盐胁迫下AM真菌对红花耐盐性的影响

在盐胁迫下,采用盆栽方法研究AM真菌对红花植株耐盐生理指标的影响,以不接种为对照。结果表明,在0、0．1％和0．2％浓度NaCl胁迫下,AM真菌促进红花幼苗的生长,接种真菌的红花叶片SOD和CAT活性、脯氨酸和可溶性蛋白的含量都高于不接种处理的,叶片细胞质膜透性和MDA含量则低于不接种处理的,结果证明AM真菌可以提高植物的耐盐性。     

Interactome-wide Analysis Identifies End-binding Protein 1 as a Crucial Component for the Speck-like Particle Formation of Activated Absence in Melanoma 2 (AIM2) Inflammasomes

Inflammasomes are cytoplasmic receptors that can recognize intracellular pathogens or danger signals and are critical for interleukin 1β production. Although several key components of inflammasome activation have been identified, there has not been a systematic analysis of the protein components found in the stimulated complex. In this study, we used the isobaric tags for relative and absolute quantification approach to systemically analyze the interactomes of the NLRP3, AIM2, and RIG-I inflammasomes in nasopharyngeal carcinoma cells treated with specific stimuli of these interactomes (H₂O₂, poly (dA:dT), and EBV noncoding RNA, respectively). We identified a number of proteins that appeared to be involved in the interactomes and also could be precipitated with anti-apoptosis-associated speck-like protein containing caspase activation and recruitment domain antibodies after stimulation. Among them, end binding protein 1 was an interacting component in all three interactomes. Silencing of end binding protein 1 expression by small interfering RNA inhibited the activation of the three inflammasomes, as indicated by reduced levels of interleukin 1β secretion. We confirmed that end binding protein 1 directly interacted with AIM2 and ASC in vitro and in vivo. Most importantly, fluorescence confocal microscopy showed that end binding protein 1 was required for formation of the speck-like particles that represent activation of the AIM2 inflammasome. In nasopharyngeal carcinoma tissues, immunohistochemical staining showed that end binding protein 1 expression was elevated and significantly correlated with AIM2 and ASC expression in nasopharyngeal carcinoma tumor cells. In sum, we profiled the interactome components of three inflammasomes and show for the first time that end binding protein 1 is crucial for the speck-like particle formation that represents activated inflammasomes.Nasopharyngeal carcinoma (NPC)¹ is a malignancy of the head and the neck that is highly prevalent in Southern China and Southeast Asia (1). Both environmental and genetic risk factors are considered to be important for the development of NPC (2, 3); among them, Epstein-Barr virus (EBV) infection of the epithelium is the most important known factor (1). In addition to the EBV-encoded oncoprotein-mediated blockade of intracellular mechanisms in EBV-associated tumors (1), chronic inflammation is considered to be an important oncogenic factor in NPC (4). Interleukin 1 beta (IL-1β), which is an inflammatory cytokine that has oncogenic effects in many tumors (5), can be detected in NPC tumor tissues (6, 7). IL-1β secretion is mediated by cytosolic protein complexes called inflammasomes, which induce IL-1β secretion by activating catalytic caspase 1 (8). However, no previous study has examined inflammasome components in NPC tumor cells or the mechanisms of inflammasome regulation in NPC.Inflammasomes are cytoplasmic receptors that act in innate immunity to recognize intracellular pathogen-associated molecular pattern (PAMP) or danger signal-associated molecular pattern (DAMP). A number of inflammasomes have been identified in recent years (9), and they can be classified into different subgroups according to their recognizing PAMP. These groups include the NOD-like receptors (NLR), which sense intracellular pathogens (e.g. bacteria, fungi, and parasites) and activate pro-caspase 1 with or without an adaptive protein called apoptosis-associated speck-like protein containing caspase activation and recruitment domain (ASC) (10). Activated caspase 1 then induces IL-1β secretion through direct cleavage of pro-IL-1β (8). Among the NLR family members, the NLRP3 inflammasome recognizes both pathogens and danger signals such as ATP or reactive oxygen species (ROS) generation (11, 12). Members of the two other subgroups, absence in melanoma 2 (AIM2) and retinoic acid-inducible gene I (RIG-I), sense cytoplasmic double-strand DNA and 5′-triphoshphate RNA, respectively, and then recruit ASC to activate pro-caspase 1 (13, 14). Although inflammasomes are important for pathogen defense in immune cells, recent studies have shown that inflammasomes also participate in tumorigenesis in colon cancer and melanoma (15–17). A previous report showed that EBV noncoding RNAs (EBERs) are recognized by RIG-I and activate signaling to induce type I IFN in EBV-infected B lymphocytes (18). This report is consistent with our recent unpublished observation that RIG-I is activated by EBERs in NPC cells. We additionally show that NLRP3 is triggered by tumor microenvironmental factors, such as ATP and ROS, and the clinical drug cisplatin; AIM2 recognizes EBV genomic DNA and is activated by irradiation in NPC cells. Although these inflammasomes play important role in NPC, the regulation and the interactome of these inflammasome complexes are not fully understood.On activation by PAMP or DAMP, the activated inflammasomes tend to aggregate in the cytosol as speck-like particles (13). Biochemical and cell biological data have indicated that the core components of the inflammasome comprise the receptor, ASC, and pro-caspase 1, but an increasing number of proteins have been identified as interacting with these complexes. For example, heat-shock protein 90 (HSP90) is essential for the function of the NLRP3 and RIG-I inflammasomes (19, 20). NLRC5, another member of the NLR family, is involved in the NLRP3 inflammasome and is required for its activity (21). Rac1, a small Rho GTPase family member, is reportedly required for NLRP3 inflammasome activation during C. pneumoniae infection (22). The S. Typhimurium effector, SopE, activates caspase 1 through Rac1 activity (23), whereas Yersinia bacteria prevent caspase 1 activation by inhibiting Rac1 activity via the effector protein, YopE (24). Notably, Rac1 regulates cytoskeletal rearrangement (25), suggesting that cytoskeletal components may participate in inflammasome activation.End-binding protein 1 (EB1), an adenomatous polyposis coli (APC)-binding protein, regulates microtubule polymerization by recruiting the plus-end tracking protein (+TIP) complex to the plus end of microtubules (26). The interaction of EB1 and the +TIP complex depends on the C-terminal (CT) domain of EB1, whereas the calponin homology (CH) domain of EB1 binds to the microtubule (26). Many studies have shown that EB1 participates in different biological processes, including mitosis, migration and signal transduction (27–29), and also that it plays an oncogenic role in cancer by affecting cell growth or migration (30, 31). However, although EB1 is known to be a cytoskeletal component that is regulated by the small GTPase, RhoA (28), its role in inflammasome activation has not yet been explored.Here, we used the isobaric tags for relative and absolute quantification (iTRAQ) approach to systemically analyze the interactomes of the NLRP3, AIM2, and RIG-I inflammasomes in NPC cell lines treated with their specific stimuli, H₂O₂, poly (dA:dT), and EBER, respectively. We characterized the interactomes of the NLRP3, AIM2, and RIG-I inflammasomes in NPC cells by proteomic analysis, and report for the first time that EB1 can directly bind to the AIM2 inflammasome and is essential for speck-like particle formation in NPC cells. Finally, we suggest some possible mechanisms for EB1-associated AIM2 inflammasome activation via microtubule polymerization and RhoA activity.     

Decreased expression of Beclin-1 and LC3 in human lung cancer

Autophagy, a conversed response to stress, has recently been studied in human cancers. Two important autophagic genes—Beclin-1 and LC3 are reported in several human cancers. However, the expressions of Beclin-1 and LC3 in lung cancer have not yet been investigated. In the present study, we investigated the expression of Beclin-1 and LC3, and the relationship between the expression profile and the clinical or pathological changes in human lung cancer. 40 primary lung cancer patients are involved in present study. mRNA expressions of Beclin-1 and LC3-II were detected by Real Time PCR and the protein levels were assessed by immunohistochemistry and western blot. Relative lower expressions of Beclin-1 and LC3-II mRNA were found in the lung cancer tissues compared to counterpart normal tissues. Consistently, the lower amount of Beclin-1 and LC3-II protein was found in lung cancer tissues. However, the expressions of Beclin-1 and LC3-II in lung cancer tissues were not affected by patients’ age, gender, smoking, histological type, lymph node metastasis and tumor-node-metastasis (TNM) stage. Both mRNA and protein levels of Beclin-1 and LC3-II were significantly decreased in lung cancer tissues which suggested that autophagy may be involved in the pathogenesis of lung cancer.     

Aberrant alterations of the expressions and S-nitrosylation of calmodulin and the downstream factors in the brains of the rodents during scrapie infection

The aberrant alterations of calmodulin (CaM) and its downstream substrates have been reported in some neurodegenerative diseases, but rarely described in prion disease. In this study, the potential changes of Ca²⁺/CaM and its associated agents in the brains of scrapie agent 263K-infected hamsters and the prion infected cell line SMB-S15 were evaluated by various methodologies. We found that the level of CaM in the brains of 263K-infected hamsters started to increase at early stage and maintained at high level till terminal stage. The increased CaM mainly accumulated in the regions of cortex, thalamus and cerebellum of 263K-infected hamsters and well localization of CaM with NeuN positive cells. However, the related kinases such as total and phosphorylated forms of CaMKII and CaMKIV, as well as the downstream proteins such as CREB and BDNF in the brain of 263K-infected hamsters were decreased. Further analysis showed a remarkable increase of S-nitrosylated (SNO) form of CaM in the brains of 263K-infected hamsters. Dynamic analysis of S-nitrosylated CaM showed the SNO form of CaM abnormally increases in a time-dependent manner during prion infection. Compared with that of the normal partner cell line SMB-PS, the CaM level in SMB-S15 cells was increased, meanwhile, the downstream proteins, such as CaMKII, p-CaMKII, CREB, as well as BDNF, were also increased, especially in the nucleic fraction. No SNO-CaM was detected in the cell lines SMB-S15 and SMB-PS. Our data indicate an aberrant increase of CaM during prion infection in vivo and in vitro.     

Lrp6 Genotype affects Individual Susceptibility to Nonalcoholic Fatty Liver Disease and Silibinin Therapeutic Response via Wnt/β-catenin-Cyp2e1 Signaling

Background: Nonalcoholic fatty liver disease (NAFLD) is a serious threat to human health worldwide, with a high genetic susceptibility. Rs2302685, a functional germline variant of LRP6, has been recently found to associate with NAFLD risk. This study was aimed to clarify the underlying mechanism associated with rs2302685 risk and its impact on pharmacotherapy in treatment of NAFLD.Methods: Venous blood samples were collected from NAFLD and non-NAFLD patients for SNP genotyping by using mass spectrometry. The Lrp6-floxdel mouse (Lrp6^(+/-)) was generated to model the partial function associated with human rs2302685. The liver injury and therapeutic effects of silibinin were compared between Lrp6^(+/-) and Lrp6^(+/+) mice received a methionine-choline deficient (MCD) diet or normal diet. The effect of Lrp6 functional alteration on Wnt/β-catenin-Cyp2e1 signaling activities was evaluated by a series of cellular and molecular assays.Results: The T allele of LRP6 rs2302685 was confirmed to associate with a higher risk of NAFLD in human subjects. The carriers of rs2302685 had reduced level of AST and ALT as compared with the noncarriers. The Lrp6^(+/-) mice exhibited a less severe liver injury induced by MCD but a reduced response to the treatment of silibinin in comparison to the Lrp6^(+/+) mice, suggesting Lrp6 as a target of silibinin. Wnt/β-catenin-Cyp2e1 signaling together with ROS generation could be exacerbated by the overexpression of Lrp6, while decreased in response to Lrp6 siRNA or silibinin treatment under NAFLD modeling.Conclusions: The Lrp6 function affects individual susceptibility to NAFLD and the therapeutic effect of silibinin through the Wnt/β-catenin-Cyp2e1 signaling pathway. The present work has provided an underlying mechanism for human individual susceptibility to NAFLD associated with Lrp6 polymorphisms as well as a rationale for the effective use of silibinin in NAFLD patients.     

芸苔属植物自交不亲和性的分子机制

芸薹属植物自交不亲和性受单一位点的复等位基因控制,此位点命名为S位点。它决定柱头表面花粉识别的专一性。S位点糖蛋白基因（SLG）和S受体激酶基因（SRK）是控制芸薹属植物花柱自交不亲和性的两个关键因子。本文介绍了编码自交不亲和性的S位点的SLG、SRK和花粉S基因的鉴定、结构及功能,并对其信号传导途径的可能机制做了简要概述。     

猪细小病毒VP2与大肠杆菌不耐热肠毒素B亚单位在干酪乳杆菌表面共表达

将分别编码猪细小病毒(PPV)主要免疫保护性抗原VP2蛋白与大肠杆菌不耐热肠毒素B亚单位(LTB)基因插入乳酸杆菌细胞表面表达载体pPG中, 成功构建了重组表达载体pPG-VP2-LTB, 将其电转化干酪乳杆菌Lactobacillus casei 393, 获得了表达猪细小病毒VP2-LTB融合蛋白的重组乳酸菌表达系统, 经2%乳糖诱导, SDS-PAGE和Western-blot检测表明, 有大小约78 kD的蛋白得到了表达, 具有与天然病毒蛋白一样的抗原特异性, 全细胞ELISA结果表明, LTB同