双斑蟋若虫后足的再生观察

研究蟋蟀若虫是否具有再生的能力,在实验室内对直翅目蟋蟀科的双斑蟋Gryllus bimaculata de Geer若虫后足的再生情况进行初步观察。结果表明:双斑蟋若虫的后足确实具有再生的能力,且再生足的伸长生长与正常足一样,均出现于虫体蜕皮时。再生的过程主要分为3个阶段:突起期、“肢芽”期、短小足期。实验结果还表明:再生能力与断肢发生的时间及断肢部位有关;断肢部位离肢体越远,再生能力越强;断肢发生的时间越早,再生能力越强。     

绵羊fertilin β基因编码区的钓取与结构分析

Fertilin β与精卵的结合和融合有密切关系。为探讨fertilin β蛋白在绵羊受精过程中的作用机理, 采用RACE技术, 首次钓取了该基因的编码区。结果绵羊fertilin β基因的编码区cDNA全长为2,217 bp。同源性分析显示, 绵羊的fertilin β氨基酸序列与牛、猪和人的fertilin β具有79.4%、66.7%和58.1%的同源性。系统发育分析表明, 绵羊fertilin β与牛属于同一分支, 并且也显示了绵羊和牛分类地位最近, 这和传统的分类一致。Fertilin β蛋白结构域分析显示, 绵羊fertilin β去整合素识别序列为TDE, 与牛的序列相同。除了上述三肽序列外, 紧随X-D/E-E的ECD保守序列, 从而形成了X-D/E-ECD五肽保守序列, 在绵羊fertilin β中该五肽序列为TDECE。     

The JAK–STAT Pathway Is Critical in Ventilator-Induced Diaphragm Dysfunction

Mechanical ventilation (MV) is one of the lynchpins of modern intensive-care medicine and is life saving in many critically ill patients. Continuous ventilator support, however, results in ventilation-induced diaphragm dysfunction (VIDD) that likely prolongs patients’ need for MV and thereby leads to major associated complications and avoidable intensive care unit (ICU) deaths. Oxidative stress is a key pathogenic event in the development of VIDD, but its regulation remains largely undefined. We report here that the JAK–STAT pathway is activated in MV in the human diaphragm, as evidenced by significantly increased phosphorylation of JAK and STAT. Blockage of the JAK–STAT pathway by a JAK inhibitor in a rat MV model prevents diaphragm muscle contractile dysfunction (by ~85%, p < 0.01). We further demonstrate that activated STAT3 compromises mitochondrial function and induces oxidative stress in vivo, and, interestingly, that oxidative stress also activates JAK–STAT. Inhibition of JAK–STAT prevents oxidative stress-induced protein oxidation and polyubiquitination and recovers mitochondrial function in cultured muscle cells. Therefore, in ventilated diaphragm muscle, activation of JAK–STAT is critical in regulating oxidative stress and is thereby central to the downstream pathogenesis of clinical VIDD. These findings establish the molecular basis for the therapeutic promise of JAK–STAT inhibitors in ventilated ICU patients.     

Extremely Stable Soluble High Molecular Mass Multi-Protein Complex with DNase Activity in Human Placental Tissue

Human placenta is an organ which protects, feeds, and regulates the grooving of the embryo. Therefore, identification and characterization of placental components including proteins and their multi-protein complexes is an important step to understanding the placenta function. We have obtained and analyzed for the first time an extremely stable multi-protein complex (SPC, ∼1000 kDa) from the soluble fraction of three human placentas. By gel filtration on Sepharose-4B, the SPC was well separated from other proteins of the placenta extract. Light scattering measurements and gel filtration showed that the SPC is stable in the presence of NaCl, MgCl2, acetonitrile, guanidinium chloride, and Triton in high concentrations, but dissociates efficiently in the presence of 8 M urea, 50 mM EDTA, and 0.5 M NaCl. Such a stable complex is unlikely to be a casual associate of different proteins. According to SDS-PAGE and MALDI mass spectrometry data, this complex contains many major glycosylated proteins with low and moderate molecular masses (MMs) 4–14 kDa and several moderately abundant (79.3, 68.5, 52.8, and 27.2 kDa) as well as minor proteins with higher MMs. The SPC treatment with dithiothreitol led to a disappearance of some protein bands and revealed proteins with lower MMs. The SPCs from three placentas efficiently hydrolyzed plasmid supercoiled DNA with comparable rates and possess at least two DNA-binding sites with different affinities for a 12-mer oligonucleotide. Progress in study of placental protein complexes can promote understanding of their biological functions.     

Engineering of the Saccharomyces cerevisiae yeast strain with multiple chromosome-integrated genes of human alpha-fetoprotein and its high-yield secretory production, purification, structural and functional characterization

Alpha-fetoprotein (AFP) is a biological drug candidate of high medicinal potential in the treatment of autoimmune diseases, cancer, and regenerative medicine. Large-scale production of recombinant human alpha-fetoprotein (rhAFP) is desirable for structural and functional studies and applied research. In this study we cloned and expressed in the secreted form wild-type glycosylated human rhAFP and non-glycosylated mutant rhAFP(0) (N233S) in the yeast strain Saccharomyces cerevisiae with multiple chromosome-integrated synthetic human AFP genes. RhAFP and rhAFP(0) were successfully produced and purified from the culture liquids active naturally folded proteins. Elimination of the glycosylation by mutation reduced rhAFP(0) secretion about threefold as compared to the wild-type protein showing critical role of the N-linked glycan for heterologous protein folding and secretion. Structural similarity of rhAFP and rhAFP(0) with natural embryonic eAFP was confirmed by circular dichroism technique. Functional tests demonstrated similar type of tumor suppressive and immunosuppressive activity for both recombinant species rhAFP and rhAFP(0) as compared to natural eAFP. It was documented that both types of biological activities attributed to rhAFP and rhAFP(0) are due to the fast induction of apoptosis in tumor cells and mitogen-activated lymphocytes. Despite the fact that rhAFP and rhAFP(0) demonstrated slightly less effective tumor suppressive activity as compared to eAFP but rhAFP(0) had produced statistically notable increase in its ability to induce inhibition of in vitro lymphocyte proliferation as compared to the glycosylated rhAFP and eAFP. We conclude that N-linked glycosylation of rhAFP is required for efficient folding and secretion. However the presence of N-linked sugar moiety was shown to be unimportant for tumor suppressive activity but was critically important for its immunoregulative activity which demonstrates that different molecular mechanisms are involved in these two types of biological functional activities attributed to AFP.     

高血压患者用药的依从性

高血压是人类健康面临的一个重要挑战,对患者及其家庭和社会造成了巨大的疾病负担。药物治疗是控制高血压的重要手段,合理应用抗高血压药能使90%的患者血压处于正常范围,但实际高血压的控制率仍很低,且发病率逐年上升,究其根本原因,是由于患者用药依从性不良。本文综述了高血压患者用药依从性的概念、现状及评估方法,并对其影响因素进行了详细的阐述,指出应积极干预及加强健康教育,以最大限度提高患者的用药依从性,降低高血压带来的靶器官损害,并进一步对寻求更准确、客观的用药依从性评估方法做出展望。     

白簕叶挥发油的化学成分

用GCMS联用技术首次研究了云南西双版纳白簕叶挥发油的化学成分,并应用色谱峰面积归一化法计算各成分的相对含量。一共分离出108个峰,确认了其中的81种成分,所鉴定的组分占挥发油总量的96.50%,主要是萜烯类及其含氧衍生物等。     

垂体瘤转化基因1(PTTG1)的表达变化对人前列腺癌细胞LNCaP生长增殖的影响

垂体肿瘤转化基因1(PTTG1)具有促进肿瘤生长和转移的作用.通过上调或下调基因表达的策略,观察PTTG1基因对人前列腺癌细胞株LNCaP细胞生长增殖的影响.利用PCR技术分离出PTTG1全长cDNA,分别正向和反向插入真核表达载体pIRES2-EGFP,重组载体分别命名为正义PTTG1-S/pIRES2-EGFP(即pI-P-S)和反义PTTG1-AS/pIRES2-EGFP(即pI-P-AS),将这两种重组载体稳定转染LNCaP细胞,通过流式细胞仪和MTT法分别检测了细胞周期和细胞增殖的情况.转染正义PTTG1后处于S期和G2期的细胞明显增加,细胞生长增殖能力增强;相反,转染反义PTTG1后处于S期和G2期细胞明显减少,细胞生长增殖能力减弱(P<0.05).结果表明,PTTG1能明显改变人前列腺癌细胞株LNCaP的细胞周期和细胞生长增殖能力,它的异常表达可能参与前列腺癌细胞生长增殖过程.     

Recombinant human erythropoietin with additional processable protein domains: Purification of protein synthesized in <Emphasis Type="Italic">Escherichia coli</Emphasis> heterologous expression system

Three variants of human recombinant erythropoietin (rhEPO) with additional N-terminal protein domains were obtained by synthesis in an Escherichia coli heterologous expression system. These domains included (i) maltose-binding protein (MBP), (ii) MBP with six histidine residues (6His) in N-terminal position, (iii) s-tag (15-a.a. oligopeptide derived from bovine pancreatic ribonuclease A) with N-terminal 6His. Both variants of the chimeric protein containing MBP domain were prone to aggregation under nondenaturing conditions, and further purification of EPO after the domain cleavage by enterokinase proved to be impossible. In the case of 6His-s-tag-EPO chimeric protein, the products obtained after cleavage with enterokinase were successfully separated by column chromatography, and rhEPO without additional domains was obtained. Results of MALDI-TOF mass spectrometry showed that after refolding 6His-s-tag-EPO formed a structure similar to that of one of native EPO with two disulfide bonds. Both 6His-s-tag-EPO and rhEPO without additional protein domains purified after proteolysis possessed the same biological activity in vitro in the cell culture.     

4种桦树幼苗耐盐性分析与评价

耐盐植物引种和培育是开发利用盐碱地的主要方式,具有重要的研究价值。本试验以哈萨克斯坦引进的吉尔吉斯白桦（Betula kirghisorum）、欧洲白桦（B.pendula）、毛枝桦（B.pubescens）和本地的白桦（B.platyphylla Suk.）1年生幼苗为试验材料,于2014年7月在东北林业大学进行中性盐（NaCl）和碱性盐（NaHCO₃）的胁迫试验,测定生长量、光合参数和叶绿素含量,并通过因子分析法,对比评价4种桦树幼苗的耐盐碱能力,筛选出综合性状优良的桦树树种,为耐盐植物引种和培育提供有价值的数据。结果表明：随着盐浓度的升高,桦树幼苗的高生长和光合效率受到显著抑制,而当浓度≥0.5%时,大部分幼苗枯死。株高增长量、基径增长量、净光合速率（P_n）、光能利用效率（SUE）、羧化效率（CUE）、表观量子效率（AQY）及叶绿素含量之间的相关性多数达到了显著水平;最后利用因子分析法分别构建了0.1% NaCl、0.3% NaCl、0.1% NaHCO3和0.3% NaHCO₃胁迫处理的综合评价公式,并分别筛选出了综合性状相对优良的单株,其中NaCl胁迫下较优单株为32、33、34、35;NaHCO₃胁迫下较优单株为262、263、264、35。综合比较认为,吉尔吉斯白桦对低中浓度的中性盐的抗性最强,本地对照白桦对低中浓度碱性盐的抗性最强,而吉尔吉斯白桦和毛枝桦对高浓度碱性盐抗性较强。