EB病毒潜伏膜蛋白1通过结合TRAFs调控NF-κB

为了探讨EB病毒潜伏膜蛋白1(LMP1)的致瘤机制,对鼻咽癌中LMP1激活重要的核转录因子NF-κB机制进行了研究.首先,采用免疫共沉淀-蛋白质印迹在稳定表达LMP1的鼻咽癌细胞系HNE2-LMP1中证实LMP1与TRAF1,2,3结合形成免疫共沉淀复合物,进一步以野生型LMP1及其三种突变体的鼻咽癌细胞系LMP1（野生型,wt）、HNE2-LMP1 del187～351(CTAR1缺失型)、HNE2-LMP1(1～231)(CTAR2缺失型)、HNE2-LMP1(1～187)(羧基端胞浆区缺失型)、HNE2-pSG5(空白载体型)为材料,结合NF-κB报道基因质粒（pGL2-NF-κB-luc）的荧光素酶活性表达分析NF-κB的活性,证实:较之母细胞, 野生型LMP1活化NF-κB达13.8倍, LMP1(1～187)几乎不活化NF-κB,LMP1(1～231)活化NF-κB达4.9倍, LMP1(del187～351)活化NF-κB达9.1倍;TRAF1过表达升高LMP1(wt)及LMP1(1～231)介导的NF-κB活性,而对LMP1(del 187～351)活化NF-κB无影响;TRAF3过表达或TRAF3负显性突变体抑制LMP1(wt)及LMP1(1～231)介导的NF-κB活性,而不影响LMP1(del 187～351)活化NF-κB; TRAF2过表达升高LMP1(wt)、LMP1 (1～231)及LMP1(del 187～351)介导的NF-κB活性.这些结果表明：鼻咽癌中LMP1通过TRAF1、TRAF2或TRAF3调控NF-κB,TRAF1和TRAF3主要通过CTAR1发挥作用,TRAF2的作用主要是通过CTAR1和CTAR2介导的.     

EB病毒LMP1 CTAR1、CTAR2的表达促使人鼻咽癌细胞HNE2增殖

探讨EB病毒LMP1不同结构域在鼻咽癌中的致瘤作用,为阐明鼻咽癌分子发病机理,寻找治疗鼻咽癌的分子靶提供实验依据。以转染空白载体为对照,利用电穿孔转染方法,建立稳定表达LMP1不同突变体的鼻咽癌细胞系HNE2-LMP1(1～815)、HNE2-LMP1(1～231)、HNE2-LMP1△187～351,并以这些细胞系为材料,用MTT法检测增殖期活细胞,BrdU掺入法检测细胞增殖状况,比较各组细胞的软琼脂集落形成率和裸鼠成瘤能力,以观察LMP1不同的结构域对鼻咽癌细胞生长的影响。LMP1(1～231)和LMP1△187～351在体外明显促进HNE2细胞增殖,HNE2-LMP1(1～231)、HNE2-LMP1△187～351平均吸光度(A)比值、BrdU掺入率、软琼脂集落形成率均高于HNE2-pSG5与HNE2(P<0 01),而HNE2-LMP1(1～187)与HNE2-pSG5、HNE2相比,这些指标无明显差别。HNE2-LMP1△187～351和HNE2-LMP1(1～231)的裸鼠成瘤潜伏期、倍增时间与平均瘤重明显高于HNE2-pSG5鼻咽癌细胞系,其差异有显著的统计学意义(P<0 05)。而HNE2-LMP1(1～187)、HNE2-pSG5和HNE2鼻咽癌细胞系在潜伏期、倍增时间与平均瘤重方面两两比较,差异无显著的统计学意义(P>0 05)。EB病毒LMP1CTAR1和CTAR2对HNE2细胞生长有明显促进作用,提示EB病毒LMP1可能在鼻咽癌的发生发展中起着重要的作用。     

EB病毒LMP1-CTAR3对NP69细胞增殖和蛋白质表达的影响

为了探讨EB病毒潜伏性膜蛋白1(LMP1)第三个功能活性区域(CTAR3)在鼻咽上皮细胞NP69中的转化作用机制,采用逆病毒感染的方法,将浓缩的逆病毒RV-LMP1和RV-LMP1△232～351分别感染鼻咽上皮细胞NP69,建立NP69-LMP1与NP69-LMP1△232～351稳定表达细胞系.通过绘制生长曲线、平皿克隆形成试验和软琼脂集落形成试验比较野生型和突变型LMP1对NP69细胞增殖的影响,运用蛋白质组学方法鉴定NP69-LMP1与NP69-LMP1△232～351细胞间的差异表达蛋白,选用实时荧光定量RT-PCR与Western blot对其中部分蛋白质点差异表达进行验证.结果发现:a.突变型LMP1△232～351促NP69细胞增殖的能力较野生型LMP1明显降低(n=3,P<0.05);b.鉴定了LMP1-CTAR3在NP69细胞中参与调节的16个蛋白质(表达上调的蛋白质8个,下调的8个).c.实时荧光定量RT-PCR和Western blot证实了部分上述蛋白质的差异表达.以上结果说明,LMP1-CTAR3是其发挥促细胞增殖的重要活性部位,可能通过参与调节G蛋白和异柠檬酸脱氢酶等蛋白质的表达而起作用.     

EB病毒LMP1及其CTAR1、CTAR2导入人HNE2鼻咽癌细胞的研究

用电穿孔转染法,建立稳定表达野生型LMP1及其不同突变体的鼻咽癌细胞系,并以这些细胞系为材料,用MTT法检测增殖期活细胞,观察LMP1及其不同的结构域对鼻咽癌细胞生长的影响。结果得到了LMP1及其三种突变体、空白载体表达的鼻咽癌细胞系;HNE2－LMP1（野生型）、HNE2－LMP1△185-351(CTAR1缺失型）、HNE2－LMP1（1－231）（CTAR2缺失型）、HNE2－LMP1（1－185）（羧基端胸浆区缺失型）、HNE2－pSG5(空载体型）。进一步证实HNE2－LMP1、HNE2－LMP1（1－231）、HNE2－LMP1△185－351平均吸光度（A）比值高于对照组HNE2－pSG5及HNE2（P＜0.01）。这提示：EB病毒LMP1及其LMP1（1－231）和LMP1△185－351在体外明显促进HNE2细胞增殖。结果表明EB病毒LMP1可能在鼻咽癌中发挥着重要的作用。     

EB病毒潜伏膜蛋白1通过TRAF/TRADD激活JNK信号途径

为了探讨在鼻咽癌细胞中EB病毒编码的潜伏膜蛋白1(LMP1)激活c-Jun氨基端激酶(JNK)信号途径的分子机制,利用可调控表达LMP1的鼻咽癌细胞系L7,蛋白质印迹检测,发现LMP1能够促进JNK的活化;利用稳定表达LMP1的鼻咽癌细胞系HNE₂-LMP1及其三种突变体HNE₂-LMP1ΔCTAR1、HNE₂-LMP1ΔCTAR2、HNE₂-LMP1ΔCTAR1,2及LMP1阴性的HNE₂为材料,采用蛋白质印迹和报告基因法分析JNK和活化蛋白1(AP1)活化情况,结果显示HNE₂-LMP1和HNE₂-LMP1ΔCTAR1中磷酸化JNK蛋白表达量和AP1活性都无显著差异,而与HNE₂-LMP1ΔCTAR2、HNE₂-LMP1ΔCTAR1,2、阴性对照HNE2及空白载体转染细胞的JNK蛋白表达和AP1活性具有显著差异;进一步比较转染TRAF、TRADD显性负性突变体鼻咽癌细胞系HNE₂-LMP1中磷酸化的JNK量和AP1活性,结果显示：TRAF-DN和TRADD-DN的导入使活化的JNK蛋白和AP-1活性显著降低,二者间无显著差异,提示TRAF和TRADD可能参与了LMP1对JNK和AP-1的活化.以上结果提示在鼻咽癌细胞系中LMP1功能结构域CTAR2通过结合TRAF/TRADD激活JNK从而活化重要的转录因子AP1.     

EB病毒潜伏膜蛋白1通过结合TRAFs调控NF-κB

为了探讨EB病毒潜伏膜蛋白1（LMP1）的致瘤机制,对鼻咽癌中LMP1激活重要的核转录因子NF－κB机制进行了研究,首先,采用免疫共沉淀－蛋白质印迹在稳定表达LMP1的鼻咽癌细胞系HNE2－LMP1中证实LMP1与TRAF1,2,3结合形成免疫共沉淀复合物,进一步以野生型LMP1及其三种突变体的鼻咽癌细胞系LMP1（野生型, wt),HNE2－LMP1 del187-351(CTAR1缺失型）,HNE2－LMP1（1－231）,（CTAR2缺失型）,HNE2－LMP1（1－187）（羰基端胞浆区缺失型）,HNE2－pSG5(空白载体型）为材料,结合NF－κB报道基因质粒（pG12-NF-B-luc)的荧光素酶活性表达分析NF－κB的活性,证实：较之母细胞,野生型LMP1活化NF－B达13．8倍,LMP1（1－187）几乎不活化NF－kb,LMP1(1-231)活化NF－kB 达4．9倍,LMP1（del187-351)活化NFκB达9．1倍,TRAF1过表达升高LMP1（ wt)及LMP1（1－231）介导的NF－κB活性,而对LMP1（del187-351)活化NFκB无影响,TRAF3过表达或TRAF3负显性突变体抑,制LMP1（wt)及LMP1（1－231）介导的NF－κB活性而不影响LMP1（del187－351）活化NF－κB,TRAF2过表达升高LMP1（wt),LMP1(1-231)及LMP1（del 187-351)介导的NF－kB活性,这些结果表明：鼻咽癌中LMP1通过TRAF1,TRAF2或TRAF3调控NF－kB,TRAF1和TRAF3主要通过CTAR1发挥作用,TRAF2的作用主要是通过CTAR1和CTAR2介导的。     

Epstein-Barr病毒潜伏性膜蛋白1 CTAR3缺失突变体的构建与功能分析

[目的]探讨Epstein-Barr病毒潜伏性膜蛋白1(LMP1)促细胞转化的主要活性部位及其作用机制.[方法]采用PCR方法重组LMP1羧基末端活化域3(aa232-aa351)对应密码子缺失突变体(LMP1△232-351),将突变型LMP1△232-351和野生型LMP1(LMP1WT)分别导入永生化的鼻咽上皮细胞NP69中,比较二者对细胞的转化作用.同时,构建含JAK3启动子序列的荧光素酶表达质粒(pGL-2/JAK3-LUC),将LMP1△232-351与LMP1WT分别与含有JAK3启动子序列或NF-kB结合序列启动子的荧光酶表达质粒共转染293细胞(用pLNSX质粒作对照),比较二者活化JAK3启动子或转录因子NF-KB的功能.[结果](1)LMP1△232-351促NP69细胞转化的能力较LMP1WT显著降低(n=3,p<0.01).(2)LMP1WT能明显呈浓度依赖陛活化JAK3启动子,而LMP1△232-351上调能力几乎丧失.[结论]LMP1羧基末端活化域3(aa232-aa351)是LMP1的重要活性部位之一,其促细胞转化的作用与JAK3蛋白表达调节有关.     

鼻咽癌细胞中EB病毒编码的潜伏膜蛋白1活化cyclinD1的表达

为了探讨EB病毒编码的潜伏膜蛋白1(EBV-LMP1)促进细胞增殖,参与EBV相关疾病致瘤的分子机制,研究了LMP1在鼻咽癌细胞中调节cyclinD1表达,进而影响细胞周期行进及细胞恶性表型改变,并初步确定了LMP1发挥该功能的结构域.利用已建株的Tet-on-LMP1-HNE2鼻咽癌细胞系,蛋白质印迹实验分析LMP1诱导cyclinD1蛋白质表达的表达动力学,包括时间效应及剂量效应;利用三种LMP1功能区缺失的突变体及野生型LMP1,以载体型细胞为对照,确定LMP1活化cyclinD1表达的结构域.同时结合基因诱导表达及反义寡聚核酸技术阻断基因表达的实验方法,进一步确定LMP1上调的cyclinD1功能,即对细胞周期行进及细胞恶性表型的影响.结果表明LMP1确实可以诱导cyclinD1的表达（2～4倍）,且诱导具有时间依赖性及剂量依赖性;利用三种LMP1功能区缺失的突变体及野生型LMP1,以载体型细胞为对照,结合报道基因分析法,确定与空白载体细胞系比较,野生型LMP1从转录水平可反式激活cyclinD1报道基因活性约11.2倍,其中CTAR1及CTAR2均可活化cyclinD1表达,但以CTAR2为主,与野生型LMP1诱导cyclinD1反式激活活性比较,CTAR1缺失导致cyclinD1报道基因活性下降23.6%,CTAR2缺失导致cyclinD1活性下降约80.7%,C端均缺失时cyclinD1活性只有野生型的17.7%.流式细胞仪分析显示,强力霉素诱导后cyclinD1高表达的细胞停留于G0/G1期明显减少,较未经诱导的细胞,从66.42%减至56.55%,而进入S期及G2/M期的细胞明显增多.在稳定表达LMP1的细胞中,与导入正义LMP1比较,导入反义LMP1 PS-ODNs及反义cylinD1,可以使细胞软琼脂集落形成率明显降低(从30.48%分别降至15.21%,21.76%).EBV-LMP1可以活化cyclinD1的表达,且发挥这种功能的结构域以CTAR2为主,活化的cyclinD1参与细胞周期行进,抑制LMP1及cyclinD1的表达均可导致细胞软琼脂集落形成率降低.     

携带绿色荧光报告基因的EBV-LMP1真核表达载体构建与鼻咽癌细胞中的表达

潜伏膜蛋白1(LMP1)是由EB病毒编码的致瘤蛋白,众多研究表明LMP1蛋白可通过NF-κB、p38 MAPK、c-JNK等多条重要信号通路引起鼻咽癌细胞的生物学行为改变。我们从EB病毒阳性的B95-8狨猴淋巴瘤细胞中克隆EB病毒LMP1 c DNA,构建携带绿色荧光基因的真核表达质粒p IRES2-Zs-Green1-LMP1,通过脂质体转染的方法将质粒导入鼻咽癌细胞株CNE1、CNE2中,利用质粒所携带的绿色荧光蛋白表达粗略计算转染的效率,通过免疫细胞化学(ICC)、RT-PCR、Western-Blot检测该质粒的表达。本实验室所构建的p IRES2-Zs-Green1-LMP1表达质粒能在鼻咽癌细胞内表达LMP1蛋白,为后续的实验研究奠定基础。     

EB病毒LMP1-CTAR3对NP69细胞增殖和蛋白质表达的影响

为了探讨EB病毒潜伏性膜蛋白 1(LMP1)第三个功能活性区域(CTAR₃)在鼻咽上皮细胞NP69中的转化作用机制,采用逆病毒感染的方法,将浓缩的逆病毒RV-LMP1和RV-LMP1^Δ232～351分别感染鼻咽上皮细胞NP69,建立NP69-LMP1与NP69-LMP1^Δ232～351稳定表达细胞系．通过绘制生长曲线、平皿克隆形成试验和软琼脂集落形成试验比较野生型和突变型LMP1对NP69细胞增殖的影响,运用蛋白质组学方法鉴定NP69-LMP1与NP69-LMP1^Δ232～351细胞间的差异表达蛋白,选用实时荧光定量RT-PCR与Western blot对其中部分蛋白质点差异表达进行验证．结果发现：a．突变型LMP1^Δ232～351促NP69细胞增殖的能力较野生型LMP1明显降低(n=3,P < 0.05);b．鉴定了LMP1-CTAR₃在NP69细胞中参与调节的16个蛋白质(表达上调的蛋白质8个,下调的8个)．c．实时荧光定量RT-PCR和Western blot证实了部分上述蛋白质的差异表达．以上结果说明,LMP1-CTAR₃是其发挥促细胞增殖的重要活性部位,可能通过参与调节G蛋白和异柠檬酸脱氢酶等蛋白质的表达而起作用．     

Preparation of hair for measurement of elements by inductively coupled plasma-mass spectrometry (ICP-MS)

The preparation of hair for the determination of elements is a critical component of the analysis procedure. Open-beaker, closedvessel microwave, and flowthrough microwave digestion are methods that have been used for sample preparation and are discussed. A new digestion method for use with inductively coupled plasma-mass spectrometry (ICP-MS) has been developed. The method uses 0.2 g of hair and 3 mL of concentrated nitric acid in an atmospheric pressurelow-temperature microwave digestion (APLTMD) system. This preparation method is useful in handling a large numbers of samples per day and may be adapted to hair sample weights ranging from 0.08 to 0.3 g. After digestion, samples are analyzed by ICP-MS to determine the concentration of Li, Be, B, Na, Mg, Al, P, S, K, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ge, As, Se, Rb, Sr, Zr, Mo, Pd, Ag, Cd, Sn, Sb, I, Cs, Ba, Pt, Au, Hg, Tl, Pb, Bi, Th, and U. Benefits of the APLTMD include reduced contamination and sample handling, and increased precision, reliability, and sample throughput.     

日本北海道虻类雌性外生殖器形态(双翅目：虻科)(英文)

【目的】利用日本北海道虻类评估和验证外生殖器在分类学上的意义。【方法】将虻类成虫标本浸渍在生理盐水中并置于双目显微镜下通过针和镊子在培养皿中进行解剖并绘图,观察第9背板、第10背板、尾叶、第8腹板、受精囊、受精囊管及生殖叉器的形态特征。【结果】在日本北海道共记录了虻科(Tabanidae) 3亚科7属38种。我们观察并描述了3亚科其中的6属24种的雌性外生殖器的主要特征。亚科之间存在明显差异;然而在一般情况下属之间很难建立一种方法来确定共同点;种之间只有在斑虻属Chrysops中有相似之处,其他属中则比较多样化。因此,亚科鉴定根据第9背板、第8腹板及受精囊足以进行区分,属及种鉴定需要结合第9背板、第10背板、尾叶、第8腹板、受精囊、受精囊管及生殖叉器各自的特征组合在一起才能区分开来。我们也制作了虻类外生殖器的检索表。【结论】和许多其他昆虫一样,外生殖器是虻科的重要分类特征,对于促进分类学和系统学的发展具有重要意义。本研究首次对分布在日本北海道的虻科雌性外生殖器进行了系统研究。     

Ecological importance of sedges: a survey of the Australasian Cyperaceae genus Lepidosperma

Background

Sedges (Cyperaceae) form an important ecological component of many ecosystems around the world. Sword and rapier sedges (genus Lepidosperma) are common and widespread components of the southern Australian and New Zealand floras, also occurring in New Caledonia, West Papua, Borneo, Malaysia and southern China. Sedge ecology is seldom studied and no comprehensive review of sedge ecology exists. Lepidosperma is unusual in the Cyperaceae with the majority of species occurring in dryland habitats.

Scope

Extensive review of ecological literature and field observations shows Lepidosperma species to be important components of many ecosystems, often dominating understorey and sedge-rich communities. For the first time, a detailed ecological review of a Cyperaceae genus is presented.

Conclusions Lepidosperma

species are long-lived perennials with significant abundance and persistence in the landscape. Speciation patterns in the genus are of considerable interest due to complex biogeographical patterns and a high degree of habitat specificity. Potential benefits exist for medicinal products identified from several Lepidosperma species. Over 178 organisms, including 26 mammals, 42 birds, six reptiles, five amphibians, eight arachnids, 75 insects, three crustaceans and 13 fungi, are found to be dependent on, or making use of, Lepidosperma species. A significant relationship exists between Lepidosperma species and the moth genus Elachista. Implications for the conservation and ecology of both sedges and associated species are discussed.     

外泌体在白血病中的重要调控作用

目前,白血病复发是患者死亡的主要原因之一。肿瘤细胞和微环境的相互作用,以及隐匿在骨髓中的肿瘤干细胞,促进了白血病的复发和向淋巴组织的转移,因此白血病的治疗、转移和复发问题受到广泛关注。外泌体是由绝大多数细胞分泌的双层脂质膜囊泡,可以调控细胞间的交流和信息传递。在白血病细胞、基质细胞和内皮细胞之间的相互联系中都涉及到外泌体,白血病细胞来源的外泌体存在于白血病患者的血浆中,能把其携带的白血病相关抗原及微小RNA呈递给靶细胞,促进白血病肿瘤细胞的增殖,有助于肿瘤细胞实现免疫逃避,保护白血病细胞抵抗化疗药物导致的细胞毒性作用,促进血管生成及肿瘤细胞的迁移。因此,外泌体与白血病的转移、治疗及预后密切相关,可以用来检测和监测白血病的进展。本文综述了外泌体的来源、形成与分泌机制,以及外泌体在白血病发生前、发展中、预后和免疫治疗中所扮演的重要角色。     

Frequency of cyanogenesis in tropical rainforests of far north Queensland, Australia

BACKGROUND AND AIMS: Plant cyanogenesis is the release of toxic cyanide from endogenous cyanide-containing compounds, typically cyanogenic glycosides. Despite a large body of phytochemical, taxonomic and ecological work on cyanogenic species, little is known of their frequency in natural plant communities. This study aimed to investigate the frequency of cyanogenesis in Australian tropical rainforests. Secondary aims were to quantify the cyanogenic glycoside content of tissues, to investigate intra-plant and intra-population variation in cyanogenic glycoside concentration and to appraise the potential chemotaxonomic significance of any findings in relation to the distribution of cyanogenesis in related taxa. METHODS: All species in six 200 m(2) plots at each of five sites across lowland, upland and highland tropical rainforest were screened for cyanogenesis using Feigl-Anger indicator papers. The concentrations of cyanogenic glycosides were accurately determined for all cyanogenic individuals. KEY RESULTS: Over 400 species from 87 plant families were screened. Overall, 18 species (4.5 %) were cyanogenic, accounting for 7.3 % of total stem basal area. Cyanogenesis has not previously been reported for 17 of the 18 species, 13 of which are endemic to Australia. Several species belong to plant families or orders in which cyanogenesis has been little reported, if at all (e.g. Elaeocarpaceae, Myrsinaceae, Araliaceae and Lamiaceae). A number of species contained concentrations of cyanogenic glycosides among the highest ever reported for mature leaves-up to 5.2 mg CN g(-1) d. wt, for example, in leaves of Elaeocarpus sericopetalus. There was significant variation in cyanogenic glycoside concentration within individuals; young leaves and reproductive tissues typically had higher cyanogen content. In addition, there was substantial variation in cyanogenic glycoside content within populations of single species. CONCLUSIONS: This study expands the limited knowledge of the frequency of cyanogenesis in natural plant communities, includes novel reports of cyanogenesis among a range of taxa and characterizes patterns in intra-plant and intra-population variation of cyanogensis.     

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.^1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression^3-6 and other characteristics of cultures at steady-state equilibrium.⁷ Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.⁸ A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.^9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. We present here the design and operation of a relatively simple, low cost array of miniature chemostats—or ministats—and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.     

Fine Structure of Sperm of Ekphymatodera thomasoni (Heteroderinae,Nemata)

Fine structure of developing sperm of the monospecific genus, Ekphymatodera, was compared with other Heteroderinae as part of a study to recognize diversity and phylogenetically informative characters within the subfamily. Sperm of Ekphymatodera originate from germ cells connected to a central rachis, a character which is shared with Globodera, but not with other Heteoderinae. In Ekphymatodera, and cyst-forming genera, a layer of cortical microtubules lies just beneath the surface of the plasma membrane. Sperm of Ekphymatodera are unique among Heteroderinae examined by the presence of spiral surface elevations on the filopodia, a character that may prove to be a synapomorphy for Sarisoderini. Fibrous bodies are abundant in spermatids; however, they do not persist in sperm of Ekphymatodera as they do in Meloidodera and Verutus. The male gonad of Ekphymatodera is lined by epithelial cells, which are greatly enlarged near the ejaculatory canal. These enlarged cells contain vesicles with concentric lamellar inclusions, not observed in other genera of the subfamily. Sperm of Heteroderinae are rich in diversity, and examination of additional representative species may indicate new phylogenetically informative characters.     

A phylogenetic analysis of 100+ genera and 50+ families of euasterids based on morphological and molecular data with notes on possible higher level morphological synapomorphies

 A data matrix of 143 morphological and chemical characters for 142 genera of euasterids according to the APG system was compiled and complemented with rbcL and ndhF sequences for most of the genera. The data were subjected to parsimony analysis and support was assessed by bootstrapping. Strict consensus trees from analyses of morphology alone and morphology + rbcL + ndhF are presented. The morphological data recover several groups supported by molecular data but at the level of orders and above relationships are only superficially in agreement with molecular studies. The analyses provide support for monophyly of Gentianales, Aquifoliales, Apiales, Asterales, and Dipsacales. All data indicate that Adoxaceae are closely related to Dipsacales and hence they should be included in that order. The trees were used to assess some possible morphological synapomorphies for euasterids I and II and for the orders of the APG system. Euasterids I are generally characterised by opposite leaves, entire leaf margins, hypogynous flowers, “early sympetaly” with a ring-shaped corolla primordium, fusion of stamen filaments with the corolla tube, and capsular fruits. Euasterids II often have alternate leaves, serrate-dentate leaf margins, epigynous flowers, “late sympetaly” with distinct petal primordia, free stamen filaments, and indehiscent fruits. It is unclear which of these characters represent synapomorphies and symplesiomorphies for the two groups, respectively, and there are numerous expections to be interpreted as reversals and parallelisms. Received August 28, 2000 Accepted August 7, 2001     

Literature based species occurrence data of birds of northeast India

The northeast region of India is one of the world's most significant biodiversity hotspots. One of the richest bird areas in India, it is an important route for migratory birds and home to many endemic bird species. This paper describes a literature-based dataset of species occurrences of birds of northeast India. The occurrence records documented in the dataset are distributed across eleven states of India, viz.: Arunachal Pradesh, Assam, Bihar, Manipur, Meghalaya, Mizoram, Nagaland, Sikkim, Tripura, Uttar Pradesh and West Bengal. The geospatial scope of the dataset represents 24 to 29 degree North latitude and 78 to 94 degree East longitude, and it comprises over 2400 occurrence records. These records have been collated from scholarly literature published between1915 and 2008, especially from the Journal of the Bombay Natural History Society (JBNHS). The temporal scale of the dataset represents bird observations recorded between 1909 and 2007. The dataset has been developed by employing MS Excel. The key elements in the database are scientific name, taxonomic classification, temporal and geospatial details including geo-coordinate precision, data collector, basis of record and primary source of the data record. The temporal and geospatial quality of more than 50% of the data records has been enhanced retrospectively. Where possible, data records are annotated with geospatial coordinate precision to the nearest minute. This dataset is being constantly updated with the addition of new data records, and quality enhancement of documented occurrences. The dataset can be used in species distribution and niche modeling studies. It is planned to expand the scope of the dataset to collate bird species occurrences across the Indian peninsula.     

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.