Carbohydrate analysis by a phenol-sulfuric acid method in microplate format

Among many colorimetric methods for carbohydrate analysis, the phenol-sulfuric acid method is the easiest and most reliable method. It has been used for measuring neutral sugars in oligosaccharides, proteoglycans, glycoproteins, and glycolipids. This method is used widely because of its sensitivity and simplicity. In its original form, it required 50-450 nmol of monosaccharides or equivalent for analysis and thus is inadequate for precious samples. A scaled-down version requiring only 10-80 nmol of sugars was reported previously. We have now modified and optimized this method to use 96-well microplates for high throughput, to gain greater sensitivity, and to economize the reagents. This modified and optimized method allows longer linear range (1-150 nmol for Man) and excellent sensitivity. Moreover, our method is more convenient, requiring neither shaking nor covering, and takes less than 15 min to complete. The speed and simplicity of this method would make it most suitable for analyses of large numbers of samples such as chromatographic fractions.     

Production and characterization of neutralizing monoclonal antibodies that recognize an epitope in domain 2 of Bacillus anthracis protective antigen

Antibodies against the protective antigen (PA) of Bacillus anthracis play a key role in response to infection by this important pathogen. The aim of this study was to produce and characterize monoclonal antibodies (mAbs) specific for PA and to identify novel neutralizing epitopes. Three murine mAbs with high specificity and nanomolar affinity for B. anthracis recombinant protective antigen (rPA) were produced and characterized. Western immunoblot analysis, coupled with epitope mapping using overlapping synthetic peptides, revealed that these mAbs recognize a linear epitope within domain 2 of rPA. Neutralization assays demonstrate that these mAbs effectively neutralize lethal toxin in vitro.     

Dual Role of α-Secretase Cleavage in the Regulation of γ-Secretase Activity for Amyloid Production

Processing of the amyloid precursor protein (APP) by β- and γ-secretases generates pathogenic β-amyloid (Aβ) peptides associated with Alzheimer disease (AD), whereas cleavage of APP by α-secretases precludes Aβ formation. Little is known about the role of α-secretase cleavage in γ-secretase regulation. Here, we show that α-secretase-cleaved APP C-terminal product (αCTF) functions as an inhibitor of γ-secretase. We demonstrate that the substrate inhibitory domain (ASID) within αCTF, which is bisected by the α-secretase cleavage site, contributes to this negative regulation because deleting or masking this domain turns αCTF into a better substrate for γ-secretase. Moreover, α-secretase cleavage can potentiate the inhibitory effect of ASID. Inhibition of γ-secretase activity by αCTF is observed in both in vitro and cellular systems. This work reveals an unforeseen role for α-secretase in generating an endogenous γ-secretase inhibitor that down-regulates the production of Aβ. Deregulation of this feedback mechanism may contribute to the pathogenesis of AD.     

A unique set of 11,008 onion expressed sequence tags reveals expressed sequence and genomic differences between the monocot orders Asparagales and Poales

Enormous genomic resources have been developed for plants in the monocot order Poales; however, it is not clear how representative the Poales are for the monocots as a whole. The Asparagales are a monophyletic order sister to the lineage carrying the Poales and possess economically important plants such as asparagus, garlic, and onion. To assess the genomic differences between the Asparagales and Poales, we generated 11,008 unique ESTs from a normalized cDNA library of onion. Sequence analyses of these ESTs revealed microsatellite markers, single nucleotide polymorphisms, and homologs of transposable elements. Mean nucleotide similarity between rice and the Asparagales was 78% across coding regions. Expressed sequence and genomic comparisons revealed strong differences between the Asparagales and Poales for codon usage and mean GC content, GC distribution, and relative GC content at each codon position, indicating that genomic characteristics are not uniform across the monocots. The Asparagales were more similar to eudicots than to the Poales for these genomic characteristics.     

长江春大豆核心种质构建及分析

利用长江春大豆初选核心种质SSR(simple sequence repeat）标记和农艺性状表型等基础数据,对用不同个体取样方法以及不同数据类型建立的核心种质进行评价,目的是确定中国大豆（Glycine max）核心种质的最佳取样策略提供依据,结果表明,根据SSR分子数据聚类,采用类内随机取样,类内以遗传相似性系数取样以及仅依据遗传相似性系数取样都可用于大豆核心种质构建,但是综合不同评价参数发现,以类内随机取样最佳,类内按遗传相似性系数取样次之,单独以遗传相似性系数取样较差。分析不同SSR等位变异保留比例的遗传多样性指数发现,当保留90%和80%的SSR等位变异时,核心种质具有更高的遗传多样性,由于与SSR分子数据种质遗传关系评价的不一致性,农艺性状等基础数据虽然可用来构建核心种质,但其SSR分子水平代表性相对较低,本研究结果还表明,用不同方法或同一方法不同重复次数取样建立的核心种质具有异质性,且这种异质性随核心种质取样比例的降低而增大,因此,虽然可依据不同数据类型确定相应的方法建立核心种质,但综合表型和分子数据建立的核心种质更具有代表性。     

Identification of new human cadherin genes using a combination of protein motif search and gene finding methods

We have combined protein motif search and gene finding methods to identify genes encoding proteins containing specific domains. Particularly, we have focused on finding new human genes of the cadherin superfamily proteins, which represent a major group of cell-cell adhesion receptors contributing to embryonic neuronal morphogenesis. Models for three cadherin protein motifs were generated from over 100 already annotated cadherin domains and used to search the complete translated human genome. The genomic sequence regions containing motif "hits" were analyzed by eukaryotic GeneMark.hmm to identify the exon-intron structure of new genes. Three new genes CDH-J, PCDH-J and FAT-J were found. The predicted proteins PCDH-J and FAT-J were classified into protocadherin and FAT-like subfamilies, respectively, based on the number and organization of cadherin domains and presence of subfamily-specific conserved amino acid residues. Expression of FAT-J was shown in almost all tested tissues. The exon-intron organization of CDH-J was experimentally verified by PCR with specifically designed primers and its tissue-specific expression was demonstrated. The described methodology can be applied to discover new genes encoding proteins from families with well-characterized structural and functional domains.     

粳稻穗角与稻米品质的相关性及稻米品质遗传分析

测定了粳稻直立穗品种丙8979与弯曲穗品种C堡杂交组合的P1、P2及其重组自交系349个株系的穗角和10个稻米品质性状, 分析了穗角与稻米品质性状之间的相关性, 并运用主基因+多基因混合遗传模型, 对稻米品质10个性状进行了遗传分析。结果表明,穗角与糙米率、整精米率、垩白粒率、垩白度、糊化温度、胶稠度和直链淀粉含量均无显著相关; 与精米率呈显著正相关(r=0.124*); 与粒长和长宽比均呈极显著正相关(相关系数分别为0.470**和0.241**)。糙米率、精米率和直链淀粉含量均受2对主基因+多基因控制, 2对主基因具有累加作用和加性×加性的上位性作用; 整精米率、粒长、长宽比和胶稠度受2对加性-上位性主基因+多基因控制;垩白粒率、垩白度和糊化温度均受3对加性-上位性主基因+多基因控制。糙米率、精米率、整精米率、垩白粒率、垩白度和糊化温度6个品质性状以主基因遗传为主,粒长、长宽比、胶稠度和直链淀粉含量4个性状以多基因遗传为主。     

虾夷扇贝雄核发育单倍体的人工诱导研究

人工雄核发育,是利用遗传失活的卵子与正常精子受精,再利用染色体加倍技术使受精卵恢复为二倍体的遗传操作技术。利用该技术可大大加速品系纯化的过程,对遗传育种学研究有重要意义。目前有关鱼类雄核发育的研究已见于鲤( Gyprnus carpio)1,2、虹鳟( Oncorhynchus mykiss)3、红点溪鳟(Salvelinus fontinalis)4、泥鳅(Misgurnus anguilicaudatus)5,6、斑马鱼(Danio rerio)7、马苏大麻哈鱼(Oncorhynchus masou)8等种类。人工诱导贝类雄核发育的研究资料在国内外都很少,目前只有栉孔扇贝和太平洋牡蛎雄核发育诱导条件及其发育早期荧光显微观察方面的报道9,10。虾夷扇贝(Patinopecten yessoensis) 原产于日本,目前已成为北方沿海地区一种重要的增养殖种类,具有很高的经济价值,同时也是目前海洋贝类育种研究的对象之一。本实验首次研究了紫外线照射对虾夷扇贝卵子遗传失活的影响,成功诱导出雄核发育单倍体,为今后开展虾夷扇贝雄核发育二倍体的人工诱导奠定了基础,现将其结果予以报道。       

两个水稻GDP-甘露糖-3’，5＇-异构酶基因特征及表达模式的研究

GDP-甘露糖-3’,5’-异构酶（GME）可以催化GDP-甘露糖转化为左旋GDP-半乳糖,该反应对于高等植物体内抗坏血酸的合成是非常重要的．但目前在分子水平上还没有对GME基因进行研究的报道．通过逆转录PCR（RT-RCR）技术从水稻成熟叶片中克隆到两个GME基因的cDNA序列,并与其他植物物种中的GMEs进行比对,结果显示,GME基因在所有植物物种中高度保守,尽管进化树分析表明单子叶植物GMEs和双子叶植物GMEs在进化上相互独立．同时,分析这两个水稻GME基因的剪切模式揭示了二者也存在高度相似性．采用半定量RT-PCR技术对两个GME基因在不同组织和不同胁迫条件下的表达模式进行研究表明,OsGME1基因在冷胁迫条件下表达水平上调,这和先前水稻冷胁迫蛋白质组学研究的结果是一致的．而OsGME2和OsGME1基因在用赤霉素处理条件下表达水平均下调,暗示赤霉素可能通过调节GME基因的表达来调控植物体内的抗坏血酸合成．