哺乳动物营养代谢的时间生物学研究进展

时间生物学主要是研究生物体内生理和行为的时间机制的学科,而这种机制主要是由生物钟调控的。研究表明,营养代谢的各个方面如葡萄糖转运、糖原异生、脂质合成及降解、氧化磷酸化等作用都受到生物钟核心转录机制的调控,并具有时间敏感性;相反,代谢信号也可以反馈调节生物钟系统,包括生物钟基因表达和行为活动。生物钟的紊乱会造成诸如心血管疾病、肥胖、糖尿病等多种疾病。本文从代谢与生物钟的相互关系、各类营养信号和营养素对生物钟的作用以及生物钟与营养代谢相关疾病的关系等多方面综述了哺乳动物营养代谢的时间生物学研究进展。     

Shotgun cross-linking analysis for studying quaternary and tertiary protein structures

We developed a new approach that employs a novel computer algorithm for the sensitive and high-throughput analysis of tertiary and quaternary interaction sites from chemically cross-linked proteins or multi-protein complexes. First, we directly analyze the digests of the chemically cross-linked proteins using only high-accuracy LC-MS/MS data. We analyze these data using a computer algorithm, we term X!Link, to find cross-links between two peptides. Our algorithm is rapid, taking only a few seconds to analyze approximately 5000 MS/MS spectra. We applied this algorithm to analyze cross-linked sites generated chemically using the amino specific reagent, BS3, in both cytochrome c and the mitochondrial division dynamin mutant, Dnm1G385D, which exists as a stable homodimer. From cytochrome c, a well-established test protein, we identified a total of 31 cross-links, 21 interpeptide and 10 intrapeptide cross-links, in 257 MS/MS spectra from a single LC-MS/MS data set. The high sensitivity of this technique is indicated by the fact that all 19 lysines in cytochrome c were detected as a cross-link product and 33% of all the Lys pairs within 20 A were also observed as a cross-link. Analysis of the cross-linked dimeric form of Dnm1G385D identified a total of 46 cross-links, 38 interpeptide and 8 intrapeptide cross-links, in 98 MS/MS spectra in a single LC-MS/MS data set. These results represent the most abundant cross-links identified in a single protein or protein dimer to date. Statistical analysis suggests a 1% false discovery rate after optimization of filtering parameters. Further analysis of the cross-links identified using our approach indicates that careful manual inspection is important for the correct assignment of cross-linking sites when multiple cross-linkable sites or several similar sequences exist. In summary, we have developed a sensitive MS-based approach to identify peptide-peptide cross-links that does not require isotopic labeling or comparison with non-cross-linked controls, making it faster and simpler than current methodologies.     

人胸腺上皮细胞培养及其分泌产物

本文通过降低培养基中血清含量,向RPMI 1640培养基中补加三碘甲腺原氨酸而获得一种人胸腺网状上皮细胞占优势生长的培养物。在此培养基中细胞经传代培养长达90天,仍维持正常形态特征。胸腺组织在培养14天后,新生细胞的突起形成网状结构,细胞化学检查和电镜观察表明具有丰富的分泌颗粒,囊泡及张力原纤维束和桥粒等上皮细胞特征。收集合并细胞培养液,经部分纯化后检查其生物活性,表现出具有促进玫瑰花结形成和降低胸腺细胞TdT活性的作用,说明培养细胞的分泌产物具有胸腺激素活性。根据形态学,细胞化学和生物活性检测结果,我们倾向于认为该培养物主要为网状上皮细胞。     

Molecular Requirement for Sterols in Herpes Simplex Virus Entry and Infectivity

Herpes simplex virus 1 (HSV-1) required cholesterol or desmosterol for virion-induced membrane fusion. HSV successfully entered DHCR24^−/− cells, which lack a desmosterol-to-cholesterol conversion enzyme, indicating that entry can occur independently of cholesterol. Depletion of desmosterol from these cells resulted in diminished HSV-1 entry, suggesting a general sterol requirement for HSV-1 entry and that desmosterol can operate in virus entry. Cholesterol functioned more effectively than desmosterol, suggesting that the hydrocarbon tail of cholesterol influences viral entry.     

Involvement of Annexin A2 in p53 induced apoptosis in lung cancer

Tumor suppressor p53 plays important roles in cell cycle regulation, apoptosis and DNA repair in different cell types including lung cancer. There are different p53 apoptotic pathways in high and low metastatic ability lung cancer cells. However, the exactly mechanism in the pathway is still unclear. Here we found that Annexin A2, a Ca²⁺-dependent phospholipid-binding protein, is involved in p53-mediated apoptosis. First, by using mRNA differential display technique, down-regulated Annexin A2 expression was found in all cell lines transfected of Ad-p53 (adenoviral expression construct encoding wild type p53 gene) especially in highly metastatic Anip973 lung cancer cells. Then, decreased expression of Annexin A2 was further confirmed by Northern blot and Western blot analysis. At last, knock down of Annexin A2 by siRNA inhibited cellular proliferation in BE1 cell line with highly metastatic ability. Taken together, our results suggested that Annexin A2 may play roles in p53 induced apoptosis and it is also involved in regulation of cell proliferation. The authors Yun Huang, Yan Jin and Cheng-hui Yan contributed equally to this work.     

The effect of HMGB1 A box on lung injury in mice with acute pancreatitis

The objective of this study is to observe the effect of high-mobility group protein B1 A Box (HMGB1 A) box on lung injury in mice with acute pancreatitis and its effect on the level of high-mobility group protein B1 (HMGB1) in lung, to explore the mechanism. A total of 60 male Institute of Cancer Research mice were randomly divided into control group (n = 30) and treatment group (n = 30). Severe acute pancreatitis mice model was induced by 20% L-Arg intraperitoneal injection. The recombination HMGB1 A box was used in treatment after modeling. All the mice were killed under anesthesia at 24 and 48 h after the modeling injection. The level of HMGB1 and activity of myeloperoxidase (MPO) in lung were measured. The pathological changes of lung were observed. The level of HMGB1 in lung of A box treatment group decreased more significantly 24 h and 48 h after modeling compared with control group. The activity of MPO in lung of A box treatment group decreased more significantly 24 h after modeling compared with control group. The lung tissue pathologic score of A box treatment group decreased more significantly 48 h after modeling compared with control group. HMGB1 expression levels in the lungs were positively related to histological score of injured lung in acute pancreatitis. It indicates that HMGB1 A box is remarkably protective to lung injury induced by acute pancreatitis.     

鸡减蛋综合征病毒(EDSV)基因组右末端结构的分析

从中国发病鸡中分离的鸡减蛋综合征病毒（ＥｇｇＤｒｏｐＳｙｎｄｒｏｍＶｉｒｕｓ,ＥＤＳＶ）弱毒株（ＡＡ－２）,其基因组全长约为３３ｋｂ。用限制性内切酶ＨｉｎｄⅢ水解ＥＤＳＶ全基因组,构建了以ｐＢｌｕｅｓｃｒｉｐｔⅡ（ＫＳ＋）为载体的右末端片段的克隆（约４．２ｋｂ）,对其进行了序列测定和结构分析。该片段全长４１８３个碱基对（ｂｐ）,位于基因组右末端８７．３ｍ．ｕ．－１００ｍ．ｕ．。结果显示,该片段与哺乳动物腺病毒右末端Ｅ４区结构不同,与禽Ⅰ型腺病毒代表株ＣＥＬＯ右末端片段亦无同源性。本文为深入了解ＥＤＳＶ基因组结构特点,ＥＤＳＶ与其他腺病毒基因结构与功能的进化关系和ＥＤＳＶ载体的构建奠定了分子生物学基础     

FRIL蛋白体外维持脐血造血干/祖细胞的作用及细胞周期调控基因的表达

为探讨新的豆类凝集素(Flt3 receptor-interacting lectin,FRIL)体外维持脐血CD34^ 细胞的作用以及维持过程中细胞周期调控基因HTm4及HTm4S mRNA的表达及意义,我们利用FRIL维持培养脐血CD34^ 细胞,对其增殖曲线、细胞周期及集落形成能力进行常规分析,并用半定量RT—PCR法分别测定FRIL体外维持不同时间后脐血CD34^ 细胞中周期调控基因HTm4及HTm4S mRNA的表达变化。结果显示,FRIL培养的CD34^ 造血干／祖细胞的增殖趋势平缓,整个培养期间细胞增殖倍数不超过起始的3倍：14d之前,FRIL培养细胞的高增殖潜能集落形成细胞(HPP—CFC)形成集落数与FL组无差别,其后则维持高于FL的情况。细胞周期分析则显示,在28d的培养过程内,利用FRIL培养的细胞始终有80％以上维持在G0期;而周期调控基因HTm4及HTm4S在刚分离的脐血CD34^ 细胞中的表达水平较高;但培养1d后,几乎检测不到HTm4基因的表达;培养3～14d,该基因的表达回升并持续维持在高水平。而HTm4S基因的表达在第7d达最高水平,其余时间基本呈稳定表达。转染HTm4和HTm4S,亚细胞定位结果显示HTm4主要定位于核周围,而HTm4S则定位于整个胞浆,由此可能导致它们功能的区别。以上结果提示,长期培养体现出FRIL在维持造血干／祖细胞多能性上的优势;细胞周期调控基因HTm4及其新剪接子参与了FRIL体外长期维持脐血造血干／祖细胞处于静息状态的过程。     

荧光定量RT-PCR检测血管内皮细胞PAI-1 mRNA方法的建立

为了建立用荧光定量PCR检测人微血管内皮细胞纤溶酶原激活剂抑制物-1（PAI-1）mRNA表达的方法。提取人微血管内皮细总RNA,经RT-PCR获得靶基因（PAI-1）及管家基因（β-actin）的PCR产物。纯化后,作为标准品梯度稀释,采用SYBR Green I定量PCR检测,建立标准曲线。方法学考核参数为特异性、线性范围、灵敏性和重复性。分析全反式维甲酸对内皮细胞表达PAI-1 mRNA的干预效果。这种定量方法特异性好,检测的灵敏度达10^3拷贝,线性范围为10^4-10^10拷贝,循环阈值与PCR体系中起始模板量的对数值之间有着良好的线性关系（r^2〉0.990）,批内变异≤4.93%,批间变异≤9.12%。全反式维甲酸能上调内皮细胞PAI-1 mRNA的表达,且呈剂量依赖性。因此,荧光定量RT-PCR检测血管内皮细胞PAI-1基因表达的方法有助于溶栓药物药理学的研究和新药筛选。     

Down-regulated expression of the protein-tyrosine phosphatase 1B (PTP1B) is associated with aggressive clinicopathologic features and poor prognosis in hepatocellular carcinoma

The protein-tyrosine phosphatase 1B (PTP1B) is a classical non-transmembrane protein tyrosine phosphatase that plays a key role in metabolic signaling and can exert both tumor suppressing and tumor promoting effects in different cancers depending on the substrate involved and the cellular context. However, the expression level and function of PTP1B in hepatocellular carcinoma (HCC) remain unclear. In this study, PTP1B expression was detected by immunohistochemistry in normal liver tissue (n=16) and hepatocellular carcinoma (n=169). The correlations between PTP1B expression level and clinicopathologic features and patient survival were also analyzed. One hundred and eleven of 169 HCC patients (65.7%) had negative or low PTP1B expression in tumorous tissues, whereas normal tissues always expressed strong PTP1B. Decreased PTP1B expression was significantly associated with aggressive clinicopathologic features and poor prognosis. Immunohistochemistry also showed that low PTP1B expression level was correlated with high percentage of OV6(+) tumor-initiating cells (T-ICs) and high frequency of nuclear β-Catenin expression in HCC specimens. Our findings demonstrate for the first time that the loss of inhibitory effect of PTP1B may contribute to progression and invasion of HCC through activation of Wnt/β-Catenin signaling and expansion of liver T-ICs. PTP1B may serve as a valuable prognostic biomarker and potential therapeutic target in HCC.