RTNs家族研究进展

编码内质网蛋白家族的基因(RTNs)是一类广泛存在于真核生物的基因家族,它有着特殊的拓扑结构。RTNs蛋白的羧基端存在一个大约200个氨基酸的保守区(RHD),此保守区包含2个疏水区段。RTNs蛋白定位内质网膜上,高等脊椎动物中枢神经受损时,RTN4-A／Nogo-A起抑制神经生长的作用,RTNs蛋白家族其余成员的功能还不是很清楚,可能与胞内运输,细胞分裂和细胞凋亡等有关。现就RTNs成员的基本结构、表达分布、亚细胞定位、拓扑结构和RTNs蛋白的功能等进行综述。     

肝细胞中活化转录因子ATF6抑制SREBP1的转录活性

内质网膜定位的活化转录因子ATF6和SREBP1均是经过蛋白酶切水解激活,激活后的ATF6(N)和SREBP1(N)进入细胞核内,分别指导内质网膜未折叠蛋白聚集反应相关基因和脂肪酸合成相关基因的表达.研究发现,肝细胞内葡萄糖饥饿激活ATF6并抑制SREBP1的转录活性及其靶基因的表达.过表达ATF6(N)能够抑制SREBP1介导的转录及其下游基因的表达.免疫共沉淀实验显示,ATF6(N)在细胞核内结合SREBP1(N),这种结合在无糖状况下增强.不同功能区缺失分析表明,ATF6和SREBP1通过亮氨酸拉链(leucinezipper)功能区相互作用.在葡萄糖饥饿状况下,ATF6对SREBP1转录活性的抑制保证了细胞基本生命活动所需要的能量.     

聚乳酸微柱阵列型拓扑结构基底上SH-SY5Y细胞VEGF和IL-8的分泌及表达

以紫外光光刻、硅蚀刻及复制模塑技术制备了聚乳酸 (PLLA) 微柱阵列型拓扑结构基底,考察了SH-SY5Y人神经母细胞瘤细胞在拓扑结构基底上的生长及血管内皮生长因子 (VEGF) 和白细胞介素-8 (IL-8) 的分泌及表达。细胞的形态及铺展采用扫描电子显微图像进行分析,细胞在拓扑结构基底上生长24 h后的VEGF及IL-8分泌量采用ELISA进行检测,VEGF及IL-8在mRNA水平的表达量以实时定量PCR进行评价。实验中成功制备了微柱名义直径为2 μm和4 μm、微柱名义间距为2 μm和7 μm的4种拓扑结构基底。研究发现,SH-SY5Y细胞在2-2 μm (微柱名义直径-名义间距)、4-2 μm、4-7 μm拓扑结构基底上的VEGF和/或IL-8分泌量和表达较之PLLA平面基底上相应值出现上调,而在2-7 μm拓扑结构基底上VEGF及IL-8二者均表现出表达和分泌量大幅度和明显的上调。与在PLLA平面基底上相比,SH-SY5Y细胞在拓扑结构基底上表现出细胞形态 (铺展面积及圆度) 的明显变化,细胞VEGF及IL-8分泌量和表达的上调伴随细胞铺展面积的明显降低。结果表明微柱阵列型拓扑结构是影响SH-SY5Y细胞VEGF、IL-8分泌及表达的重要微环境因素,VEGF和IL-8可能构成SH-SY5Y细胞在拓扑结构基底上生长的重要分泌物标志。     

耐热碱性磷酸酯酶的功能结构域的定位

 为了确定耐热碱性磷酸酯酶 (TAPND2 7)发挥活性所必需的功能结构域 ,通过 PCR介导的诱变缺失 ,得到了 N端分别缺失 8、1 6、2 5个氨基酸的 3个缺失体 p TAPN8、p TAPN1 6和p TAPN2 5以及 C端分别缺失 1 0和 30个氨基酸的两个缺失体 p TAPC1 0和 p TAPC30 .经表达和活性测定 ,发现 p TAPN8和 TAPC1 0保持了较高的活性而其余 3个缺失体则失去酶活性 .据此 ,TAPND2 7的活性区域被定位在 8～ 465氨基酸之间 .在分离纯化的基础上测定了一些酶学性质 .发现 TAPN8和 TAPC1 0的比活没有大的改变 ,Tm 下降了 5.5℃ ;TAPN8的最适反应温度上升了1 0℃ .结果提示了 N端和 C端的这些氨基酸残基对热稳定性有一定的贡献 ,N端氨基酸残基还对酶的亲热性有贡献 .     

戊型肝炎病毒第4基因型中国株ORF2编码蛋白的抗原表位分析

以戊型肝炎病毒(HEV)第4基因型中国株ORF2编码蛋白p166Chn制备单克隆抗体(McAbs), 同时制备20个N端或C端逐渐截短的p166Chn截短蛋白, 与7种不同基因型和亚型的p166蛋白一起, 通过ELISA、免疫印迹(Western blot)以及竞争抑制实验对主要存在于我国的HEV第4基因型毒株进行抗原表位分析。结果发现所制备的McAbs与p166Chn截短蛋白的免疫反应有两种类型, 以1G10为代表的McAbs能与N端不短于aa477、C端不短于aa613的截短蛋白反应, 其针对的抗原表位是构象依赖型表位, 依赖于aa477~aa613肽链区段; 而McAb 2F11则能与N端不短于aa474、C端不短于aa617的截短蛋白反应, 其针对的抗原表位也是构象表位, 但需依赖于较长的肽链区段(aa474~aa617)。竞争抑制实验显示两类McAbs互不抑制, 进一步证实了所发现的两个抗原表位在空间位置上的不同。更有意义的是, 两类McAbs均能与其他不同HEV基因型和亚型来源的p166重组蛋白发生阳性反应, 表明这两个抗原表位是HEV基因型共同性的, 可以在世界各国分布的不同基因型HEV毒株中诱导交叉免疫。     

NaCl胁迫对构树幼苗内质网膜脂肪酸及蛋白质组成的影响

采用气相色谱法和SDS-PAGE电泳法研究了经过50～150 mmol·L-1 NaCl胁迫处理后构树[Broussonetia papyrifera (L. ) L'Hrit. ex Vent. ]组培苗根和叶片内质网膜脂肪酸和蛋白质组成的变化.结果表明,构树根和叶片内质网膜脂肪酸组成差异较大,但均为不饱和脂肪酸相对含量较高;脂肪酸基本成分为棕榈酸、硬脂酸、棕榈油酸、油酸、亚油酸及反亚油酸,但根中还含花生酸、山萮酸、木蜡酸、亚麻酸和二十碳二烯酸,叶片中还含反油酸;根中的不饱和脂肪酸指数(IUFA)大于叶片.不同浓度NaCl胁迫对构树组培苗根和叶片内质网膜脂肪酸组成和蛋白质组成均有一定的影响.在NaCl胁迫条件下,根内质网膜饱和脂肪酸相对含量呈增加趋势,不饱和脂肪酸相对含量趋于减少,且随着NaCl浓度的提高,IUFA逐渐降低;叶片内质网膜中各脂肪酸成分相对含量的变化趋势各异,但在低浓度NaCl条件下,IUFA较对照有所提高,随NaCl浓度的升高IUFA又低于对照,且叶片内质网膜IUFA的降幅小于根.根和叶片内质网膜中蛋白质组成明显不同;不同浓度NaCl胁迫除对内质网膜各蛋白质组分表达量有一定影响外,还导致根中相对分子质量70 000 的蛋白质条带消失,叶片中则出现了相对分子质量95 000 的新蛋白质条带.     

沙眼衣原体GlgA蛋白表达和分泌的调控机制

【背景】沙眼衣原体(Chlamydia trachomatis,Ct)的分泌蛋白在Ct与宿主细胞的相互作用、感染发育周期及致病过程中发挥着至关重要的作用。GlgA蛋白是课题组前期研究发现的一种新的Ct分泌蛋白,其表达和分泌的具体机制及作用还不清楚。【目的】寻找调控CtGlgA蛋白表达和分泌的分子机制,为后续Ct致病机制研究提供实验基础和新思路。【方法】采用Signal P 4.1软件对GlgA蛋白N端进行信号肽预测分析,并用细菌分泌蛋白特异性阻断剂C16和C1化合物分别或同时处理Ct感染的He La细胞,观察阻断Ⅱ型、Ⅲ型分泌途径对GlgA蛋白分泌的影响;经新生霉素处理、噬斑筛选及穿梭质粒转染技术,构建Ct质粒缺失株和缺失互补株,并鉴定质粒编码基因在两种菌株的缺失及表达情况;间接免疫荧光法观察质粒缺失对GlgA表达和分泌的影响。【结果】GlgA蛋白N端无信号肽序列,细菌Ⅱ型、Ⅲ型分泌途径特异性阻断剂C16和C1化合物不能阻断GlgA的胞浆分泌;Ct质粒缺失株CTD1的质粒编码基因pgp7丢失,且质粒编码蛋白Pgp3及基因组编码蛋白GlgA的表达和分泌现象均消失;Ct缺失互补株CTD1-pGFP::SW2重新获得pgp7基因,并恢复Pgp3蛋白和GlgA的表达和分泌。【结论】初步证实Ct糖原合酶GlgA蛋白的表达和分泌不依赖细菌Ⅱ型和Ⅲ型分泌途径,而且与衣原体质粒密切相关。     

白念珠菌14-3-3蛋白Bmh1在细胞生长和菌丝发育中的功能解析

【目的】应用Tet-off启动子研究白念珠菌唯一的14-3-3蛋白Bmh1在白念珠菌生长和菌丝发育过程中的功能。【方法】在白念珠菌URA3+菌株SN152中,我们敲除了1个BMH1基因拷贝,并用Tet-off启动子替代另一个BMH1基因拷贝的启动子,得到了可以用强力霉素(Doxycycline)控制Bmh1表达水平的菌株。然后我们通过斑点试验和形态学观察对该菌株的生长和菌丝发育表型进行了分析。通过在ras1、flo8、efg1、cph1、tec1等重要菌丝发育调控因子突变体中过表达Bmh1,我们初步研究了Bmh1在菌丝发育调控网络中的位置。最后,我们构建了一些不同C末端的Bmh1嵌合体并检测了其对白念珠菌生长和菌丝发育的影响。【结果】Doxycycline诱导Bmh1表达水平下调时严重抑制了细胞的生长。非Doxycycline诱导条件下Bmh1高表达强烈促进了细胞的菌丝发育。这一促进作用绕过了ras1、efg1、cph1和tec1等基因缺失的影响,却被flo8基因的缺失阻断。C末端缺失或更换异源C末端的所有Bmh1突变株在Doxycycline诱导时都能够正常生长,但是没有明显促进菌丝发育。【结论】验证了白念珠菌14-3-3蛋白Bmh1是细胞生长所必需的,证明了Tet-off启动子可以严密控制Bmh1的表达水平。Bmh1是一个菌丝发育的正调控因子,位于Ras1、Efg1、Cph1和Tec1的下游,Flo8的上游。Bmh1的保守结构域是细胞生长所必需的,而C末端则是生长非必需的。     

纤溶酶原K5抗血管增生活性依赖其完整Kringle结构域

根据K5蛋白(Pro451—Ala541)的结构特征和二硫键分布特点,设计K5的两个缺失突变体K5 mut1(Cys461—Cys540,保留K5 kringle环3个完整二硫键但去除N端和C端多余氨基酸)和K5 mut2 (Cys482—Cys535,打开kringle环,只保留2个二硫键).以野生型人纤溶酶原K5 cDNA为模板,用PCR方法得到编码缺失突变体的DNA片段,定向克隆入pET22b(+)质粒载体,重组体转化进大肠杆菌BL21（DE3）,诱导表达,产物经亲和层析和高浓度甘油透析纯化后进行鉴定和生物活性测定.K5 mut1蛋白特异性抑制人视网膜微血管内皮细胞增殖,且活性强度是完整的K5蛋白2倍;K5 mut2对人视网膜微血管内皮细胞无显著抑制作用.结果提示,完整的Kringle结构（包含3个二硫键）是维持人纤溶酶原K5抗血管增生活性的必需结构域,而K5分子中Kringle结构域外的N端和C端氨基酸臂则并非其活性所必需.     

内质网膜复合亚基3调控内质网应激影响人畸胎瘤细胞的存活

内质网膜蛋白复合物(endoplasmic reticulum membrane complex,EMC)在跨膜蛋白质的生物发生和膜整合中发挥重要作用.内质网膜复合亚基3 (endoplasmic reticulum membrane complex 3,EMC3)是EMC的重要组成部分,但其在生殖细胞中发挥的作用未见...     

Keeping a low profile: small hive beetle reproduction in African honeybee colonies

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Hit‐and‐run trophallaxis of small hive beetles

Some parasites of social insects are able to exploit the exchange of food between nestmates via trophallaxis, because they are chemically disguised as nestmates. However, a few parasites succeed in trophallactic solicitation although they are attacked by workers. The underlying mechanisms are not well understood. The small hive beetle (=SHB), Aethina tumida, is such a parasite of honey bee, Apis mellifera, colonies and is able to induce trophallaxis. Here, we investigate whether SHB trophallactic solicitation is innate and affected by sex and experience. We quantified characteristics of the trophallactic solicitation in SHBs from laboratory‐reared individuals that were either bee‐naïve or had 5 days experience. The data clearly show that SHB trophallactic solicitation is innate and further suggest that it can be influenced by both experience and sex. Inexperienced SHB males begged more often than any of the other groups had longer breaks than their experienced counterparts and a longer soliciting duration than both experienced SHB males and females, suggesting that they start rather slowly and gain more from experience. Successful experienced females and males were not significantly different from each other in relation to successful trophallactic interactions, but had a significantly shorter soliciting duration compared to all other groups, except successful inexperienced females. Trophallactic solicitation success, feeding duration and begging duration were not significantly affected by either SHB sex or experience, supporting the notion that these behaviors are important for survival in host colonies. Overall, success seems to be governed by quality rather than quantity of interactions, thereby probably limiting both SHB energy investment and chance of injury (<1%). Trophallactic solicitation by SHBs is a singular example for an alternative strategy to exploit insect societies without requiring chemical disguise. Hit‐and‐run trophallaxis is an attractive test system to get an insight into trophallaxis in the social insects.     

Global analysis of canola genes targeted by SHORT HYPOCOTYL UNDER BLUE 1 during endosperm and embryo development

Seed development in dicots includes early endosperm proliferation followed by growth of the embryo to replace the endosperm. Endosperm proliferation in dicots not only provides nutrient supplies for subsequent embryo development but also enforces a space limitation, influencing final seed size. Overexpression of Arabidopsis SHORT HYPOCOTYL UNDER BLUE1::uidA (SHB1:uidA) in canola produces large seeds. We performed global analysis of the canola genes that were expressed and influenced by SHB1 during early endosperm proliferation at 8 days after pollination (DAP) and late embryo development at 13 DAP. Overexpression of SHB1 altered the expression of 973 genes at 8 DAP and 1035 genes at 13 DAP. We also surveyed the global SHB1 association sites, and merging of these sites with the RNA sequencing data identified a set of canola genes targeted by SHB1. The 8‐DAP list includes positive and negative genes that influence endosperm proliferation and are homologous to Arabidopsis MINI3, IKU2, SHB1, AGL62, FIE and AP2. We revealed a major role for SHB1 in canola endosperm development based on the dynamics of SHB1‐altered gene expression, the magnitude of SHB1 chromatin immunoprecipitation enrichment and the over‐representation of eight regulatory genes for endosperm development. Our studies focus on an important agronomic trait in a major crop for global agriculture. The datasets on stage‐specific and SHB1‐induced gene expression and genes targeted by SHB1 also provide a useful resource in the field of endosperm development and seed size engineering. Our practices in an allotetraploid species will impact similar studies in other crop species.     

Susceptibility of small honey bee colonies to invasion by the small hive beetle,Aethina tumida (Coleoptera,Nitidulidae)

Weak and small honey bee colonies are supposed to be more susceptible to infestations by the small hive beetle [Aethina tumida, small hive beetle (SHB)]. To test this, we established 24 nucleus colonies [12 with and 12 without previous SHB removal (= screening)]. Four weeks later, we compared beetle numbers and the occurrence of SHB reproduction to the corresponding full‐sized colonies. Full‐sized colonies with no screening were infested with significantly more SHBs than all other groups (mean ± standard deviation = 46.9 ± 26.7). Regardless of this, none of the full‐sized colonies showed damage or evidence of SHB reproduction. In contrast, five nucleus colonies collapsed and SHB larvae were found in an additional seven colonies. Our study demonstrates that SHB infestation levels which are harmless to full‐sized colonies may have a negative impact on small nucleus colonies.     

Global warming promotes biological invasion of a honey bee pest

Climate change and biological invasions are two major global environmental challenges. Both may interact, e.g. via altered impact and distribution of invasive alien species. Even though invasive species play a key role for compromising the health of honey bees, the impact of climate change on the severity of such species is still unknown. The small hive beetle (SHB, Aethina tumida, Murray) is a parasite of honey bee colonies. It is endemic to sub‐Saharan Africa and has established populations on all continents except Antarctica. Since SHBs pupate in soil, pupation performance is governed foremost by two abiotic factors, soil temperature and moisture, which will be affected by climate change. Here, we investigated SHB invasion risk globally under current and future climate scenarios. We modelled survival and development time during pupation (=pupal performance) in response to soil temperature and soil moisture using published and novel experimental data. Presence data on SHB distribution were used for model validation. We then linked the model with global soil data in order to classify areas (resolution: 10 arcmin; i.e. 18.6 km at the equator) as unsuitable, marginal and suitable for SHB pupation performance. Under the current climate, the results show that many areas globally yet uninvaded are actually suitable, suggesting considerable SHB invasion risk. Future scenarios of global warming project a vehement increase in climatic suitability for SHB and corresponding potential for invasion, especially in the temperate regions of the Northern hemisphere, thereby creating demand for enhanced and adapted mitigation and management. Our analysis shows, for the first time, effects of global warming on a honey bee pest and will help areas at risk to prepare adequately. In conclusion, this is a clear case for global warming promoting biological invasion of a pest species with severe potential to harm important pollinator species globally.     

The SHB adapter protein is required for normal maturation of mesoderm during in vitro differentiation of embryonic stem cells

Definitive mesoderm arises from a bipotent mesendodermal population, and to study processes controlling its development at this stage, embryonic stem (ES) cells can be employed. SHB (Src homology 2 protein in beta-cells) is an adapter protein previously found to be involved in ES cell differentiation to mesoderm. To further study the role of SHB in this context, we have established ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-). Differentiating embryoid bodies (EBs) derived from these ES cell lines were used for gene expression analysis. Alternatively, EBs were stained for the blood vessel marker CD31. For hematopoietic differentiation, EBs were differentiated in methylcellulose. SHB-/- EBs exhibited delayed down-regulation of the early mesodermal marker Brachyury. Later mesodermal markers relatively specific for the hematopoietic, vascular, and cardiac lineages were expressed at lower levels on day 6 or 8 of differentiation in EBs lacking SHB. The expression of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 was also reduced in SHB-/- EBs. SHB-/- EBs demonstrated impaired blood vessel formation after vascular endothelial growth factor stimulation. In addition, the SHB-/- ES cells formed fewer blood cell colonies than SHB+/+ ES cells. It is concluded that SHB is required for appropriate hematopoietic and vascular differentiation and that delayed down-regulation of Brachyury expression may play a role in this context.     

Hepatitis B virus MHBs antigen is selectively sensitive to glucosidase-mediated processing in the endoplasmic reticulum.

Previous studies have shown that hepatitis B virus (HBV) secretion from HepG 2.2.15 cells is prevented by inhibitors of the endoplasmic reticulum (ER) glucosidase under conditions where secretion of cellular glycoproteins are not detectably affected. The 2.2.15 cells are derived from HepG2 and contain intact dimers of the viral genome. They produce and secrete infectious HBV. The secretion of the viral envelope polypeptide, MHBs, was selectively and quantitatively reduced from 2.2.15 cells in which glucosidase was inhibited, whereas the envelope polypeptide, SHBs, was relatively insensitive, being as resistant as were most host glycoproteins. Because 2.2.15 cells express all HBV ORFs, it seemed possible that the sensitivity of MHBs secretion involved its interaction with the viral nucleocapsid or other viral gene products. The work reported here showed that MHBs secretion from HepG2 cells transfected with a plasmid that expresses only the MHBs polypeptide was as sensitive to glucosidase inhibitors as it was from 2.2.15 cells. These data show that the sensitivity of the MHBs polypeptide secretion to glucosidase inhibitors is entirely encrypted within its structural gene. The reasons the MHBs polypeptide, but not SHBs, is so sensitive to glucosidase processing are discussed.     

Bees and flowers: How to feed an invasive beetle species

Invasive species may exploit a wide range of food sources, thereby fostering their success and hampering mitigation, but the actual degree of opportunism is often unknown. The small hive beetle (SHB), Aethina tumida, is a parasite of honeybee colonies endemic to sub‐Saharan Africa. SHBs have now spread on all habitable continents and can also infest colonies of other social bees. To date, the possible role of solitary bee nests as alternative hosts is unknown. Similarly, flowers as possible alternative food sources are not well understood. Here, we show that SHBs can complete an entire life cycle in association with nests of solitary bees Megachile rotundata. The data also show that flowers can serve as alternative food sources. These results support the opportunistic nature of this invasive species, thereby generating further obstacles for mitigation efforts in the field. It also suggests that SHB invasions may result in more serious consequences for endemic bee fauna than previously thought. This provides further motivation to slow down the global spread of this pest, and to improve its management in areas, where it is established.     

Behavioral responses of the small hive beetle,Aethina tumida,to odors of three meliponine bee species and honey bees,Apis mellifera scutellata

Recent studies have shown that honey bees, bumble bees, and some meliponine bee species of the genera Trigona, Meliponula, and Dactylurina are hosts of the small hive beetle (SHB) Aethina tumidaMurray (Coleoptera: Nitidulidae), a pest of honey bee colonies in various regions of the world. Olfaction has been implicated in SHB infestations of honey bee and bumble bee colonies. We used olfactometer bioassays to investigate responses of adult male and female SHBs to odors from intact colonies and separate hive components (pot honey, pot pollen, cerumen, and propolis) of three African meliponine bee species, Meliponula ferruginea (Lepeletier) (black morphospecies), M. ferruginea (reddish brown morphospecies), and Meliponula bocandei (Spinola) (Hymenoptera: Apidae). Although both sexes of the beetle strongly preferred intact colony, pot honey, and pot pollen odors, there was no evidence of attraction to propolis and cerumen odors from the three meliponine bee species. Both sexes of SHB also strongly preferred odors from honey bees, Apis mellifera L. (Hymenoptera: Apidae), over odors from the three meliponine bee species. Our results provide substantial evidence of the host potential of African meliponine bees for the SHB, and we discuss this complex association of the SHB with species within the Apidae family.     

Hepatitis B virus large and middle glycoproteins are degraded by a proteasome pathway in glucosidase-inhibited cells but not in cells with functional glucosidase enzyme

The secretion of hepatitis B virus (HBV) large (LHBs) and middle (MHBs) envelope polypeptides from tissue cultures requires proper protein folding and is prevented by inhibitors of the endoplasmic reticulum (ER) glucosidase. Using competitive inhibitors of the ER glucosidase, here it is shown that the amounts of glycosylated and unglycosylated forms of LHBs and MHBs proteins are all greatly reduced in tissue cultures producing HBV envelope glycoproteins. In contrast, the HBV small (SHBs) protein was not affected. The reduction in secretion of LHBs and MHBs proteins appears to be mediated by proteasomal degradation pathways, since it is prevented by either lactacystin or epoxomicin, two inhibitors of proteasomal degradation. Although there is no detectable proteasomal degradation of LHBs and MHBs in cells with functional glucosidase, the implications of the nearly quantitative sensitivity of glycosylated and unglycosylated forms of LHBs and MHBs proteins, with selective sparing of SHBs protein, in cells in which glucosidase is inhibited is surprising, and its implications are discussed.