营养元素对小球藻生长及胞内蛋白质和多糖含量的影响

在24～26℃、自然光照的条件下,首先考察小球藻生长与胞内蛋白质和多糖积累的关系,其次利用正交设计实验考察不同浓度的营养元素对小球藻生长及胞内蛋白质和多糖含量的影响。结果表明,小球藻中蛋白质和多糖的合成与积累主要发生在生长过程的平衡期。实验的目标不同,所需要的营养元素浓度及其优化组合不同。利于小球藻生长、胞内蛋白质和多糖积累的营养元素的优化组合分别为:C6H12O6:2g/L;KNO3:0.8g/L;NaH2PO4:0.2 g/L;C6H12O6:10 g/L;KNO3:1.6 g/L;NaH2PO4:0.2 g/L;C6H12O6:6 g/L;KNO3:1.6g/L;NaH2PO4:0.1g/L。与之对应的细胞密度、蛋白质和总糖含量分别为:6.46×107个细胞/L,655.4 mg/g,168.1mg/g。

不同木质底物诱导下白囊耙齿菌胞外木质纤维素酶活性和胞内蛋白质组的差异

【目的】研究白囊耙齿菌5种胞外分泌木质纤维素酶活性变化以及胞内蛋白质组的表达差异,探讨该菌对不同基质的利用机制,为筛选用于生物制浆、林木病腐防治和工业污染物降解的高效菌株提供科学依据。【方法】选用白桦、红皮云杉木屑作为诱导物,不加木屑为对照,研究白囊靶齿菌5种主要胞外分泌木质纤维素酶的活性以及胞内蛋白质组的表达差异。【结果】木屑诱导下的木质纤维素相关胞外酶活性大都高于对照样品,且锰过氧化物酶和漆酶活性在处理组与对照组间的差异达显著和极显著水平;木质素降解酶系中漆酶活性最强,木质素过氧化物酶次之,锰过氧化物酶最弱;纤维素降解酶系中葡聚糖外切酶活性远大于葡聚糖内切酶活性,2种酶活性总体上趋于稳定,表达量较高,受培养时间的影响变化不明显;白桦和云杉木屑处理组中检测到32个胞内明显差异表达蛋白点,2个处理组各有3个特异的差异表达蛋白点。白桦木屑诱导下的特异表达蛋白与膜蛋白、膜运输和能量提供有关,而云杉诱导组特异表达蛋白均与蛋白合成有关。【结论】不同树种木屑底物诱导下白囊耙齿菌胞外木质纤维素酶活性存在显著差异。同时胞内蛋白质的表达也存在明显差异,在分解白桦木屑时分泌蛋白的胞内外运输可能是决定木材降解能力的关键,而在分解云杉木屑时其相关分泌蛋白的大量合成将起决定作用。

化学-渗透压法温和破碎处理下大肠杆菌细胞胞内蛋白质的释放率

恰当的细胞破碎工艺是获取胞内蛋白质的必需环节,然而现有细胞破碎技术仍有较多不足。为了研究合适的细胞破碎方法,拟通过化学法与渗透压法的联用,建立化学-渗透压法温和高效破碎大肠杆菌细胞的新工艺。结果表明:(1)单用化学法处理大肠杆菌细胞,最高仅能释放37.3%的胞内蛋白质,而化学-渗透压法最高可以释放84.2%的胞内蛋白质;对渗透压处理环节进行优化后,胞内蛋白质释放率提高至93.1%及以上;(2)以优化的化学-渗透压法释放重组原动蛋白2β(PK2β)工程菌细胞的胞内蛋白质,其中70.5%~80.2%重组PK2β呈可溶性,而超声法处理的样品中不含有可溶性重组PK2β,表明化学-渗透压法不仅简便高效、成本低廉,而且有利于保留可溶性重组蛋白质,而超声破碎法会导致可溶性蛋白发生变性沉淀而增加蛋白质分离的难度。综合分析可知,化学-渗透压法在基因工程及生物工程中具有良好的应用前景。

木质纤维素降解白腐菌的蛋白质组学研究进展

木质纤维素是生物能源领域颇具潜力的原料,其复杂的结构组成是制约高效降解利用这一资源发展生物炼制的瓶颈。白腐菌作为自然界最主要的木质纤维素降解菌之一,揭示其降解的分子机制是阐明自然界木质纤维素降解过程和该生物质资源得以高效利用的基础。近年来,蛋白质组学技术在木质纤维素生物降解菌方面的研究取得许多成果,为深入研究木质纤维素的生物降解机理提供了重要信息。从木质纤维素降解白腐菌的胞内蛋白质组学、亚蛋白质组学和分泌蛋白质组学,以及木质纤维素降解过程降解酶之间的相互作用等方面综述了近年来的研究进展,以期从蛋白质组学角度阐明木质纤维素生物降解的分子机制。

真菌的蛋白质组学研究——方法与进展

蛋白质组学分析作为对生物体代谢调控分析非常有效的技术手段,在近年来被广泛应用在微生物代谢调控研究中。真菌是与人们生产、生活密切相关的一类非常重要的微生物,弄清其代谢调控机制,可为日后的生产应用奠定理论基础。本文就近年来应用在真菌蛋白质组学上的研究方法以及研究进展作一综述。

The BLOC Interactomes Form a Network in Endosomal Transport

With the identification of more than a dozen novel Hermansky-Pudlak Syndrome （HPS） proteins in vesicle trafficking in higher eukaryotes, a new class of trafficking pathways has been described. It mainly consists of three newly-defined protein com- plexes, BLOC-l, -2, and -3. Compelling evidence indicates that these complexes together with two other well-known complexes, AP3 and HOPS, play important roles in endosomal transport. The interactions between these complexes form a network in protein trafficking via endosomes and cytoskeleton. Each node of this network has intra-complex and extra-complex interactions. These complexes are connected by direct interactions between the subunits from different complexes or by indirect interactions through coupling nodes that interact with two or more subunits from different complexes. The dissection of this network facilitates the understanding of a dynamic but elaborate transport machinery in protein/membrane trafficking. The disruption of this network may lead to abnormal trafficking or defective organellar development as described in patients with Hermansky-Pudlak syndrome.

Endocytosis of FcαR is clathrin and dynamin dependent, but its cytoplasmic domain is not required

FcαR, the Fc receptor for IgA, is essential for IgA-mediated immune responses. Previous studies have shown that IgA and IgA immune complexes can be rapidly endocytosed by FcαR. However, the underlying mechanism remains unclear. Here, we investigated the endocytic pathway of FcαR in monocytic cell line, U937, that naturally express FcuR and in transfected Chinese hamster ovary （CHO）, COS-7 and Hela cells. By using selective chemical inhibitors of different endocytic pathways, overexpression of dominant-negative mutants of Eps15 and knockdown of clathrin heavy chain （CHC） via RNA interference, we demonstrated that endocytosis of FcaR was through a clathrin-mediated pathway. The endocytosed FcαR went into Rab5- and Rabll-positive endosomes. However, endocytosis of FcaR could not be blocked by a dominant-negative mutant of Rab5. We also demonstrated that endocytosis of FcαR was dynamin-dependent by overexpressing a dominant-negative mutant of dynamin. The potential endocytic motif for FcαR was also examined. Unexpectedly, we found that the entire cytoplasmic domain of FcaR was not required for the endocytic process of FcαR. We conclude that endocytosis of FcaR is clathrin- and dynamin-dependent, but is not regulated by RabS, and the endocytic motif is not located in the cytoplasmic domain of FcαR.

Endostatin promotes the anabolic program of rabbit chondrocyte

Endostatin is a natural occurred angiogenesis inhibitor derived from collagenXVIII. So far its function during the angiogenesis process of bone formation and arthropathy has not been well studied yet. The present study addresses the function of endostatin in rabbit articular chondrocytes (RAC). We found that endostatin can promote RAC adhesion and spreading as well as its proliferation. In monolayer cultured RAC, CollagenII, TIMP1 and collagenXVIII transcription were up regulated by endostatin while collagenI and MMP9 were down regulated. Moreover collagenXVIII and endostatin antigens are present at synovial fluid. These findings indicate new function of endostatin as a homeostatic factor in cartilage metabolism.

Study progress of cell endocytosis

Endocytosis is a process through which extracellular materials are transported into cell through membrane deformation. This process is not a simple step-by-step process in which a series of proteins function according to the chronological order, but rather a complex process comprising many members which are regulated precisely. The role of endocytosis is broadly divided into two categories, phagocytosis and pinocytosis, the latter is divided into four species in accordance with the size of endocytosis substances： clathrin dependent endocytosis, the diameter of clathrin-coated vesicle is 100-150 nm; caveolin dependent endocytosis, the diameter of caveolin protein-coated vesicle is 50-100 nm; macropinocytosis, the diam- eter of macropinocytosis is generally 0.5-2 μm, sometimes up to 5 μm; clathrin and caveolin independent endocytosis. Many proteins including endophilin A1, A2, A3, and endocytotic proteins B, B1a, and Blb as well as dynamin, actin and Rab protein families are involved in endocytosis and play an important role in different stages. The abnormal endocytosis may be involved in the development of certain diseases.

The non-canonical NF-κB pathway promotes NPC2 expression and regulates intracellular cholesterol trafficking

Niemann-Pick type C2(NPC2) is a lysosome luminal protein that functions in concert with NPC1 to mediate egress of lowdensity lipoprotein-derived cholesterol from lysosome. The nuclear factor kappa B subunit 2(NF-κB2) protein is a component of NF-κB transcription factor complex critically implicated in immune and inflammatory responses. Here, we report that NF-κB2 regulates intracellular cholesterol transport by controlling NPC2 expression. RNAi-mediated disruption of NF-κB2, as well as other signaling members of the non-canonical NF-κB pathway, caused intracellular cholesterol accumulation. Blockage of the non-canonical NF-κB pathway suppressed NPC2 expression, whereas Lymphotoxin β receptor(LTβR) activation or Baff receptor(BaffR) stimulation up-regulated the mRNA abundance and protein level of NPC2. Further, NF-κB2 activated NPC2 transcription through direct binding to its promoter region. We also observed cholesterol accumulation in NF-κB2-deficient zebrafish embryo and NF-κB2 mutant mice. Collectively, these data identify a regulatory role for the non-canonical NF-κB pathway in intracellular cholesterol trafficking and suggest a link between cholesterol transport and immune system.

Hsa-miR-1246, hsa-miR-320a and hsa-miR-196b-5p inhibitors can reduce the cytotoxicity of Ebola virus glycoprotein in vitro

Ebola virus （EBOV） causes a highly lethal hemorrhagic fever syndrome in humans and has been associated with mortality rates of up to 91% in Zaire, the most lethal strain. Though the viral envelope glycoprotein （GP） mediates widespread inflammation and cellular damage, these changes have mainly focused on alterations at the protein level, the role of microRNAs （miRNAs） in the molecular pathogenesis underlying this lethal disease is not fully understood. Here, we report that the miRNAs hsa-miR-1246, hsa-miR-320a and hsa-miR-196b-5p were induced in human umbilical vein endothelial cells （HUVECs） following expression of EBOV GP. Among the proteins encoded by predicted targets of these miRNAs, the adhesion-related molecules tissue factor pathway inhibitor （TFPI）, dystroglycan! （DAG1） and the caspase 8 and FADD-like apoptosis regulator （CFLAR） were significantly downregulated in EBOV GP-expressing HUVECs. Moreover, inhibition of hsa-miR-1246, hsa-miR-320a and hsa-miR-196b-5p, or overexpression of TFPI, DAG1 and CFLAR rescued the cell viability that was induced by EBOV GP. Our results provide a novel molecular basis for EBOV pathogenesis and may contribute to the development of strategies to protect against future EBOV pandemics.

Optical applications of quantum dots in biological system

This review presents a simple introduction on the unique properties and general synthesis of quantum dots (QDs) in which we lay emphasis on the optical applications in the biological system. The detection of biological molecules such as DNA, protein and enzyme, the cell-based analysis and in vivo animal imaging are mainly discussed.

Proteomic analysis of human umbilical vein endothelial cells exposed to PM_(2.5)

Exposure to fine ambient particulate matter(PM_(2.5)) is known to be associated with cardiovascular disease. To uncover the molecular mechanisms involved in cardiovascular toxicity of PM_(2.5), we investigated alterations in the protein profile of human umbilical vein endothelial cells(HUVECs) treated with PM_(2.5) using two-dimensional electrophoresis in conjunction with mass spectrometry(MS). A total of 31 protein spots were selected as differentially expressed proteins and identified by matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) MS. The results demonstrated that DNA damage and cell apoptosis are important factors contributing to PM_(2.5)-mediated toxicity in HUVECs. It is further proposed that PM_(2.5) can inhibit superoxide dismutase(SOD) activity and increase reactive oxygen species(ROS) and malonaldehyde(MDA) production in a concentration-dependent manner. Induction of apoptosis and DNA damage through oxidative stress pathways may be one of the key toxicological events occurring in HUVECs under PM_(2.5) stress. These results indicated that the toxic mechanisms of PM_(2.5) on cardiovascular disease are related to endothelial dysfunction.

Altered expression of micro RNAs in the response to ER stress

MicroRNAs, a class of small noncoding RNAs, play key roles in diverse biological and pathological processes. ER stress, resulting from the accumulation of unfolded or misfolded proteins in the ER lumen, is triggered by various physiological events and pathological insults. Here, using RNA deep sequencing analysis, we found that the expression of some microRNAs was altered in HeLa and HEK293 cells under ER stress. Protein and RNA levels of DGCR8, Drosha, Exportin-5, Dicer, and Ago2 showed no significant alteration in ER-stressed cells, which suggested that the change in microRNA expression might not be caused by the microRNA biogenesis pathway but by other, unknown factors. Real-time PCR assays confirmed that hsa-miR-423-5p was up-regulated, whereas hsa-miR-221-3p and hsa-miR-452-5p were down-regulated, in both HeLa and HEK293 cells under ER stress. Luciferase activity and Western blot assays verified that CDKN1A was a direct target of hsa-miR-423-5p and that CDKN1B was a direct target of hsa-miR-221-3p and hsamiR-452-5p. We speculated that by regulating their targets, microRNAs might function cooperatively as regulators in the adaptive response to ER stress.