta. AMPK activation in trophoblasts contributed to GLUT3 translocation and subsequent glucose metabolism, which were redirected into gluconeogenesis, resulting in deposition of glycogen and accumulation of phosphoenolpyruvate; the latter enhanced viability but compromised trophoblast invasion. However, ablation of AMPK in the mouse placenta resulted in decreased glycogen deposition and structural malformation. These data reveal a novel homeostasis between invasiveness and viability in trophoblasts, which is mechanistically relevant for switching between the ‘go’ and ‘grow’ cellular programs.

Pre‐eclampsia (PE) is associated with trophoblast AMPK hyperactivation, presumably due to LKB1 phosphorylation, and glucose uptake is consequently increased via trafficking of GLUT3 from the cytosol to the plasma membrane. Such translocation enhances glycolytic flux and redirects glucose metabolic intermediates into gluconeogenesis, resulting in PEP accumulation, which not only benefits cell survival but also suppresses invasion by repressing MMPs, and thus in turn modulates switching between the ‘go’ and ‘grow’ cellular programs.     

植物肌动蛋白研究的过去及现状

肌动蛋白作为一种骨架蛋白广泛存在于植物细胞,有重要的生理功能.综述了植物肌动蛋白的发现及研究现状,着重介绍了植物肌动蛋白的性质、结构和生理功能.     

The effect of single nucleotide polymorphism identification strategies on estimates of linkage disequilibrium

At present there is tremendous interest in characterizing the magnitude and distribution of linkage disequilibrium (LD) throughout the human genome, which will provide the necessary foundation for genome-wide LD analyses and facilitate detailed evolutionary studies. To this end, a human high-density single-nucleotide polymorphism (SNP) marker map has been constructed. Many of the SNPs on this map, however, were identified by sampling a small number of chromosomes from a single population, and inferences drawn from studies using such SNPs may be influenced by ascertainment bias (AB). Through extensive simulations, we have found that AB is a potentially significant problem in estimating and comparing LD within and between populations. Specifically, the magnitude of AB is a function of the SNP discovery strategy, number of chromosomes used for SNP discovery, population genetic characteristics of the particular genomic region considered, amount of gene flow between populations, and demographic history of the populations. We demonstrate that a balanced SNP discovery strategy (where equal numbers of chromosomes are sampled from multiple subpopulations) is the optimal study design for generating broadly applicable SNP resources. Finally, we validate our theoretical predictions by comparing our results to publicly available data from ten genes sequenced in 24 African American and 23 European American individuals.     

Unifying Fluorescence Microscopy and Mass Spectrometry for Studying Protein Complexes in Cells

We have developed and applied a method unifying fluorescence microscopy and mass spectrometry for studying spatial and temporal properties of proteins and protein complexes in yeast cells. To combine the techniques, first we produced a variety of DNA constructs that can be used for genomic tagging of proteins with modular fluorescent and affinity tags. The modular tag consists of one of the multiple versions of monomeric fluorescent proteins fused to a variety of small affinity epitopes. After this step we tested the constructs by tagging two yeast proteins, Pil1 and Lsp1, the core components of eisosomes, the large protein complexes involved in endocytosis in Saccharomyces cerevisiae, with a variety of fluorescent and affinity probes. Among the modular tags produced we found several combinations that were optimal for determining subcellular localization and for purifying the tagged proteins and protein complexes for the detailed analysis by mass spectrometry. And finally, we applied the designed method for finding the new protein components of eisosomes and for gaining new insights into molecular mechanisms regulating eisosome assembly and disassembly by reversible phosphorylation and dephosphorylation. Our results indicate that this approach combining fluorescence microscopy and mass spectrometry into a single method provides a unique perspective into molecular mechanisms regulating composition and dynamic properties of the protein complexes in living cells.Fluorescent proteins have become invaluable probes for studying molecular processes in living cells with light microscopy techniques (1–3). Proteins, organelles, and entire cells can be selectively visualized using a variety of fluorescent proteins fused to the proteins of interest (1–6). Combined with genetics and molecular biology techniques fluorescence microscopy provides an efficient tool for observing molecular phenotypes useful for dissecting the pathways of cell cycle progression and cell response to internal and external signals (7). However, understanding the mechanism controlling the properties of proteins in cells can be a challenging task, frequently requiring a comprehensive characterization of the proteins at the molecular level.The proteins tagged with green fluorescent protein (GFP)¹ can be also purified using GFP antibodies. Cheeseman and Desai (8) and Cristea et al. (9) have enriched GFP-tagged proteins and protein complexes for further detailed analysis by MS. The MS-based methods for protein analysis are fast, sensitive, and able to identify both proteins in complex protein mixtures and residues bearing post-translational modifications (10, 11). Thus, the addition of affinity purification and mass spectrometry steps enabled the researchers to study protein interactions and the post-translational modifications in the context of the protein subcellular localization. Juxtaposition of the protein localization, composition of the protein complexes, and post-translational modifications frequently yield a unique perspective of the cellular processes and the molecular mechanisms of their regulation (12, 13).Using fluorescent proteins also as affinity probes can be problematic in several instances. First of all, the good quality antibodies against the rapidly increasing number of fluorescent proteins (3, 6) are not yet readily available. Furthermore raising antibodies specifically recognizing fluorescent proteins originating from the same organism but fluorescing a different color can be difficult or even impossible because such proteins frequently differ by mutations of only a few amino acids (1–6). Thus, we seek an alternative approach to the design of tags suitable for subcellular localization and purification of proteins and protein complexes that is 1) independent of the availability of antibody to a specific form of a fluorescent protein, 2) suitable for multiplexing, i.e. simultaneous observation of subcellular localization of several proteins and affinity purification of the proteins and stably associated protein complexes, and 3) flexible and easy to modify to incorporate better versions of fluorescent proteins and affinity tags after they are discovered.One possible solution that satisfies the stated requirements is to use a modular tag containing a version of a fluorescent protein fused to an affinity epitope. In this case we can decouple requirements for both modules and optimize the performance of each one independently for fluorescence microscopy and affinity purification experiments. To our knowledge, this possibility was first realized by Thorn and co-worker (14) who have fused 3HA (three repeats of YPYDVPDYA epitope from hemagglutinin protein) and 13MYC (13 repeats of EQKLISEEDL epitope, corresponding to a stretch of the C-terminal amino acids of the human c-MYC protein) tags to several variants of fluorescent proteins. The authors have argued that the fusion of the fluorescent proteins to the affinity epitopes may enable fluorescence and immunochemical analysis but did not test this idea. Cheeseman and Desai (8) fused the S-peptide and hexahistidine epitopes to the GFP protein to enable additional tandem purification steps. Su and co-workers (15) also fused a hexahistidine tag (His₆) to GFP to purify recombinantly produced proteins. Although hexahistidine tag performs well for isolation of overexpressed recombinant proteins, it works poorly for affinity purification of low abundance, endogenously expressed proteins (16). A double affinity tag containing a single MYC epitope and hexahistidine was also used to purify recombinantly produced fluorescent proteins (6).Here we describe the design and implementation of the modular fluorescent and affinity tags. These tags contain a variety of fluorescent proteins, which can be used exclusively for obtaining subcellular visualization, and several small epitope tags that can be utilized to perform two-step affinity purification. To test the performance of the constructs produced, we tagged two yeast proteins, Pil1 and Lsp1, the core components of eisosomes, with a variety of modular tags.Eisosomes are large heterodimeric protein complexes recently discovered in Saccharomyces cerevisiae (17). There are ∼50–100 eisosomes in each mature yeast cell distributed uniformly in a characteristic dotted pattern at the cell surface periphery. Each eisosome contains ∼2000–5000 copies of Pil1 and Lsp1. It was shown that eisosomes serve as portals of endocytosis in yeast. The function of eisosomes is regulated by reversible phosphorylation (18, 19).Among the constructs tested, we found several combinations of fluorescent protein and affinity tags that were optimal for determining subcellular localization and purification of the proteins and protein complexes. We applied these tags to further investigate eisosomes and found several new protein components of the complexes and obtained new insights into molecular mechanisms regulating eisosome integrity by reversible phosphorylation and dephosphorylation. Our results indicate that an approach combining fluorescence microscopy and mass spectrometry into a single method provides a unique perspective into molecular mechanisms regulating composition and dynamic properties of the protein complexes in living cells.     

血管紧张素AT1受体抗体阳性孕鼠后代高盐饮食后血管功能障碍

血管紧张素AT1受体抗体(AT1-Ab)可损伤胎盘发育,进而导致胎儿宫内生长受限(intrauterine growth restriction,IUGR).根据胎儿源性成人疾病学说,IUGR会明显增加成人后患心血管疾病的几率.本研究旨在观察AT1-Ab阳性孕鼠后代生长至成年后血管功能有无异常.24只雌性Wistar大...     

A meta-analysis of tumor necrosis factor-alpha, interleukin-6, and interleukin-10 in preeclampsia

Inflammation may play a major role in the pathogenesis of preeclampsia (PE). In this meta-analysis, we determined whether maternal polymorphisms and serum concentrations of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) were associated with PE. All studies investigating the associations between PE and maternal polymorphisms of TNF-α-308G/A, IL-6-174G/C, and IL-10-1082A/G or serum concentrations of TNF-α, IL-6, and IL-10 were reviewed. We found that neither maternal TNF-α-308G/A (p=0.86, odds ratio [OR]=0.98, 95% confidence interval [CI], 0.76-1.25), IL-6 174G/C (p=0.14, OR=1.23, 95% CI, 0.93-1.61), nor IL-10-1082A/G (p=0.72, OR=1.07, 95% CI, 0.75-1.52) were associated with PE. On the other hand, maternal TNF-α (p<0.00001, weighted mean difference [WMD]=19.63 pg/ml, 95% CI, 18.54-20.72 pg/ml), IL-6 (p<0.00001, WMD=6.58 pg/ml, 95% CI, 5.49-7.67 pg/ml), and IL-10 (p=0.0005, WMD=19.30 pg/ml, 95% CI, 8.42-30.17 pg/ml) concentrations were significantly higher in PE patients versus controls. Our findings strengthen the clinical evidence that PE is accompanied by exaggerated inflammatory responses, but do not support TNF-α-308G/A, IL-6-174G/C, and IL-10-1082A/G as candidate susceptibility loci in PE.