Proteome-Wide Profiling of the MCF10AT Breast Cancer Progression Model

Background

Mapping the expression changes during breast cancer development should facilitate basic and translational research that will eventually improve our understanding and clinical management of cancer. However, most studies in this area are challenged by genetic and environmental heterogeneities associated with cancer.

Methodology/Principal Findings

We conducted proteomics of the MCF10AT breast cancer model, which comprises of 4 isogenic xenograft-derived human cell lines that mimic different stages of breast cancer progression, using iTRAQ-based tandem mass spectrometry. Of more than 1200 proteins detected, 98 proteins representing at least 20 molecular function groups including kinases, proteases, adhesion, calcium binding and cytoskeletal proteins were found to display significant expression changes across the MCF10AT model. The number of proteins that showed different expression levels increased as disease progressed from AT1k pre-neoplastic cells to low grade CA1h cancer cells and high grade cancer cells. Bioinformatics revealed that MCF10AT model of breast cancer progression is associated with a major re-programming in metabolism, one of the first identified biochemical hallmarks of tumor cells (the “Warburg effect”). Aberrant expression of 3 novel breast cancer-associated proteins namely AK1, ATOX1 and HIST1H2BM were subsequently validated via immunoblotting of the MCF10AT model and immunohistochemistry of progressive clinical breast cancer lesions.

Conclusion/Significance

The information generated by this study should serve as a useful reference for future basic and translational cancer research. Dysregulation of ATOX1, AK1 and HIST1HB2M could be detected as early as the pre-neoplastic stage. The findings have implications on early detection and stratification of patients for adjuvant therapy.     

The C-Terminal Domain of the mGluR1 Metabotropic Glutamate Receptor Affects Sensitivity to Agonists

Abstract: The metabotropic glutamate receptor (mGluR) subtype 1 exists as at least three variants (−1a, −1b, and −1c) generated by alternative splicing at the C-terminal domain. Fluorometric Ca²⁺ measurements were used to compare the concentration dependency of agonist-induced rises in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in human embryonic HEK 293 cells transiently expressing rat mGluR1a, mGluR1b, or mGluR1c. The rank order of agonist potencies was quisqualate ≫ (2 S, 1' S, 2' S )-2-(carboxycyclopropyl)glycine (L-CCG-I) > (1 S, 3 R )-1-aminocyclopentane-1,3-dicarboxylic acid [(1 S, 3 R )-ACPD] and did not differ among the splice variants. However, agonists were consistently more potent at mGluR1a than at mGluR1c and mGluR1b. In the same system, we characterized the agonist pharmacology of two chimeric rat mGluR3/1 receptors where the first and/or the second intracellular loop(s) and the C-terminal domain were exchanged with the corresponding mGluR1a or mGluR1c sequences and that were previously shown to mediate elevations in [Ca²⁺]_i in response to agonists. The potency of agonists was higher at the chimera having the C-terminus of mGluR1a as compared with those having the mGluR1c C-terminus. Both chimeric mGluR3/1 receptors had the same rank order of agonist potencies: L-CCG-I ≫ (1 S, 3 R )-ACPD ∼ quisqualate. These data support the hypothesis that the C-terminal domain of mGluRs plays a role in determining the potency of agonists for inducing mGluR-mediated functional responses.     

Is Yield Increase Sufficient to Achieve Food Security in China?

Increasing demand for food, driven by unprecedented population growth and increasing consumption, will keep challenging food security in China. Although cereal yields have substantially improved during the last three decades, whether it will keep thriving to meet the increasing demand is not known yet. Thus, an integrated analysis on the trends of crop yield and cultivated area is essential to better understand current state of food security in China, especially on county scale. So far, yield stagnation has extensively dominated the main cereal-growing areas across China. Rice yield is facing the most severe stagnation that 53.9% counties tracked in the study have stagnated significantly, followed by maize (42.4%) and wheat (41.9%). As another important element for production sustainability, but often neglected is the planted area patterns. It has been further demonstrated that the loss in productive arable land for rice and wheat have dramatically increased the pressure on achieving food security. Not only a great deal of the planted areas have stagnated since 1980, but also collapsed. 48.4% and 54.4% of rice- and wheat-growing counties have lost their cropland areas to varying degrees. Besides, 27.6% and 35.8% of them have retrograded below the level of the 1980s. The combined influence (both loss in yield and area) has determined the crop sustainable production in China to be pessimistic for rice and wheat, and consequently no surprise to find that more than half of counties rank a lower level of production sustainability. Therefore, given the potential yield increase in wheat and maize, as well as substantial area loss of rice and wheat, the possible targeted adaptation measures for both yield and cropping area is required at county scale. Moreover, policies on food trade, alongside advocation of low calorie diets, reducing food loss and waste can help to enhance food security.     

A novel mutation in the mitochondrial tRNAAla gene (m.5636T>C) in a patient with progressive external ophthalmoplegia

We report a heteroplasmic novel mutation m.5636T>C in the mt-tRNA^Ala in a patient with bilateral ptosis and ophthalmoparesis in whom a muscle biopsy showed cytochrome c oxdidase (COX) negative and ragged red fibers. Using laser capture microdissection we have isolated COX negative fibers and COX positive fibers from the muscle of the patient and determined that the mutation load was clearly increased in COX negative muscle fibers. Additionally, the mutated m.5636T nucleotide is conserved in all the mammal and non-mammal species analyzed and might be structurally relevant as it is located in a position involved in the formation of tertiary structure of canonical mitochondrial tRNAs.     

过氧亚硝基阴离子通过JAK/STAT信号途径促进系膜细胞纤连蛋白的合成

氧化应激是糖尿病肾病的重要发病机制之一。过氧亚硝基阴离子(peroxynitrite,ONOO–)是参与氧化应激损伤的重要成员,与糖尿病及其并发症密切相关。该文观察高糖环境下ONOO–对系膜细胞合成纤连蛋白(fibronectin,FN)的影响,并探讨其作用机制。实验中,人肾小球系膜细胞分为4组:正常对照组、高糖组、高糖+尿酸组及高糖+AG490组。培养12,24,48 h后收集细胞及其上清液、并提取细胞总蛋白。采用酶联免疫吸附实验(ELISA)检测细胞上清液中FN的含量,采用免疫细胞化学和Western blot检测NT总蛋白(ONOO–生成的生物标志物)、p-JAK2及p-STAT3蛋白的表达。结果显示,与同期正常组相比,高糖组NT总蛋白、p-JAK2及p-STAT3的表达及FN含量明显增高(P<0.05),并且随着时间的延长表达逐渐增多,以48 h组最为显著;高糖+尿酸组,NT、p-JAK2、p-STAT3及FN较高糖组明显减少(P<0.05);高糖+AG490组,p-JAK2、p-STAT3及FN较高糖组明显减少(P<0.05),但NT表达与高糖组差异无统计学意义(P>0.05)。由此可见,高糖环境下系膜细胞中存在ONOO–的过量表达,ONOO–通过JAK/STAT信号途径促进系膜细胞FN的合成。     

采用GFP标记技术研究钙调蛋白在活细胞中与细胞周期相关的分布

采用融合绿色荧光蛋白 (GFP)和钙调蛋白 (CaM)的方法来研究钙调蛋白在细胞周期不同阶段的分布 .首先 ,发现CaM在细胞内的分布随细胞周期的不同而改变 .CaM在G1期主要分布于细胞质中 ,在S期开始向细胞核内转移 ,并于G2期高度集中于细胞核内 .其次 ,G2期细胞核内的CaM似乎与有丝分裂的启动有关 ,因为此时抑制CaM的活性可同时抑制核膜破裂及染色质凝缩 .最后 ,发现在进入有丝分裂后 ,CaM主要集中于纺锤体靠近两极的地方 .此时 ,抑制CaM的活性会引起纺锤体结构的破坏 .同时讨论了CaM的这些特异性分布与细胞周期调控之间的关系 .     

Antibodies targeting the PfRH1 binding domain inhibit invasion of Plasmodium falciparum merozoites

Invasion by the malaria merozoite depends on recognition of specific erythrocyte surface receptors by parasite ligands. Plasmodium falciparum uses multiple ligands, including at least two gene families, reticulocyte binding protein homologues (RBLs) and erythrocyte binding proteins/ligands (EBLs). The combination of different RBLs and EBLs expressed in a merozoite defines the invasion pathway utilized and could also play a role in parasite virulence. The binding regions of EBLs lie in a conserved cysteine-rich domain while the binding domain of RBL is still not well characterized. Here, we identify the erythrocyte binding region of the P. falciparum reticulocyte binding protein homologue 1 (PfRH1) and show that antibodies raised against the functional binding region efficiently inhibit invasion. In addition, we directly demonstrate that changes in the expression of RBLs can constitute an immune evasion mechanism of the malaria merozoite.     

Double-strand hydrolysis of DNA by a magnesium(II) complex with diethylenetriamine

The development of artificial nucleases that hydrolyze DNA or RNA is of great interest in molecular biology, biotechnology, and medicine. We now report that a magnesium(II) complex of diethylenetriamine (Mg-dien) can effectively promote the double-stranded cleavage of plasmid DNA and the dideoxynucleotide dApdA under physiological conditions of pH and temperature. Experiments performed in the presence of hydrogen peroxide, radical scavengers, or under rigorously anaerobic conditions indicate that DNA cleavage mediated by Mg-dien occurs via a hydrolytic path. Mg-dien efficiently hydrolyzes supercoiled pBR322 DNA and the pseudo-first-order rate constant at 37 degrees C and pH 8.0 is estimated to be 1.60 h(-1). The dinucleotide dApdA hydrolysis, with Mg-dien at 170 microM, shows a rate enhancement factor of ca. 5 x 10(8). 1H and 31P(1H) NMR studies show that Mg-dien effectively hydrolyzes 5'-dAMP to give deoxyadenosine and inorganic phosphate. While Mg2+ has been found at the catalytic sites of many natural nucleases, Mg-dien appears to be the first synthetic Mg2+-containing system capable of hydrolyzing dideoxynucleotides and DNA and thus may provide a simple model system to assist mechanistic studies of naturally occurring nucleases.     

Origin and evolution of the RIG-I like RNA helicase gene family

Background  

The DExD/H domain containing RNA helicases such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) of invading viruses. The RIG-I and MDA5 proteins differentially recognise conserved PAMPs in double stranded or single stranded viral RNA molecules, leading to activation of the interferon system in vertebrates. They share three core protein domains including a RNA helicase domain near the C terminus (HELICc), one or more caspase activation and recruitment domains (CARDs) and an ATP dependent DExD/H domain. The RIG-I/MDA5 directed interferon response is negatively regulated by laboratory of genetics and physiology 2 (LGP2) and is believed to be controlled by the mitochondria antiviral signalling protein (MAVS), a CARD containing protein associated with mitochondria.