乙型脑炎病毒DNA初免-蛋白加强策略在小鼠模型上的免疫效应评价

为了评价基因Ⅰ型乙型脑炎病毒prM-E DNA疫苗与prM和EⅢ融合抗原亚单位疫苗采用DNA初免-蛋白加强免疫策略对小鼠的免疫效果,本研究将prM-E融合基因插入到pVAX1真核表达载体中,构建重组表达载体prM-E-pVAX1作为DNA疫苗进行初免,利用原核表达系统获得的prM和EⅢ融合抗原作为亚单位疫苗进行加强免疫。将32只4−6周龄雌性BALB/c小鼠随机分成4组,设置prM-E-pVAX1 DNA疫苗组、DNA初免-蛋白加强免疫组、prM和EⅢ融合抗原亚单位疫苗组及pVAX1载体对照组,通过ELISA检测血清中特异性抗体水平;通过噬斑减少中和试验滴定中和抗体滴度;通过细胞因子表达丰度和淋巴细胞增殖试验分析不同疫苗免疫组诱导产生的细胞免疫反应。结果表明,用DNA初免-蛋白加强策略免疫的小鼠诱导产生的中和抗体滴度略高于prM和EⅢ融合抗原亚单位疫苗免疫组,显著高于prM-E-pVAX1 DNA疫苗免疫组。DNA初免-蛋白加强策略在小鼠模型中诱导产生了有效的Th1/Th2型免疫反应,特别是显著诱导了Th1型细胞免疫反应。本研究为预防流行性乙型脑炎提供了新的免疫策略和理论参考依据。     

In-depth Profiling and Quantification of the Lysine Acetylome in Hepatocellular Carcinoma with a Trapped Ion Mobility Mass Spectrometer

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide with limited therapeutic options. Comprehensive investigation of protein posttranslational modifications in HCC is still limited. Lysine acetylation is one of the most common types of posttranslational modification involved in many cellular processes and plays crucial roles in the regulation of cancer. In this study, we analyzed the proteome and K-acetylome in eight pairs of HCC tumors and normal adjacent tissues using a timsTOF Pro instrument. As a result, we identified 9219 K-acetylation sites in 2625 proteins, of which 1003 sites exhibited differential acetylation levels between tumors and normal adjacent tissues. Interestingly, many novel tumor-specific K-acetylation sites were characterized, for example, filamin A (K865), filamin B (K697), and cofilin (K19), suggesting altered activities of these cytoskeleton-modulating molecules, which may contribute to tumor metastasis. In addition, we observed an overall suppression of protein K-acetylation in HCC tumors, especially for enzymes from various metabolic pathways, for example, glycolysis, tricarboxylic acid cycle, and fatty acid metabolism. Moreover, the expression of deacetylase sirtuin 2 (SIRT2) was upregulated in HCC tumors, and its role of deacetylation in HCC cells was further explored by examining the impact of SIRT2 overexpression on the proteome and K-acetylome in Huh7 HCC cells. SIRT2 overexpression reduced K-acetylation of proteins involved in a wide range of cellular processes, including energy metabolism. Furthermore, cellular assays showed that overexpression of SIRT2 in HCC cells inhibited both glycolysis and oxidative phosphorylation. Taken together, our findings provide valuable information to better understand the roles of K-acetylation in HCC and to treat this disease by correcting the aberrant acetylation patterns.     

Screening of rice cultivars for Cr-stress response by using the parameters of seed germination,morpho-physiological and antioxidant analysis

Rice is the most important crop for the majority of population across the world with sensitive behavior toward heavy metals such as chromium (Cr) in polluted regions. Although, there is no information on the Cr resistance phenotyping in rice. Herein, two different groups of rice cultivars (normal, and hybrid) were used, each group with 14 different rice cultivars. Firstly, seed germination analysis was conducted by evaluating various seed germination indices to identify the rice cultivars with greatest seed germination vigor. Furthermore, exposure of chromium (Cr) toxicity to 28 different rice varieties (NV1-NV14, HV1-HV14) caused noticeable plant biomass reduction. Subsequently, NV2, NV6, NV10, NV12, NV13 (normal type), HV1, HV4, HV8, and HV9 (hybrid types) were pragmatic as moderately sensitive varieties, while NV3, NV4, NV9, and NV14 (normal type), HV3, HV6, HV7, and HV13 were observed as moderately tolerant. Although, NV7, and HV10 were ranked most sensitive cultivars, and NV11, and HV14 were considered as most tolerant varieties as compared to the other rice (both groups) genotypes. Afterward, Cr induced reduction in chlorophyll pigments were significantly lesser in HV14 relative to NV11, NV7, and especially HV10, and as a result HV14 modulated the total soluble sugar level as well as reduced ROS accumulation, and MDA contents production by stimulating the antioxidant defense mechanism conspicuously which further reduced the electrolyte leakage as well. Our outcomes provide support to explore the Cr tolerance mechanism in cereal crops as well as knowledge about rice breeding with increased tolerance against Cr stress.     

Litterfall Production Along Successional and Altitudinal Gradients of Subtropical Monsoon Evergreen Broadleaved Forests in Guangdong, China

Evaluation of litterfall production is important for understanding nutrient cycling, forest growth, successional pathways, and interactions with environmental variables in forest ecosystems. Litterfall was intensively studied during the period of 1982–2001 in two subtropical monsoon vegetation gradients in the Dinghushan Biosphere Reserve, Guangdong Province, China. The two gradients include: (1) a successional gradient composed of pine forest (PF), mixed pine and broadleaved forest (MF) and monsoon evergreen broadleaved forest (BF), and (2) an altitudinal gradient composed of Baiyunci ravine rain forest (BRF), Qingyunci ravine rain forest (QRF), BF and mountainous evergreen broadleaved forest (MMF). Mean annual litterfall production was 356, 861 and 849 g m⁻² for PF, MF and BF of the successional gradient, and 1016, 1061, 849 and 489 g m⁻² for BRF, QRF, BF and MMF of the altitudinal gradient, respectively. As expected, mean annual litterfall of the pioneer forest PF was the lowest, but rapidly increased over the observation period while those in other forests were relatively stable, confirming that forest litterfall production is closely related to successional stages and growth patterns. Leaf proportions of total litterfall in PF, MF, BF, BRF, QRF and MMF were 76.4%, 68.4%, 56.8%, 55.7%, 57.6% and 69.2%, respectively, which were consistent with the results from studies in other evergreen broadleaved forests. Our analysis on litterfall monthly distributions indicated that litterfall production was much higher during the period of April to September compared to other months for all studied forest types. Although there were significant impacts of some climate variables (maximum and effective temperatures) on litterfall production in some of the studied forests, the mechanisms of how climate factors (temperature and rainfall) interactively affect litterfall await further study.     

水稻病程相关PR1家族蛋白质在叶片生长及与白叶枯病菌互作反应中的表达

病程相关(PR)蛋白质经常被用作抗病反应的分子标记。利用免疫印迹(WB)技术检测了7个PR1家族蛋白质在水稻(Oryza sativa)叶片生长及与白叶枯病菌互作反应过程中的表达,发现6个PR1家族蛋白质在叶片生长中有表达。检测PR1蛋白质在Xa21介导的抗白叶枯病过程中的表达,结果显示PR1#052、PR1#072、PR1#073和PR1#121四个蛋白质在抗病反应后期呈上调或诱导表达,PR1#071则表达下调。进一步比较它们在抗病、感病和对照(Mock)反应中的表达丰度,发现在抗病和感病反应中的变化幅度均明显大于对照反应,推测这些PR蛋白质在水稻-白叶枯病菌互作反应中发挥作用。另外,对PR1基因上游启动子区的cis元件进行了分析。该研究初步揭示了水稻PR1家族蛋白质的表达谱,为进一步了解PR1蛋白质的功能提供了线索。     

Activation of Ras/Erk pathway by a novel MET-interacting protein RanBPM

MET is a receptor protein-tyrosine kinase (RPTK) for hepatocyte growth factor (HGF), which is a multifunctional cytokine controlling cell growth, morphogenesis, and motility. MET overexpression has been identified in a variety of human cancers. Oncogenic missense mutations of the tyrosine kinase domain of the MET gene have been identified in human papillary renal cell carcinomas. In this study, RanBPM, also known as RanBP9, is identified as a novel interacting protein of MET through yeast two-hybrid screen. RanBPM contains a conserved SPRY (repeats in splA and RyR) domain. We demonstrate that RanBPM can interact with MET in vitro and in vivo, and the interaction can be strengthened by HGF stimulation. RanBPM interacts with the tyrosine kinase domain of MET through its SPRY domain. We show that RanBPM can induce GTP-Ras association and Erk phosphorylation and elevate serum response element-luciferase (SRE-LUC) expression, indicating that RanBPM can activate the Ras-Erk-SRE pathway. We demonstrate that RanBPM, which itself is not a guanine exchange protein, stimulates Ras activation by recruiting Sos. On the cellular level, A704 cells, a human renal carcinoma cell line, transfected with RanBPM exhibit increased migration ability. Our data suggest that RanBPM, functioning as an adaptor protein for the MET tyrosine kinase domain, can augment the HGF-MET signaling pathway and that RanBPM overexpression may cause constitutive activation of the Ras signaling pathway.     

用鱼血清转氨酶监测水污染的研究

以三硝基甲苯(INT)、六六六、滴滴涕(DDT)、对硫磷(E-605)、氯化汞分别进行白鲢鱼种的急性致毒实验,与对照组相比,鱼血清谷草转氨酶活性显著增加;对硫磷还引起血清谷丙转氨酶活性的升高。血清转氨酶活性增加的程度与氯化汞浓度相关。不同种类的我国淡水鲤科鱼类、不同鱼龄、不同水体以及短期饥饿、惊扰及网箱饲养对血清转氨酶活性没有影响,但水温升高或溶氧低于1ppm会使鱼血清谷草转氨酶活性升高。水温与鱼血清谷草转氨酶活性有相关性。       

c-erbB-2单克隆抗体A18在乳腺癌中表达特性的研究

目的：研究一株自制的c-erbB-2单克隆抗体A18在乳腺癌中表达的特性。方法：应用免疫组织化学SP法、蛋白质印迹分析法和荧光激活的流式细胞分选检测技术检测A18在635例乳腺癌组织、100例癌旁乳腺组织、不表达c-erbB-2的NIH／3T3（鼠成纤维细胞）和NE91（转表皮生长因子受体基因的NR6鼠成纤维面积）细胞株及高表达c-erbB-2的T6－17（转c-erbB-2－基因的NIH／3T3细胞）和SKBR3（人乳腺癌细胞）细胞株中的表达状况,并与市售进口c-erbB-2抗体（MaximBiotech产品）进行了平行对照研究。A18系采用细胞表面区域表位包埋法免疫小鼠制备而成,,结果：A18阳性染色定位于细胞膜,部分伴微弱的细胞浆着色,无明显非特异性染色,A18和进口抗体对NIH／3T3、NE91细胞均呈阴性,T6－17、SKBR3细胞均呈阳性,在乳腺癌组织中,A18的阳性率为60．3％,明显高于癌旁乳腺组织的5．0％,A18与进口同类单抗的阴性、阳性及总符合率分别为84．0％、82．8％及83．2％,与进口同类多抗的阴性,阳性及总符合率分别为88．0％、90．2％和89．5％。A1和进口同类单抗与乳腺癌临床病理特征的关系一致。经反复冻融7次或4℃保存10个月A18效价仍保持在3μg/ml。结论：A18特异性强,定位准确,效价高而稳定、可用于临床乳腺癌的检测。     

秦巴山区野生百合(Lilium spp.)的育种应用研究

为探索秦巴山区野生百合资源在百合育种中的应用方向及途径, 获得具有其独特遗传背景的育种材料,将秦巴山区野生百合的抗病毒及独特观赏特性等优良遗传性状逐渐渗到栽培品种中去。该研究借助切割柱头杂交及胚抢救技术,选取6种秦巴山区野生百合［岷江百合(Lilium gegale)、宜昌百合(L. leucanthum)、山丹（L. pumilum）、野百合（L. brownii）、宝兴百合（L. duchartrei）、川百合（L. davidii）］以及亚洲百合（Asiatic hybrids,AA）品种‘Elite’、东方百合（Oriental hybrids,OO）品种‘Sorbonne’、‘Siberia’和‘Marlon’、OT百合（Oriental × Trumpet hybrids,OT）品种‘Yelloween’、‘Serano’、‘Corel′door’进行了32组共计263朵花的（品）种间杂交,并针对膨大变软的果实剥离可供离体培养的胚及胚囊进行胚抢救。结果表明：（1）不同杂交组合坐果率、胚获得及萌发率呈现出较大差异,综合坐果率为11.4%,对30个膨大的果实中共计38个可供离体培养的胚及胚囊进行胚抢救,有7株最终萌发。（2）以野生百合为父母本的12组杂交组合中,6组获得了膨大果实,得到6株杂交后代。（3）栽培百合做母本,野生百合做父本的20组杂交组合中,共9组获得了膨大果实,除‘Elite’ × 山丹可直接收获种子外,共得到1株杂交后代。（4）以岷江百合及宜昌百合为亲本的远缘杂交TT × AA及回交OT × TT成功获得杂种后代。（5）秦巴山区6种野生百合在远缘杂交中获得育种后代的几率存在较大差异,宜昌百合和岷江百合获得后代几率较高,宝兴百合获得后代几率较低,野百合未获得后代。以上结果表明,岷江百合和宜昌百合为母本的TT × AA杂交和宝兴百合为父本的TT × AA杂交以及岷江百合和宜昌百合为父本的OT回交,为三种百合的育种利用提供了新途径,野百合的育种应用途径需要继续探索。     

The inhibitory effect of BSP-A1/-A2 on protein kinase C and tyrosine protein kinase

Bovine seminal plasma contains a group of similar proteins, namely BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins), and they are secreted by the seminal vesicles. In our study, we purified the BSP-A1/-A2 through affinity chromatography and found for the first time that BSP-A1/-A2 can inhibit the activity of protein kinase C (PKC) and tyrosine protein kinase (TPK). The inhibition was dose dependent. When the PKC and TPK activities are expressed as the logarithm of percentage activity taking the activity in the absence of the BSP-A1/-A2 as 100%, there is a linear relationship between the their activities and the dose of BSP-A1/-A2.