Caspase-2 Short Isoform Interacts with Membrane-Associated Cytoskeleton Proteins to Inhibit Apoptosis

Caspase-2 (casp-2) is the most conserved caspase across species, and is one of the initiator caspases activated by various stimuli. The casp-2 gene produces several alternative splicing isoforms. It is believed that the long isoform, casp-2L, promotes apoptosis, whereas the short isoform, casp-2S, inhibits apoptosis. The actual effect of casp-2S on apoptosis is still controversial, however, and the underlying mechanism for casp-2S-mediated apoptosis inhibition is unclear. Here, we analyzed the effects of casp-2S on DNA damage induced apoptosis through “gain-of-function” and “loss-of-function” strategies in ovarian cancer cell lines. We clearly demonstrated that the over-expression of casp-2S inhibited, and the knockdown of casp-2S promoted, the cisplatin-induced apoptosis of ovarian cancer cells. To explore the mechanism by which casp-2S mediates apoptosis inhibition, we analyzed the proteins which interact with casp-2S in cells by using immunoprecipitation (IP) and mass spectrometry. We have identified two cytoskeleton proteins, Fodrin and α-Actinin 4, which interact with FLAG-tagged casp-2S in HeLa cells and confirmed this interaction through reciprocal IP. We further demonstrated that casp-2S (i) is responsible for inhibiting DNA damage-induced cytoplasmic Fodrin cleavage independent of cellular p53 status, and (ii) prevents cisplatin-induced membrane blebbing. Taken together, our data suggests that casp-2S affects cellular apoptosis through its interaction with membrane-associated cytoskeletal Fodrin protein.     

Two independent growth factor-generated signals regulate c-fos and c-myc mRNA levels in Swiss 3T3 cells

Polypeptide growth factors that stimulate cell proliferation bind to cell surface receptors and activate intracellular signal transduction pathways. One major signalling pathway, initiated by phosphatidylinositol (PI) turnover, involves activation of protein kinase C. Some polypeptide growth factors, including mitogens that activate protein kinase C, induce a rapid increase in expression of the proto-oncogenes, c-myc and c-fos. In order to characterize the signal transduction pathways responsible for proto-oncogene activation, we treated Swiss 3T3 cells with the tumor promoter phorbol dibutyrate to generate cells deficient in protein kinase C. These cells were then stimulated with platelet extract, bombesin, or epidermal growth factor (EGF) and the levels of c-myc and c-fos mRNA were determined. Platelet extract or bombesin, which stimulate PI turnover, were substantially weaker inducers of c-myc and c-fos mRNA levels in the protein kinase C-depleted cells, although some variability with platelet extract was noted. EGF, which does not stimulate PI turnover in several cell systems, was by contrast a potent inducer of both proto-oncogenes whether or not the cells were deficient in protein kinase C. Pretreatment of cells with phorbol dibutyrate caused little or no change in the basal levels of c-myc or c-fos mRNA, but led to a small but significant increase in basal levels of ornithine decarboxylase mRNA. These results demonstrate that EGF and growth factors that activate PI turnover induce expression of the c-myc and c-fos proto-oncogenes through different pathways.     

Salt-induced cooperativity in ATPase activity of plasma membrane-enriched fractions from cultured Citrus cells: kinetic evidence

ATPase activity was studied in plasma membrane-enriched fractions prepared from cultured Citrus sinensis L. cv. Osbeck cells. In general, properties of the plasma membrane ATPase from cultured cells, such as optimal pH and temperature. V_max and K_m were similar to those already observed in higher plants. The effects of high salt concentrations on ATPase activity were studied in membrane fractions derived from salt-sensitive and salt-tolerant cells grown in the presence or absence of salt. NaCl did not have an in vivo effect on V_max and the apparent K_m value for ATP. However, high concentrations of NaCl, or KCl, added in vitro, induced cooperativity in the enzyme and reduced the affinity of the enzyme for its substrate. Isoosmolar concentrations of sucrose or choline chloride failed to do so. Our results suggest that the plasma membrane ATPase of Citrus cells has more than one substrate-binding site on the native form of the enzyme which interact in the presence of salt and act independently in its absence.     

肝胆显像螯合物—氮基三乙酸单酰苯胺及其类似物的合成

本课题合成四个氮基三乙酸单酰苯胺(IDA)类及其类似物肝胆显像螯合物。(1)用家兔做了肝胆显像扫描实验,其显像效果较好。(2)～(3)为未见报道的化合物。     

Research Progress on Anti-Inflammatory Mechanisms of Black Ginseng

In recent years, black ginseng, a new type of processed ginseng product, has attracted the attention of scholars globally. Ginsenoside and ginseng polysaccharide, the main active substances of black ginseng, have been shown to carry curative effects for many diseases. This article focuses on the mechanism of their action in anti-inflammatory response, which is mainly divided into three aspects: activation of immune cells to exert immune regulatory response; participation in inflammatory response-related pathways and regulation of the expression level of inflammatory factors; effect on the metabolic activity of intestinal flora. This study identifies active anti-inflammatory components and an action mechanism of black ginseng showing multi-component, multi-target, and multi-channel characteristics, providing ideas and a basis for a follow-up in-depth study of its specific mechanism.     

Intravital lipid droplet labeling and imaging reveals the phenotypes and functions of individual macrophages in vivo

Macrophages play pivotal roles in the maintenance of tissue homeostasis. However, the reactivation of macrophages toward proinflammatory states correlates with a plethora of inflammatory diseases, including atherosclerosis, obesity, neurodegeneration, and bone marrow (BM) failure syndromes. The lack of methods to reveal macrophage phenotype and function in vivo impedes the translational research of these diseases. Here, we found that proinflammatory macrophages accumulate intracellular lipid droplets (LDs) relative to resting or noninflammatory macrophages both in vitro and in vivo, indicating that LD accumulation serves as a structural biomarker for macrophage phenotyping. To realize the staining and imaging of macrophage LDs in vivo, we developed a fluorescent fatty acid analog-loaded poly(lactic-co-glycolic acid) nanoparticle to label macrophages in mice with high efficiency and specificity. Using these novel nanoparticles, we achieved in situ functional identification of single macrophages in BM, liver, lung, and adipose tissues under conditions of acute or chronic inflammation. Moreover, with this intravital imaging platform, we further realized in vivo phenotyping of individual macrophages in the calvarial BM of mice under systemic inflammation. In conclusion, we established an efficient in vivo LD labeling and imaging system for single macrophage phenotyping, which will aid in the development of diagnostics and therapeutic monitoring. Moreover, this method also provides new avenues for the study of lipid trafficking and dynamics in vivo.Supplementary key words: macrophage, inflammation, lipid droplet, nanoparticle delivery, in vivo imaging, fatty acid analog, bone marrow, systemic inflammation, lipid trafficking, biomarker

Macrophages, a type of immune cells, almost reside in all tissues of body, from the skin to the bone marrow (BM) (1). Macrophages have remarkable plasticity, and they can be activated into specific subtypes by modifying their physiology and functions in response to local environmental cues. Activated macrophages are commonly divided into proinflammatory killing subtype and anti-inflammatory repairing subtype. Proinflammatory macrophages responding to bacteria, IFN-γ, and lipopolysaccharide (LPS) are involved in host defense and inflammation, whereas anti-inflammatory macrophages responding to interleukin-4 (IL-4), IL-10, and IL-13 play a pivotal role in tissue homeostasis and remodeling (2). Increasing evidence indicates that the reactivation of macrophages toward proinflammatory states under diverse kinds of stress is correlated with a plethora of inflammatory diseases, such as atherosclerosis, diabetes, obesity, rheumatoid arthritis, neurodegeneration, and BM failure syndromes (3, 4). Thus, characterization of macrophage activation status and the underlying molecular mechanism in situ will help elucidate their functions in these diseases; however, in vivo analysis of the macrophage activation status in their native multicellular microenvironment is challenging.Although lipid droplets (LDs) have been initially described as intracellular fat storage organelles in adipocytes, increasing studies indicate that myeloid cells also form LDs under inflammation and stress (5, 6). Macrophages, as the effector cells of innate immunity, are found to form LDs to support their host defense when exposed to pathogens, such as parasites, bacteria, and viruses (7, 8, 9, 10, 11). However, abnormal LD accumulation in tissue-resident macrophages correlates with the pathogenesis of various inflammatory diseases. For instance, foam cells in atherosclerotic lesions can maintain the local inflammatory response by secreting proinflammatory cytokines (12, 13, 14). Moreover, LD-accumulating microglia contribute to neurodegeneration by producing high levels of reactive oxygen species (ROS) and secreting proinflammatory cytokines (15). These findings indicate that LD accumulation might be a hallmark of macrophages with proinflammatory functions.In this study, based on the typical activation of in vitro BM-derived macrophages, we find that proinflammatory M_{(LPS + IFN-γ)} macrophages are characterized by LD accumulation, whereas resting macrophages and anti-inflammatory M_(IL-4) and M_(IL-10) macrophages do not contain any LDs. These features also hold for Matrigel plug-recruited macrophages and tissue-resident macrophages in mice. These findings demonstrate that LD accumulation could serve as a morphological index to distinguish proinflammatory macrophages from others.It is feasible to distinguish LD-containing cells using imaging techniques, which has translational potential for identification of proinflammatory macrophages in vivo. However, current techniques for LD visualization are traditional in vitro staining method, and in vivo staining and imaging of LD in individual macrophages remains a challenge. Through nanocarrier screening, we selected the poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) as nanocarrier to deliver the lipophilic carbocyanine dye (DiI_C18(5) solid (1,1''-dioctadecyl-3,3,3'',3''-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt) [DiD]) and lipid staining dye (C1-BODIPY 500/510-C12) into macrophages. Using these dual fluorescence-labeled PLGA NPs, we achieved in situ and in vivo functional identification of single macrophages in various tissues under systemic or local inflammatory stress. Collectively, this study establishes an efficient in vivo labeling and imaging system of intracellular LDs for phenotyping the activation status and functions of individual macrophages in their dynamic niche, which is pivotal for disease diagnosis and preclinical research.     

乙酸合成途径阻断及NADH氧化酶表达对于谷氨酸棒杆菌生产乙偶姻的影响

【目的】研究乙酸合成途径阻断及NADH氧化酶表达对于谷氨酸棒杆菌生产乙偶姻的影响。【方法】在谷氨酸棒杆菌CGF2中异源表达als SD操纵子构建乙偶姻生产菌株CGT1,考察敲除乙酸生成途径cat和pqo对乙偶姻的影响。然后引入短乳杆菌的NADH氧化酶,在优化的溶氧条件下研究其对乙偶姻产量的影响。【结果】CGT1在摇瓶发酵中可积累6.27 g/L乙偶姻,敲除cat使乙偶姻产量显著提高30.94%,达到8.21 g/L;双敲除cat和pqo没有进一步提高产量。通过优化发酵的溶氧水平,乙偶姻产量达到10.06 g/L。在高溶氧水平下引入NADH氧化酶导致菌株的生长和糖代谢速率提高,但乙偶姻产量略有降低。在分批补料发酵中,重组菌株乙偶姻产量达到40.51 g/L,产率为0.51 g/(L?h)。【结论】在谷氨酸棒杆菌中阻断乙酸合成途径cat能够有效提高乙偶姻产量,NADH氧化酶在高溶氧水平下表达不利于乙偶姻的合成,需要进一步调节表达水平以确定其效果。     

盐生荒漠地表水热与二氧化碳通量的季节变化及驱动因素

以古尔班通古特沙漠南缘原始盐生荒漠为对象,利用涡度相关法,对原始盐生荒漠地表水热、二氧化碳通量进行了连续观测,对通量的季节变化、浅层土壤水分条件改变对盐生荒漠植物群落水汽、二氧化碳通量以及水分利用效率的影响进行了系统的分析.结果表明:净辐射通量、潜热通量和二氧化碳通量都具有明显的季节变化趋势,而显热通量的季节变化不明显.在有效能量的分配上,显热通量是有效能量的主要输出项.在降水影响期和非影响期,二氧化碳通量没有明显的变化;而在非降水影响期潜热通量明显降低,表明土壤水分处于亏缺状态,但二氧化碳通量并没有降低的趋势,而与前期保持高度的一致性.以此可以推断,该荒漠盐生植物群落并不以降水为主要水分来源,降水后水汽通量和二氧化碳通量变化的不一致性是该原始盐生荒漠独特植物特征决定的.降水影响期原始盐生荒漠植物群落的水分利用效率低于非影响期,是由于降水后土壤蒸发迅速增加,而植物蒸腾与光合并未随之增加造成的.     

基于小波神经网络的麦蚜发生程度预测模型

【目的】建立基于小波神经网络病虫害预测预报模型,对提前采取防病防虫措施、减少农作物病虫害损失、提高农作物产量与质量具有重要意义。【方法】本研究以山西省运城市芮城县1980-2014年麦蚜发生程度和气象因子数据为基础,采用主成分分析法从40个基础气象因子中整合形成9个新的自变量输入模型,采用试凑法筛选隐含层节点数,用1980-2009年的数据进行网络训练,对2010-2014年麦蚜发生程度进行回测,建立了以Morlet小波函数为传递函数的小波神经网络模型,并与以Sigmoid函数为传递函数的BP神经网络模型进行了比较。【结果】小波和BP神经网络两种模型对训练样本的平均拟合精度均有10年以上超过80%,两者MAPE 值分别为 89.83% 和83.07%,MSE 值分别为0.0578和0.6192。【结论】两个模型都能较好地描述麦蚜发生程度;从预测精度和模型的稳定性来看,小波神经网络好于BP神经网络。