Enzyme-assisted extraction of flavonoids from Ginkgo biloba leaves: improvement effect of flavonol transglycosylation catalyzed by Penicillium decumbens cellulase

We report a novel enzyme-involved approach to improve the extraction of flavonoids from Ginkgo biloba, in which the enzyme is employed not only for cell wall degradation, but also for increasing the solubility of target compounds in the ethanol-water extractant. Penicillium decumbens cellulase, a commercial cell wall-degrading enzyme with high transglycosylation activity, was found to offer far better performance in the extraction than Trichoderma reesei cellulase and Aspergillus niger pectinase under the presence of maltose as the glycosyl donor. TLC, HPLC and MS analysis indicated that P. decumbens cellulase could transglycosylate flavonol aglycones into more polar glucosides, the higher solubility of which led to improved extraction. The influence of glycosyl donor, pH, solvent and temperature on the enzymatic transglycosylation was investigated. For three predominant flavonoids in G. biloba, the transglycosylation showed similar optimal conditions, which were therefore used for the enzyme-assisted extraction. The extraction yield turned to be 28.3mg/g of dw, 31% higher than that under the pre-optimized conditions, and 102% higher than that under the conditions without enzymes. The utilization of enzymatic bifunctionality described here, naming enzymatic modification of target compounds and facilitation of cell wall degradation, provides a novel approach for the extraction of natural compounds from plants.     

MicroRNA-30a sensitizes tumor cells to cis-platinum via suppressing beclin 1-mediated autophagy

Autophagy is activated in cancer cells during chemotherapy and often contributes to tumor chemotherapy resistance. In this study, we characterized the role of microRNA-30a (miR-30a) in the coordination of cancer cell apoptosis and autophagy, which determines the sensitivity of cancer cells to chemotherapy. First, the autophagy activity in cancer cells increased after cis-dichloro-diamine platinum (cis-DDP) or Taxol treatment, as indicated by the enhanced expression of beclin 1, a key regulator of autophagy, and increased number of LC3-positive autophagosomes. Second, miRNA screening using a TaqMan probe-based quantitative RT-PCR assay identified that miR-30a, a miRNA that targets beclin 1, was significantly reduced in tumor cells by cis-DDP treatment. Forced expression of miR-30a significantly reduced beclin 1 and the autophagy activity of tumor cells induced by cis-DDP. Third, the blockade of tumor cell autophagy activity by miR-30a expression or 3-methyladenine significantly increased tumor cell apoptosis induced by cis-DDP treatment. Finally, an in vivo tumor implantation mouse model clearly showed that elevation of miR-30a in implanted tumor cells by administration of the recombinant lentivirus expressing miR-30a strongly enhanced cis-DDP-induced apoptosis of tumor cells. In conclusion, our results demonstrate for the first time that miR-30a can sensitize tumor cells to cis-DDP via reducing beclin 1-mediated autophagy and that increasing miR-30a level in tumor cells represents a novel approach to enhance the efficacy of chemotherapy during cancer treatment.     

Apelin-13 induces ERK1/2 but not p38 MAPK activation through coupling of the human apelin receptor to the Gi2 pathway

Apelin signaling to the family of mitogen-activated protein kinases (MAPKs), such as extracellular-regulated kinases 1/2 (ERK1/2) and p38 MAPK, through the coupling of apelin receptor (APJ) to G-protein, mediates important pathophysiological responses. Although apelin fragments have been reported to induce ERK1/2 activation through G_i-protein, the intracellular pathways by which APJ activates these MAPKs are only partially understood. Here, using stably transfected human embryonic kidney 293 (HEK293) cells overexpressing human APJ (HEK293-apelinR), we showed that apelin-13 signaling leads to ERK1/2 and p38 MAPK pathways through APJ activation. It was found in HEK293-apelinR cells that ERK1/2 activation was initiated by apelin-13 at 5 min, with the peak of activation occurring at 15 min, and a return to the basal level within 60 min. The activation of ERK1/2 appeared to be dose-dependent with a significant activation being observed at 10 nM apelin-13 and maximal activation at 100 nM. However, phosphorylated-p38 MAPK was not detected in HEK293-apelinR cells treated with apelin-13. We also shown that the apelin-13-induced ERK1/2 activation requires a coupling with pertussis toxin-sensitive G-protein, and that overexpression of dominant-negative G_i2 completely inhibits the apelin-13-induced ERK1/2 activation. In addition, treatment with apelin-13 resulted in a concentration-dependent reduction of forskolin-stimulated cAMP production. It is therefore suggested that apelin-13 activates ERK1/2 but not p38 MAPK, which involves the coupling of APJ to the G_i2 cascade. In conclusion, the ERK1/ 2, but not p38 MAPKpathway is activated by apelin-13 through coupling of human APJ to G_i2-protein, which contributes to cellular responses.     

渗透胁迫对杜氏盐藻胞内甘油含量及相关酶活性影响

杜氏盐藻(Dunaliella salina)是一种抗渗透能力强的单细胞绿藻, 甘油在其渗透调节过程中发挥重要作用。本实验对5种不同NaCl浓度条件下, 盐藻的生长、细胞内甘油含量及甘油代谢相关酶的活性变化进行了测定。结果表明, NaCl浓度过高或过低均影响盐藻的生长; 高渗胁迫条件下甘油含量迅速增加,3-磷酸甘油磷酸酶的活性和二羟丙酮还原酶催化二羟丙酮转化为甘油的活性明显增加; 而低渗胁迫条件下的甘油含量会迅速降低, 3-磷酸甘油磷酸酶的活性丧失, 二羟丙酮还原酶催化甘油转化为二羟丙酮的活性增加。基于此实验结果, 我们对盐藻渗透胁迫条件下细胞内的甘油代谢过程与其抗渗透胁迫能力的相关性进行了探讨。     

The C terminus of p53 family proteins is a cell fate determinant

The p53 tumor suppressor is the most commonly mutated gene in human cancers. The ability of p53 to induce cell cycle arrest, apoptosis, DNA repair, and other p53-dependent activities is well known; however, the mechanism by which p53 induces a specific activity over another is unclear. Here, we showed that stringent regulation of and by p53 family isoforms facilitates differential target gene expression and thus determines cell fate. Through the use of engineered deletion mutants, we found that activation domain 2 is required for induction of the proapoptotic target gene insulin-like growth factor binding protein 3 (IGFBP3) by p53 and that the basic domain inhibits induction of this gene by p53. Thus, for the first time we provide evidence that the basic domain of p53 is inhibitory in vivo as has been determined in vitro. We also showed that the in vivo inhibitory activity of the basic domain depends upon activation domain 1, such that combined deletion of activation domain 1 and the basic domain was required to alleviate the inhibition by the basic domain. Importantly, deletion of the inhibitory functional domains, namely N-terminal activation domain 1 and the C-terminal basic domain, is paralleled in nature. We found that the IGFBP3 promoter was activated by p53(DeltaNDeltaBD), which mimics a naturally occurring N- and C-terminally truncated human p53 isoform, and by p53AS, a C-terminally truncated murine p53 isoform generated through alternative splicing, but not by full-length human or murine p53. In addition, we found that the C termini of p63 and p73 inhibit the induction of IGFBP3, such that C-terminally truncated p63 and p73 isoforms induce the expression of IGFBP3, whereas full-length ones cannot. We also demonstrated that IGFBP3 is an important effector of the apoptosis induced by N- and C-terminally truncated p53, such that knockdown of IGFBP3 by using an IGFBP3 neutralizing antibody or IGFBP3 small interfering RNA partially rescues the cell death induced by N- and C-terminally truncated p53. In addition, we identified that histone deacetylase activity, not p53 DNA binding ability, governs the regulation of IGFBP3 by full-length p53 family proteins, as inhibition of histone deacetylases restores the induction of IGFBP3 by exogenous full-length p53, p63, and p73 proteins. Furthermore, we found that activation of p53 or inhibition of histone deacetylases alone was not sufficient to induce IGFBP3; however, combined treatment endowed endogenous p53 with this activity. To better understand the significance of this regulation, we performed a microarray study and identified several target genes differentially regulated by full-length p53 and p53 lacking the N-terminal activation domain 1 and the C-terminal basic domain. Taken together, our data suggest a novel mechanism by which p53 family proteins differentially regulate gene expression and provide an insight for designing a combined therapy for cancer treatment.     

Rapamycin attenuates BAFF‐extended proliferation and survival via disruption of mTORC1/2 signaling in normal and neoplastic B‐lymphoid cells

B cell activating factor from the TNF family (BAFF) stimulates B‐cell proliferation and survival, but excessive BAFF promotes the development of aggressive B cells leading to malignant and autoimmune diseases. Recently, we have reported that rapamycin, a macrocyclic lactone, attenuates human soluble BAFF (hsBAFF)‐stimulated B‐cell proliferation/survival by suppressing mTOR‐mediated PP2A‐Erk1/2 signaling pathway. Here, we show that the inhibitory effect of rapamycin on hsBAFF‐promoted B cell proliferation/survival is also related to blocking hsBAFF‐stimulated phosphorylation of Akt, S6K1, and 4E‐BP1, as well as expression of survivin in normal and B‐lymphoid (Raji and Daudi) cells. It appeared that both mTORC1 and mTORC2 were involved in the inhibitory activity of rapamycin, as silencing raptor or rictor enhanced rapamycin's suppression of hsBAFF‐induced survivin expression and proliferation/viability in B cells. Also, PP242, an mTORC1/2 kinase inhibitor, repressed survivin expression, and cell proliferation/viability more potently than rapamycin (mTORC1 inhibitor) in B cells in response to hsBAFF. Of interest, ectopic expression of constitutively active Akt (myr‐Akt) or constitutively active S6K1 (S6K1‐ca), or downregulation of 4E‐BP1 conferred resistance to rapamycin's attenuation of hsBAFF‐induced survivin expression and B‐cell proliferation/viability, whereas overexpression of dominant negative Akt (dn‐Akt) or constitutively hypophosphorylated 4E‐BP1 (4EBP1‐5A), or downregulation of S6K1, or co‐treatment with Akt inhibitor potentiated the inhibitory effects of rapamycin. The findings indicate that rapamycin attenuates excessive hsBAFF‐induced cell proliferation/survival via blocking mTORC1/2 signaling in normal and neoplastic B‐lymphoid cells. Our data underscore that rapamycin may be a potential agent for preventing excessive BAFF‐evoked aggressive B‐cell malignancies and autoimmune diseases.     

外源基因表达造成的群体感应细菌生存压力的建模分析

外源基因的表达及其对细菌种群的影响对于群体感应系统和合成生物学产业的研究具有重要意义。然而,人们对于表达外源蛋白的细菌本身的行为模式仍然知之甚少。为了研究菌落生长和外源基因表达的过程究竟受到哪些因素的影响,文中测量了受Lux类受体调控的外源基因在N-酰基高丝氨酸内酯 (N-acyl homoserine lactone,N-AHL) 信号分子诱导下的表达,并模拟了其对细菌种群动态的影响。文中建立了一个假设性的数学模型,对信号分子诱导表达下细菌种群生长受影响的现象进行了分析。先前的研究通常将细菌种群生长受群体感应系统影响的现象归咎于合成群体感应信号分子的消耗与N-AHL信号分子的毒性,文中提供了对于这种生存压力的另一种可能的解释。     

应激颗粒的形成与生物学意义

真核细胞对外界压力刺激会做出一系列应答反应,如暂停蛋白质翻译系统,从而使细胞能更好地适应环境压力。通过应激颗粒（stress granules,SG）的形成包裹未被翻译的mRNA是该适应性调节的重要方式。研究表明,环境压力导致eIF2α上游激酶的激活从而磷酸化eIF2α,翻译起始受阻,随后,TIA-1、TTP等蛋白迅速与mRNP结合聚集成SG,并在微管蛋白的帮助下进一步向细胞核聚集,形成成熟的SG。当压力消失,SG依赖微管及其动力蛋白进行解聚,释放包裹的mRNA及蛋白。细胞内成熟的SG在转录后调节中发挥重要作用,并且通过其组成蛋白在肿瘤凋亡、病毒侵染、免疫、炎症反应及由蛋白错误折叠引起的疾病中发挥作用。该文首次综述了压力颗粒研究进展,为充分认识SG的病理生理性调节功能提供参考。     

黑线仓鼠CRH基因的编码序列及系统进化分析

为探究黑线仓鼠(Cricetulus barabensis)繁殖功能与促肾上腺皮质激素释放激素(CRH)基因结构的关系,参考近缘种的cDNA序列设计引物,采用RT-PCR法克隆得到了黑线仓鼠CRH基因的部分序列,克隆得到长度为1 112bp,为外显子1和外显子2的部分序列,包括全部编码区序列564 bp;编码区共编码187个氨基酸,GenBank登录号为JQ416143.利用编码区序列构建系统进化树,结果显示,黑线仓鼠与大鼠、小鼠亲缘关系最近,与其他哺乳动物亲缘关系较近,而与原鸡亲缘关系最远,此结果与物种的进化关系相一致.同时对其编码的蛋白质进行一级结构分析及二级、三级结构预测,得到了CRH的信号肽序列和41个氨基酸组成.本研究首次报道了黑线仓鼠的CRH基因序列,为进一步探究CRH基因奠定基础,对系统分析CRH的功能具有重要的参考价值,同时该cDNA序列可作为物种亲缘关系或遗传距离研究的理想标记.