基于RNA-seq技术对乌头属铁棒锤中自交不亲和S基因的挖掘与分析

以毛茛科乌头属铁棒锤(Aconitum pendulum N.Busch)2个品系‘蓝花铁棒锤’(‘WSYB1’)和‘黄花铁棒锤’(‘WSYY1’)为材料,对其进行转录组测序(RNA-seq),采用生物信息学方法鉴定其中可能存在的花柱S基因(self-incompatibility gene)和花粉S基因,并对它们的序列特征进行分析。结果显示,转录组中共鉴定出2个在雌蕊中特异或高表达的花柱S基因(ApSRNase)和2个在雄蕊中特异表达的花粉S基因(ApSLF)。与耧斗菜(Aquilegia coerulea James)相似,铁棒锤中也存在S-RNase(S locus ribonucleases)和SLF(S locus F-box)控制的S-RNase类的自交不亲和系统,而不存在sS(stigma S-determinant)和pS(pollen S-determinant)控制的罂粟科类型的自交不亲和系统。     

模拟氮沉降对长白山苔原灌草混合群落中植物光合特性的影响

由于草本植物持续上侵长白山灌木苔原,形成了强烈的灌草群落种间竞争。本研究以牛皮杜鹃-小叶章群落（Comm.Rhododendron aureum-Deyeuxia purpurea）为对象,根据小叶章的入侵程度设置4种盖度差异显著的样方（无、轻度、中度、重度入侵）,并设3个施氮水平（自然状态、添加11.8 kgN·hm^-2·a^-1及添加23.6 kgN·hm^-2·a^-1）,进行原位氮沉降模拟实验,监测灌木牛皮杜鹃和草本植物小叶章光合特性的差异和变化趋势,研究小叶章入侵苔原带的内在生理机制。结果显示：（1）小叶章净光合速率大于牛皮杜鹃,小叶章盖度越高、其叶绿素含量越高,而牛皮杜鹃叶绿素含量降低,随着小叶章入侵程度的增加,其净光合速率增强;（2）施氮可以提高牛皮杜鹃和小叶章的叶绿素含量和净光合速率,促进植物生长,但小叶章的增幅更大,从而增强了小叶章的竞争优势;（3）施氮和小叶章入侵具有复合作用,小叶章盖度越大,对其施氮导致小叶章净光合速率与叶绿素含量的增幅越大,而牛皮杜鹃的增幅减小。所以小叶章的成功入侵可能与其具有较高的净光合速率有关,并且施氮有利于提高小叶章的净光合速率,随着氮沉降的继续增加,更有利于小叶章的生长并提高其竞争力。     

不同采收期罗布麻和白麻药材化学成分的差异分析

建立高效液相色谱法,分别测定不同采收期的新疆产罗布麻和白麻药材中绿原酸、金丝桃苷、异槲皮苷、三叶豆苷、紫云英苷、芦丁和白麻苷的含量,进而分析2种药材的最佳采收期。发现不同采收期的新疆产罗布麻和白麻药材中绿原酸、金丝桃苷、异槲皮苷、三叶豆苷、紫云英苷、芦丁和白麻苷的含量存在一定差异,并且罗布麻和白麻呈现出不同特点。以《中国药典》中罗布麻含量测定项下的金丝桃苷为指标,罗布麻和白麻的最佳采收期都是6、7月;但是,以7个成分的总含量和6个黄酮的总含量为指标,罗布麻的最佳采收期是8、9月,白麻的最佳采收期仍是6、7月。该结果可为确定罗布麻和白麻药材的采收时间提供一定参考。     

Glycosylation-independent ERAD pathway serves as a backup system under ER stress

During endoplasmic reticulum (ER)–associated degradation (ERAD), terminally misfolded proteins are retrotranslocated from the ER to the cytosol and degraded by the ubiquitin-proteasome system. Misfolded glycoproteins are recognized by calnexin and transferred to EDEM1, followed by the ER disulfide reductase ERdj5 and the BiP complex. The mechanisms involved in ERAD of nonglycoproteins, however, are poorly understood. Here we show that nonglycoprotein substrates are captured by BiP and then transferred to ERdj5 without going through the calnexin/EDEM1 pathway; after cleavage of disulfide bonds by ERdj5, the nonglycoproteins are transferred to the ERAD scaffold protein SEL1L by the aid of BiP for dislocation into the cytosol. When glucose trimming of the N-glycan groups of the substrates is inhibited, glycoproteins are also targeted to the nonglycoprotein ERAD pathway. These results indicate that two distinct pathways for ERAD of glycoproteins and nonglycoproteins exist in mammalian cells, and these pathways are interchangeable under ER stress conditions.     

Insulin responsiveness of glucose transporter 4 in 3T3-L1 cells depends on the presence of sortilin

Insulin-dependent translocation of glucose transporter 4 (Glut4) to the plasma membrane of fat and skeletal muscle cells plays the key role in postprandial clearance of blood glucose. Glut4 represents the major cell-specific component of the insulin-responsive vesicles (IRVs). It is not clear, however, whether the presence of Glut4 in the IRVs is essential for their ability to respond to insulin stimulation. We prepared two lines of 3T3-L1 cells with low and high expression of myc₇-Glut4 and studied its translocation to the plasma membrane upon insulin stimulation, using fluorescence-assisted cell sorting and cell surface biotinylation. In undifferentiated 3T3-L1 preadipocytes, translocation of myc₇-Glut4 was low regardless of its expression levels. Coexpression of sortilin increased targeting of myc₇-Glut4 to the IRVs, and its insulin responsiveness rose to the maximal levels observed in fully differentiated adipocytes. Sortilin ectopically expressed in undifferentiated cells was translocated to the plasma membrane regardless of the presence or absence of myc₇-Glut4. AS160/TBC1D4 is expressed at low levels in preadipocytes but is induced in differentiation and provides an additional mechanism for the intracellular retention and insulin-stimulated release of Glut4.Adipocytes, skeletal muscle cells, and some neurons respond to insulin stimulation by translocating intracellular glucose transporter 4 (Glut4) to the plasma membrane. In all these cells, the insulin-responsive pool of Glut4 is localized in small membrane vesicles, the insulin-responsive vesicles (IRVs; Kandror and Pilch, 2011 ; Bogan, 2012 ). The protein composition of these vesicles has been largely characterized (Kandror and Pilch, 2011 ; Bogan, 2012 ). The IRVs consist predominantly of Glut4, insulin-responsive aminopeptidase (IRAP), sortilin, low-density-lipoprotein receptor–related protein 1 (LRP1), SCAMPs, and VAMP2. Glut4, IRAP, and sortilin physically interact with each other, which might be important for the biogenesis of the IRVs (Shi and Kandror, 2007 ; Shi et al., 2008 ). In addition, the IRVs compartmentalize recycling receptors, such as the transferrin receptor and the IGF2/mannose 6-phosphate receptor, although it is not clear whether these receptors represent obligatory vesicular components or their presence in the IRVs is explained by mass action (Pilch, 2008 ), inefficient sorting, or other reasons.Deciphering of the protein composition of the IRVs is important because it is likely to explain their unique functional property: translocation to the plasma membrane in response to insulin stimulation. Even if we presume that IRV trafficking is controlled by loosely associated peripheral membrane proteins, the latter should still somehow recognize the core vesicular components that create the “biochemical individuality” of this compartment. In spite of our knowledge of the IRV protein composition, however, the identity of the protein(s) that confer insulin sensitivity to these vesicles is unknown.Insulin responsiveness of the IRVs was associated with either IRAP or Glut4. Thus it was shown that Glut4 interacted with the intracellular anchor TUG (Bogan et al., 2003 , 2012 ), whereas IRAP associated with other proteins implemented in the regulation of Glut4 translocation, such as AS160 (Larance et al., 2005 ; Peck et al., 2006 ), p115 (Hosaka et al., 2005 ), tankyrase (Yeh et al., 2007 ), and several others (reviewed in Bogan, 2012 ). Results of these studies, or at least their interpretations, are not necessarily consistent with each other, as the existence of multiple independent anchors for the IRVs is, although possible, unlikely.Ablation of the individual IRV proteins has also led to controversial data. Thus knockout of IRAP decreases total protein levels of Glut4 but does not affect its translocation in the mouse model (Keller et al., 2002 ). On the contrary, knockdown of IRAP in 3T3-L1 adipocytes has a strong inhibitory effect on translocation of Glut4 (Yeh et al., 2007 ). In yet another study, knockdown of IRAP in 3T3-L1 adipocytes did not affect insulin-stimulated translocation of Glut4 but increased its plasma membrane content under basal conditions (Jordens et al., 2010 ). By the same token, total or partial ablation of Glut4 had various effects on expression levels, intracellular localization, and translocation of IRAP (Jiang et al., 2001 ; Abel et al., 2004 ; Carvalho et al., 2004 ; Gross et al., 2004 ; Yeh et al., 2007 ). Knockdown of either sortilin or LRP1 decreased protein levels of Glut4 (Shi and Kandror, 2005 ; Jedrychowski et al., 2010 ).One model that might explain these complicated and somewhat inconsistent results is that depletion of either major integral protein of the IRVs disrupts the network of interactions between vesicular proteins and thus decreases the efficiency of protein sorting into the IRVs (Kandror and Pilch, 2011 ). Correspondingly, the remaining IRV components that cannot be faithfully compartmentalized in the vesicles are either degraded (Jiang et al., 2001 ; Keller et al., 2002 ; Abel et al., 2004 ; Carvalho et al., 2004 ; Shi and Kandror, 2005 ; Yeh et al., 2007 ; Jedrychowski et al., 2010 ) or mistargeted (Jiang et al., 2001 ; Jordens et al., 2010 ), depending on experimental conditions and types of cells used in these studies. In other words, knockdown of any major IRV component may decrease vesicle formation along with insulin responsiveness. Thus, in spite of a large body of literature, the identity of protein(s) that confer insulin responsiveness to the IRVs is unknown.Here we used a gain-of-function approach to address this question. Specifically, we attempted to “build” functional IRVs in undifferentiated 3T3-L1 preadipocytes by forced expression of the relevant proteins. Undifferentiated preadipocytes do not express Glut4 or sortilin and lack IRVs (ElJack et al., 1999 ; Shi and Kandror, 2005 ; Shi et al., 2008 ). Correspondingly, IRAP, which is expressed in these cells, shows low insulin response (Ross et al., 1998 ; Shi et al., 2008 ). We found that ectopic expression of increasing amounts of Glut4 in undifferentiated preadipocytes does not lead to its marked translocation to the plasma membrane upon insulin stimulation. On the contrary, sortilin expressed in undifferentiated preadipocytes was localized in the IRVs and was translocated to the plasma membrane in response to insulin stimulation. Moreover, upon coexpression with Glut4, sortilin dramatically increased its insulin responsiveness to the levels observed in fully differentiated adipocytes. Thus sortilin may represent the key component of the IRVs, which is responsible not only for the formation of vesicles (Shi and Kandror, 2005 ; Ariga et al., 2008 ; Hatakeyama and Kanzaki, 2011 ), but also for their insulin responsiveness. It is worth noting that sortilin levels are significantly decreased in obese and diabetic humans and mice (Kaddai et al., 2009 ). We thus suggest that sortilin may be a novel and important target in the fight against insulin resistance and diabetes.Our experiments also demonstrate that undifferentiated preadipocytes lack a mechanism for the full intracellular retention of Glut4 that can be achieved by ectopic expression of AS160/TBC1D4.     

WDNM1 is Associated with Differentiation and Apoptosis of Mammary Epithelial Cells

In this study, we show that expression of the Westmead DMBA8 nonmetastatic cDNA 1 (WDNM1) gene was increased upon SFM and/or TNFα treatment, with a corresponding increase in apoptotic cells, and gradually decreased following re-stimulation with serum in HC11 mammary epithelial cells. TNFα induced WDNM1 expression showed the NFκB-dependent mechanism since it's expression was abrogated in IκBαM (super-repressor of NFκB)-transfected cells, but not those transfected with control vector. Furthermore, overexpression of WDNM1 suppressed growth and differentiation, and accelerated apoptosis of HC11 cells. Thus, our results demonstrate that WDNM1 gene expression, regulated by the TNFα-NFκB signal pathway, is associated with HC11 cell apoptosis.     

Interacting effects of water temperature and dietary protein level on hematological parameters in Nile tilapia juveniles,Oreochromis niloticus (L.) and mortality under Streptococcus iniae infection

Based on central composite rotatable experimental design and response surface method, the interacting effects of temperature (20 °C–34 °C) and dietary protein level (25%–50%) on hematological parameters including red blood cell (RBC), white blood cell (WBC) and hemoglobin (Hb) of juvenile Oreochromis niloticus were studied under laboratory conditions. The experiment lasted for 7 weeks. After the feeding trial, fish were challenged with Streptococcus iniae and mortality was recorded for within 8 days. Results showed that the linear and quadratic effects of temperature on RBC, WBC and Hb were highly significant (P < 0.01). When the dietary protein level was 25%–50%, the RBC, WBC and Hb were increased firstly and then decreased, but the linear and quadratic effects of protein level were insignificant (P > 0.05). The interacting effects of temperature and protein level on RBC and Hb were significant (P < 0.05). The regression equations of RBC, WBC and Hb toward the two factors of interest were established, with the coefficients of determination being 0.870, 0.836 and 0.881, respectively (P < 0.01). These equations could be used for prediction in practice. After the challenge, the mortalities for the combinations of 22.1 °C/28.7% and 20.0 °C/37.5% were significantly higher than 27.0 °C/37.5% (P < 0.05). The optimal temperature/dietary protein level combination was obtained at 27.9 °C/38.1% at which the lowest mortality (13.76%) was attained. This value was close to the optimal temperature/dietary protein level combination (29.4 °C/41.9%) for the greatest levels of RBC (2.560 × 10⁶ μL^?1), WBC (270.648 × 10³ μL^?1) and Hb (92.851 g L^?1). The results of this study indicated that preferred temperature/dietary protein level combination might strengthen the non-specific immunity and reduce susceptibility to S. iniae.     

Expression characterization and activity analysis of a cathepsin B from Pacific abalone Haliotis discus hannai

Cathepsin B (EC 3.4.22.1) is a member of lysosomal cysteine protease and has a papain-like fold. In mammals, it is involved in protein degradation and other physiological processes including immune response. However, little is known about the function of cathepsin B in mollusks. In this study, we identified and analyzed a cathepsin B homolog (HdCatB) from Pacific abalone (Haliotis discus hannai), an economically important mollusk species cultured in East Asia. HdCatB is composed of 336 amino acid residues and its mature form is predicted to start at residue 86. HdCatB possesses typical domain architecture of cathepsin B and contains a propeptide region and a cysteine protease domain, the latter containing the four active site residues (Q108, C114, H282, and N302) that are conserved in many different organisms. HdCatB shares 40–60% overall sequence identities with the cathepsin Bofa number of vertebrates and invertebrates and is phylogenetically very close to mollusk cathepsin B. Quantitative real time RT-PCR analysis revealed that HdCatB expression occurred in multiple tissues and was upregulated by bacterial infection. Recombinant HdCatB purified from Escherichia coli exhibited apparent protease activity, which was optimal at 45 °C and pH 6.0. These results indicate that HdCatB is a bioactive protease that is likely to be implicated in the immune response of abalone during bacterial infection.     

Thr28突变对虎纹捕鸟蛛毒素-Ⅳ钠通道活性的影响

虎纹捕鸟蛛毒素-Ⅳ(HWTX-Ⅳ)是从虎纹捕鸟蛛粗毒中分离纯化到的一种新型多肽类神经毒素,能明显抑制表达于大鼠背根神经节细胞的河豚毒素敏感型(TTX-S)钠通道.为了更好地研究该毒素的结构与功能之间的关系,采用芴甲氧羰基(Fmoc)固相多肽化学合成法合成了用谷氨酸(Glu)替代HWTX-Ⅳ第28位苏氨酸残基的突变体T28D-HWTX-Ⅳ,线性多肽合成产物经反相高效液相色谱(HPLC)分离纯化后进行谷胱甘肽氧化复性.复性产物采用基质辅助激光解析飞行时间质谱(MALDI-TOF/TOF MS)技术鉴定分子质量,通过全细胞膜片钳电生理技术测定其电压门控钠通道药理学活性.当第28位Thr残基被Glu取代后,突变体T28D-HWTX-Ⅳ对表达于大鼠DRG细胞膜上的TTX-S钠通道的IC50值约为362 nmol/L,对TTX-S钠通道的抑制活性比天然HWTX-Ⅳ(IC50值=30 nmol/L)下降了约12倍,显示第28位的Thr残基是HWTX-Ⅳ与TTX-S型钠通道相互作用的关键活性残基.目前的研究为进一步探索HWTX-Ⅳ的结构与功能关系及新型镇痛药物的研发奠定了基础.