PDZ-binding motifs are unable to ensure correct polarized protein distribution in the absence of additional localization signals

The C-terminal PDZ-binding motifs are required for polarized apical/basolateral localization of many membrane proteins. To determine the specificity of the PDZ-binding motifs in establishing cellular distribution, we utilized a 111-amino acid region from the C-terminus of cystic fibrosis transmembrane conductance regulator (CFTR) that is able to direct apical localization of fused reporter proteins. Substitution of the C-terminal PDZ-binding motif of CFTR with corresponding motifs necessary for basolateral localization of other membrane proteins did not lead to the redistribution of the fusion protein to the basolateral membrane. Instead, some fusion proteins remained localized to the apical membrane, whereas others showed no specific distribution. The specificity of the PDZ-based interactions was substantially increased when specific amino acids located upstream of the classical PDZ-binding motifs were included. However, even the presence of a longer C-terminal motif from a basolateral protein could not ensure basolateral distribution of the fusion protein. Our results indicate that the C-terminal PDZ-binding motifs are not the primary signals for polarized protein distribution, although they are required for targeting and/or stabilization of protein at the given location.     

Phosphorylation of glucosamine-6-phosphate synthase is important but not essential for germination and mycelial growth of Candida albicans

A site-directed mutagenesis of the GFA1 gene encoding Candida albicans glucosamine-6-phosphate (GlcN-6-P) synthase afforded its GFA1S208A version. A product of the modified gene, lacking the putative phosphorylation site for protein kinase A (PKA), exhibited all the basic properties identical to those of the wild-type enzyme but was no longer a substrate for PKA. Comparison of the C. albicans Deltagfa1/GFA1 and Deltagfa1/GFA1S208A cells, grown under conditions stimulating yeast-to-mycelia transformation, revealed that the latter demonstrated lower GlcN-6-P synthase specific activity, decreased chitin content and formed much fewer mycelial forms. All these findings, as well as the observed effects of specific inhibitors of protein kinases, suggest that a loss of the possibility of GlcN-6-P synthase phosphorylation by PKA strongly reduces but not completely eliminates the germinative response of C. albicans cells.     

水分胁迫对牛心朴子叶片光合色素及叶绿素荧光的影响

研究了水分胁迫对牛心朴子叶片光合色素及叶绿素荧光动力学参数的影响。结果表明,在长期的水分胁迫中,牛心朴子叶片的叶绿素a(Chl a)、叶绿素b(Chl b)和类胡萝卜素(Car)含量没有下降或下降不明显。直到处理末期才显著下降;叶片叶绿素荧光动力学参数Fo、Fm、Fv、Fv／Fm变化不大,在处理末期各处理Fo降低,轻度、重度水分胁迫的Fm、Fv、Fv／Fm升高。说明K期水分胁迫后牛心朴子的光合功能受到影响,但牛心朴子仍表现出较强的适应干旱的能力。     

三江平原草甸白浆土种稻后土壤理化性质变化

土壤质量变化与更新是农业发展和土壤管理的判断准则.白浆土是三江平原主要水田土壤,但旱田改水田后缺乏对其土壤质量变化规律的研究.本研究以不同种稻年限白浆土为调查对象,探讨其土壤理化性质演变特征.结果表明: 白浆土种稻后,耕层(厚16~23 cm)和犁底层(厚6~8 cm)土壤有机碳、还原物质总量增加,耕层深度随种稻年限增加呈逐渐增加趋势,犁底层无明显变化,心土层(厚20 cm)与旱田无显著差异;土壤中Fe²⁺和Mn²⁺有向下迁移现象,但只迁移到犁底层;耕层和犁底层土壤固相比率比种稻前增加,犁底层固相比率由47.8%增加到70.0%,容重由1.22 g·cm^-3增加到1.77 g·cm^-3,土壤孔隙总量降低,微孔隙比例增加,白浆土种稻后有黏粒淋溶淀积现象.白浆土种稻后,土壤物理和化学性质的变化特征与水稻土的演变规律既有一致性,又有其特殊性.     

毛状副冠对高山植物喉毛花的适应意义

高山地区生境极端,却拥有许多形态特化的植物,非生物因素在塑造花部性状及其进化过程中发挥着重要作用。该研究选取龙胆科典型高山植物喉毛花(Comastoma pulmonarium)为对象,探究其毛状副冠在多雨、强辐射的极端高山环境中的适应意义及其对植物雌雄繁殖适合度的影响。结果表明:通过比较自然状态和人工去除副冠的花,毛状副冠有效减少了雨水对花粉的冲刷(t=2.61,P0.05),提高了受精比率(t=2.05,P0.05),但是对种子的质量,即种子重量和种子萌发率的影响不显著。另外,花粉浸泡在蒸馏水中后,其萌发率显著低于蔗糖溶液中(t=30.67,P0.001),表明毛状副冠能够有效减小雨水浸泡对花粉活力的影响;同时,与自然状态相比,去除毛状副冠后的花,经太阳暴晒后,其花粉萌发率同样极显著地降低(t=9.89,P0.001),表明毛状副冠有效地避免了太阳辐射对花粉质量产生的不利影响。该研究结果表明喉毛花的毛状副冠结构是应对高山恶劣环境的一种适应策略,对植株的雌性和雄性繁殖成功都具有重要意义,从而进一步证实了非生物因素在植物花部特征演化中的重要作用。     

EFFICIENT PRODUCTION OF Staphylococcus simulans LYSOSTAPHIN IN A BENCHTOP BIOREACTOR BY RECOMBINANT Escherichia coli

Lysostaphin is an enzyme with bactericidal activity against Staphylococcus aureus and other staphylococcal species. In spite of many advantages and promising results of preliminary research, the enzyme is still not widely used in medicine, veterinary medicine, or as a food preservative. One of the most important factors limiting application of the enzyme in clinical or technological practice is the high cost of its production. In this study we have determined the optimal conditions for lysostaphin production in a 5-L batch bioreactor. The enzyme production was based on a heterologous, Escherichia coli expression system designated as pBAD2Lys and constructed earlier in our laboratory. An evident influence of physicochemical conditions of the process (areation, pH and temperature) and composition of the growing media on the amount and activity of produced enzyme was noticed. Efficiency of production of about 13,000 U/L has been achieved in the optimal conditions of the production process: low aeration (400 rpm of mechanical stirrer), pH 6, and temperature 37°C in classical LB medium. Further, about twofold improvement in the production efficiency of the enzyme was achieved as a result of modification of composition of growing media. Finally, more than 80,000 units of lysostaphin were obtained from one (batch) bioreactor with 3 L of culture of E. coli TOP10F’ transformed with pBAD2Lys plasmid. To the best of our knowledge, this is the most efficient method of production of recombinant lysostaphin in E. coli expression systems described to date.     

转mapk双链RNA干扰表达载体黄瓜对根际土壤细菌多样性的影响

随着RNA干扰技术的发展,通过植物表达病原物特异的dsRNA来防治植物病害的转基因作物越来越多.转根结线虫mapk双链RNA表达载体的黄瓜能够通过RNA干扰作用沉默线虫的mapk基因,对根结线虫具有良好的防治效果.为了评价该转基因黄瓜的安全性,明确mapk双链RNA干扰表达载体转基因黄瓜植株对根际土壤细菌多样性的影响,采用16S rRNA基因克隆文库方法对非转基因黄瓜和转基因黄瓜土壤细菌群落多样性进行分析.结果表明,非转基因黄瓜土壤细菌文库包含124个OTU(可操作分类单元),转基因黄瓜土壤细菌文库包含122个OTU.2个文库共同拥有的OTU为115个.2个文库都包含1 3个类群细菌,Acidobacteria、Actinobacteria、Armatimonadetes、Bacteroidetes、candidate division BRC1、Chloroflexi、Firmicutes、Gemmatimonadetes、Nitrospira、Planctomycetes、Proteobacteria、Verrucomicrobia、unclassified_Bacteria. 其中 Proteobacteria、Bacteroidetes 、Chloroflexi和Acidobacteria是优势菌群.其他细菌类群数量相对较少.在纲分类水平上,两个文库包含的细菌类群一致,且各类群细菌比例差异不大.在Acidobacteria门中,Acidobacteria_Gp6为优势菌群.在Bacteroidetes门中,Sphingobacteria纲细菌数量最多.在Chloroflexi门中,unclassified Chloroflexi细菌最多.在Proteobacteria门中,其中Betaproteobacteria纲的细菌数量最多.从多样性指数角度分析,两种土壤细菌群落的Shannon、Simpson和Chao值差异不大.总体来看,两种土壤细菌类群差异不显著,转基因黄瓜未对根际土壤细菌群落产生明显影响.     

小陇山国家级自然保护区华山松种群更新动态分析

以种群生命表及生存分析理论为基础,将林木依胸径大小分级,以林木径级结构代替年龄结构,采用分段匀滑技术编制小陇山国家级自然保护区华山松种群静态生命表,对其种群年龄结构、死亡率曲线、损失度曲线、存活曲线、生存函数曲线及其幼苗的更新动态、死亡率和原因进行分析。结果表明:该种群属于增长型,表现出前期增长,中期稳定,后期衰退的特点;该种群的整个生命过程,显示了三次死亡高峰,其中幼苗死亡率较高,以后的死亡率低而平缓,符合种群的自然动态规律,也指出"天保工程"实施的意义和效果;该研究指示了华山松的动态以及种群的变化趋势,对保护区已经实施的生态监测和森林保护有着十分重要的意义。     

过表达miR-191抑制猪ADSCs向脂肪细胞分化

在脂肪组织发育过程中存在许多差异表达的microRNAs（miRNAs）,而 miR-191 是从实验室前期Solexa测序结果中筛选出的差异表达miRNAs之一.本研究对不同物种miR-191的成熟及前体序列进行同源性分析,并检测其在猪不同发育阶段各组织中的表达情况,发现miR-191的成熟及前体序列在不同物种间都非常保守,通过real-time qPCR检测发现,miR=191在猪脂肪组织不同发育阶段呈高丰度差异表达,其与Solexa测序结果一致.为了更好地研究miR-191在脂肪细胞分化过程中的功能,通过基因克隆的方法,利用Ad-Easy体系,构建过表达miR 191的腺病毒载体,转染 293A细胞,成功包装出重组 miR-191腺病毒并能高效侵染猪脂肪基质细胞（adipose derived stromal cells, ADSCs）.通过real time qPCR检测表明,感染重组 miR-191 腺病毒后的ADSCs能大量表达miR-191|油红O染色可以观察到,过表达miR=191的猪ADSCs在诱导分化后与对照组相比,脂滴明显减少. 进一步研究发现,miR-191过表达的猪ADSCs中,SREBP-1C（sterol regulatory element binding protein=1C）和LPL（lipoprotein lipase）的mRNA水平显著降低,脂解关键基因HSL（hormone sensitive lipase）和ATGL（adipose triglyceride lipase）mRNA水平显著升高. 同时,Western印迹结果显示,过表达miR-191的猪ADSCs在诱导分化过程中,PPARγ（peroxisome proliferator-activated receptor γ）、C/EBPα（CCAAT/enhancer binding protein α）和A-FABP（adipocyte fatty acid binding protein）的蛋白表达水平显著下调. 实验表明,过表达miR-191抑制了猪ADSCs的分化过程. 这为深入研究miR-191在猪ADSCs分化过程中的调控机制奠定基础.     

黄芪甲苷Ⅳ通过影响β-actin对内皮型一氧化氮合酶的调节作用

目的：研究黄芪甲苷Ⅳ（AS-Ⅳ）对体外培养脐静脉内皮细胞中内皮型一氧化氮合酶（eNOS）的调节作用及可能的机制。方法：培养人脐静脉内皮细胞系EA-Hy926,用AS-Ⅳ进行干预,同时给予或不给予骨架蛋白β—actin聚合稳定剂phalloidin,用免疫共沉淀方法检测eNOS与单体8-actin结合状态的变化,用L-3H．精氨酸转化为L-SH-瓜氨酸的同位素法测定eNOS活性,I^125环-磷酸鸟苷（cGMP）放射免疫法检测细胞内cGMP水平,Westernblotting方法检测细胞中eNOS和蛋白激酶B（Akt）磷酸化水平,总蛋白水平。结果：（1）AS-IV作用10min后,细胞内单体β—actin与eNOS的结合明显增加（P〈0．05或P〈0．01）,预先给予phalloidin显著抑制了AS．IV引起的两者结合的增加（P〈0．01）。②AS—IV明显增加了eNOS活性（P〈0．05）、cGMP含量（P〈0．01）、eNOSSer-1177磷酸化水平（P〈0．01）、AktSer-473磷酸化水平（P〈0．001）,预先给予phalloidin明显降低了AS—IV引起的eNOS活性（P〈0．05）、cGMP含量（P〈0．01）和磷酸化水平的增加（P〈0．01）,但对Akt的磷酸化没有影响。结论：单体β—actin与eNOS的结合在AS-IV激活eNOS的过程中起着不可或缺作用,其主要是通过促进Akt对eNOSSer—1177的磷酸化来实现的。