不同氮素供应下水稻酚类物质代谢关键酶基因差异表达

运用实时荧光定量PCR技术探讨了不同供氮条件下强化感与弱化感水稻苯丙烷代谢途径中9个关键酶基因的表达差异。结果表明,与正常氮素供应相比,低氮胁迫引起强化感水稻‘P1312777’中与酚类代谢途径相关的9个关键酶基因表达量均上调,表达量增幅在1.9～5.4倍之间,且以PAL基因上调倍数最大。而弱化感水稻‘Lemont’则相反,只有2个基因(苯丙氨酸裂解酶基因和肉桂酰CoA基因)表达上调,但上调倍数分别是强化感水稻对应的基因的22%和74%,其余的7个基因表达均下调,降幅在29%～72%之间,表明低氮胁迫诱发的水稻化感抑草能力增强与其体内酚类物质合成代谢增强有关。     

三个玉米合成群体选系的配合力及杂种优势分析

采用NCⅡ遗传交配设计,通过在黑龙江省哈尔滨和泰来的两点试验,以玉米自交系Mo17、B73、444和丹340为测验种,对从群体品综1号、中综3号和陕综5号选育的18份自交系进行配合力及杂种优势分析,以探讨群体选系在我国东北早熟玉米区的利用途径。结果表明,供试自交系间一般配合力存在较大差异;陕综5号群体选系HR14、HR17和HR15、中综3号选系HR9和HR8、品综1号选系HR4的一般配合力较高。在供试的72个组合中HR15×丹340、HR17×丹340、HR9×Mo17、HR14×丹340、HR7×B73、HR8×B73、HR6×444、HR5×丹340产量的特殊配合力及对照优势较高,表现出较高的利用潜力。依据特殊配合力及对照优势分析,中综3号选系与旅大红骨群、陕综5号选系与兰卡斯特群、品综1号选系与瑞德群遗传关系较近。结合育种实践,在我国北方早熟春玉米区陕综5号×旅大红骨、中综3号×瑞德或兰卡斯特、品综1号×旅大红骨或唐四平头可能组成较大利用潜力的杂种优势模式。     

Tissue expression and cellular localization of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA in male mice

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an ubiquitous antioxidant enzyme, but the exact expression pattern in mammalian tissues is still unknown. The expression and cellular localization of PHGPx mRNA were examined in male mice using real time-polymerase chain reaction and in situ hybridization techniques. The rank order of PHGPx mRNA expression across tissues exhibiting substantial levels of expression was:testes ≫ heart > cerebrum ≥ ileum > stomach = liver = jejunum ≥ epididymis. In testes, PHGPx mRNA was highly expressed in spermiogenic cells and Leydig cells. The signal was also expressed in the molecular layer, Purkinje cell layer, and white matter of cerebellum, the pituicytes of neurohypophysis, the parafollicular cells and follicular basement membrane of thyroid, the exocrine portion of pancreas, the tubular epithelium of kidney, the smooth muscle cells of arteries, and the red pulp of spleen. In the gastrointestinal tract, PHGPx mRNA expression was mainly observed in the keratinized surface epithelium of forestomach, the submucosal glands and serosa layers, and further the Paneth cells of intestines. PHGPx mRNA appeared to be ubiquitously expressed in the parenchyma of heart, liver, and lung. These results indicate that PHGPx exhibits a cell- and tissue-specific expression pattern in mice.     

In-depth analysis of the adipocyte proteome by mass spectrometry and bioinformatics

Adipocytes are central players in energy metabolism and the obesity epidemic, yet their protein composition remains largely unexplored. We investigated the adipocyte proteome by combining high accuracy, high sensitivity protein identification technology with subcellular fractionation of nuclei, mitochondria, membrane, and cytosol of 3T3-L1 adipocytes. We identified 3,287 proteins while essentially eliminating false positives, making this one of the largest high confidence proteomes reported to date. Comprehensive bioinformatics analysis revealed that the adipocyte proteome, despite its specialized role, is very complex. Comparison with microarray data showed that the mRNA abundance of detected versus non-detected proteins differed by less than 2-fold and that proteomics covered as large a proportion of the insulin signaling pathway. We used the Endeavour gene prioritization algorithm to associate a number of factors with vesicle transport in response to insulin stimulation, a key function of adipocytes. Our data and analysis can serve as a model for cellular proteomics. The adipocyte proteome is available as supplemental material and from the Max-Planck Unified Proteome database.     

In situ proteolysis for protein crystallization and structure determination

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.     

Selective extraction of free astaxanthin from Haematococcus culture using a tandem organic solvent system

A novel tandem solvent process of dodecane and methanol was developed for the selective extraction of free astaxanthin from red encysted Haematococcus culture. The process consists of dodecane extraction for astaxanthin mixture from the culture (stage 1) and methanol extraction for free astaxanthin from the dodecane extract (stage 2). In the first stage, astaxanthin mixture was directly extracted to dodecane from the culture broth without cell harvest process, followed by a rapid separation of the dodecane extract and the culture medium containing cell debris by simple settling. In the second stage, free astaxanthin was selectively collected to methanol from the dodecane extract, accompanied with saponification of astaxanthin-esters by the addition of NaOH to methanol. During saponification, use of the optimum NaOH concentration (0.02 M) and low temperature (4 degrees C) reaction minimized the degradation of free astaxanthin, resulting in a total recovery yield of free astaxanthin of over 85%. The free-astaxanthin-containing methanol extract was also simply separated from dodecane by gravity settling, after which the astaxanthin-free dodecane was effectively recycled to the first stage, yielding a stable extractability of astaxanthin mixture during repeated extraction. Our results indicate the potential of the proposed tandem solvent process as an alternative extraction technology for the high-value antioxidant Haematococcus astaxanthin.     

Oxidation of organoarsenicals and antimonite by a novel flavin monooxygenase widely present in soil bacteria

Arsenic can be biomethylated to form a variety of organic arsenicals differing in toxicity and environmental mobility. Trivalent methylarsenite (MAs(III)) produced in the methylation process is more toxic than inorganic arsenite (As(III)). MAs(III) also serves as a primitive antibiotic and, consequently, some environmental microorganisms have evolved mechanisms to detoxify MAs(III). However, the mechanisms of MAs(III) detoxification are not well understood. In this study, we identified an arsenic resistance (ars) operon consisting of three genes, arsRVK, that contribute to MAs(III) resistance in Ensifer adhaerens ST2. ArsV is annotated as an NADPH-dependent flavin monooxygenase with unknown function. Expression of arsV in the arsenic hypersensitive Escherichia coli strain AW3110Δars conferred resistance to MAs(III) and the ability to oxidize MAs(III) to MAs(V). In the presence of NADPH and either FAD or FMN, purified ArsV protein was able to oxidize both MAs(III) to MAs(V) and Sb(III) to Sb(V). Genes with arsV-like sequences are widely present in soils and environmental bacteria. Metagenomic analysis of five paddy soils showed the abundance of arsV-like sequences of 0.12–0.25 ppm. These results demonstrate that ArsV is a novel enzyme for the detoxification of MAs(III) and Sb(III) and the genes encoding ArsV are widely present in soil bacteria.     

脑血管内皮细胞敲除Prmt5导致脑血管损伤及星形胶质细胞活化*

目的: 研究蛋白质精氨酸甲基转移酶5(protein arginine methyltransferase 5,Prmt5)在小鼠脑血管发育、稳态维持中的功能,并考察脑血管内皮细胞特异性敲除Prmt5后对中枢神经系统的影响。方法: 利用脑血管内皮细胞特异性表达SP-A-Cre转基因小鼠和Prmt5条件基因打靶小鼠交配,构建脑血管内皮细胞特异性Prmt5敲除小鼠。利用H-E染色、免疫荧光染色、激光散斑成像、Sulfo-NHS-Biotin染料灌注等方法评价脑血管内皮细胞特异性Prmt5敲除小鼠脑血管结构、脑血流量、血脑屏障渗透性等;利用实时定量PCR进一步检测补体C1q(complement C1q,C1q)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素-1β(interleukin 1β,IL-1β)等细胞因子的表达水平。通过免疫荧光、Western blot等检测胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、S100钙结合蛋白β(S100 calcium-binding protein β protein,S100β)和补体C3(complement C3,C3)的表达,检测小鼠皮层、丘脑和小脑中星形胶质细胞活化水平。结果: 脑血管内皮细胞特异性敲除Prmt5导致血管损伤, C1q、TNF-α和IL-1β等炎症因子表达水平上调,活化星形胶质细胞比例明显增加。结论: 脑血管内皮细胞中Prmt5在小鼠脑血管稳态维持中发挥了重要功能。