Improved immunomatrix methods to detect protein:protein interactions

Immunoprecipitation (IP) and coimmunoprecipitation (co-IP) are key techniques for studying protein-protein interactions. These methods utilize immobilized Protein A or Protein G to isolate antibody-bound target antigens. The main disadvantage of traditional IP and co-IP is that the conditions used to elute the precipitated antigen also release the antibody thus contaminating the antigen and destroying the antibody support. To overcome these problems, we describe two methods to generate a reusable antibody support by cross-linking the antibody to immobilized Protein A or Protein G, or by coupling it directly to the resin (see Scheme 1). Antibody cross-linking can be done in 1 h while antibody coupling requires 4 h. IP or co-IP is accomplished by incubating the antibody resin with the protein sample. Washes and elutions are carried out in a spin column to reduce resin loss and decrease assay time. Target proteins are eluted with 0.1 M glycine (pH 2.8) and the resin-bound antibody is re-equilibrated in phosphate-buffered saline (PBS) for reuse. Our studies have demonstrated that the immobilization efficiency for the antibody coupling method was similar for several species of antibody. Furthermore, we illustrate that using both methods of antibody immobilization yield IP and co-IP results similar to traditional protocols but eliminate the antibody heavy and light chain contamination.     

cis-Isomers of Cytokinins Predominate in Chickpea Seeds throughout Their Development

Trans-isomers of cytokinins (CK) are thought to predominate and have greater biological activity than corresponding cis-isomers in higher plants. However, this study demonstrates a system within which the predominant CK are cis-isomers. CK were measured at four developmental stages in developing chickpea (Cicer arietinum L. cultivar Kaniva) seeds by gas chromatography-mass spectrometry. Concentrations were highest at an early endospermic fluid stage and fell considerably when the cotyledons expanded. The cis-isomers of zeatin nucleotide ([9R-MP]Z), zeatin riboside ([9R]Z), and zeatin (Z) were present in greater concentrations than those of corresponding trans-isomers: (trans)[9R-MP]Z, (trans)[9R]Z, (trans)Z, or dihydrozeatin riboside. Dihydrozeatin, dihydrozeatin nucleotide, and the isopentenyl-type CK concentrations were either low or not detectable. Root xylem exudates also contained predominantly cis-isomers of [9R-MP]Z and [9R]Z. Identities of (cis)[9R]Z and (cis)Z were confirmed by comparison of ion ratios and retention indices, and a full spectrum was obtained for (cis)[9R]Z. Tissues were extracted under conditions that minimized the possibility of RNase hydrolysis of tRNA following tissue disruption, being a significant source of the cis-CK. Since no isomerization of (trans)[²H]CK internal standards occurred, it is unlikely that the cis-CK resulted from enzymic or nonenzymic isomerization during extraction. Although quantities of total CK varied, similar CK profiles were found among three different chickpea cultivars and between adequately watered and water-stressed plants. Developing chickpea seeds will be a useful system for investigating the activity of cis-CK or determining the origin and metabolism of free CK.     

C1 inhibitor gene sequence facilitates frameshift mutations.

Mutations disrupting the function or production of C1 inhibitor cause the disease hereditary angioneurotic edema. Patient mutations identified an imperfect inverted repeat sequence that was postulated to play a mechanistic role in the mutations. To test this hypothesis, the inverted repeat was cloned into the chloramphenicol acetyltransferase gene in pBR325 and its mutation rate was studied in four bacterial strains. These strains were selected to assay the effects of recombination and superhelical tension on mutation frequency. Mutations that revert bacteria to chloramphenicol resistance (Cmr) were scored. Both pairs of isogenic strains had reversion frequencies of approximately 10(-8). These rare reversion events in bacteria were most often a frameshift that involved the imperfect inverted repeat with a deletion or a tandem duplication, an event very similar to the human mutations. Increased DNA superhelical tension, which would be expected to enhance cruciform extrusion, did not accentuate mutagenesis. This finding suggests that the imperfect inverted repeat may form a stem-loop structure in the single-stranded DNA created by the duplex DNA melting prior to replication. Models explaining the slippage can be drawn using the lagging strand of the replication fork. In this model, the formation of a stem-loop structure is responsible for bringing the end of the deletion or duplication into close proximity.     

NaCl、Na2 SO4 和Na2GO3 胁迫对小麦叶片自由基含量及质膜透性的比较研究

 分别用浓度为25mmol／L、50mmol／L、100 mmol／L和200 MMOL／l的Nacl、Na2SO4和Na2CO3的营养液培养小麦4d,较之不含盐的营养液,其自由基含量上升,产生速率增加,叶片质膜透性增加。不同盐的影响也不同,在低浓度时,NaCl的影响大于Na2SO4,高浓度时,NaCl影响小于Na2CO3,Na2CO3的影响最为显著。实验结果也表现出小麦叶片自由基含量和质膜透性呈现较好的相关性。因此可认为,盐胁迫促使自由基含量增加,自由基通过过氧化作用影响质膜透性,从而影响植物的生长。     

国家重点保护野生植物的保护现状及潜在分布区预测分析

野生植物是自然生态系统的重要组成部分,中国是野生植物种类最丰富的国家之一。研究国家重点保护野生植物的分布特征、保护现状以及潜在分布区,对于制定与支持生物多样性保护策略具有重要意义。该研究基于1 032种(隶属于129科315属)国家重点保护野生植物,利用前5%丰富度算法识别其热点地区,并与自然保护区叠加评估其保护成效、确定保护空缺,进而运用MaxEnt模型预测了国家重点保护野生植物的潜在分布区分布与变化趋势。结果表明:(1)中国南部和西南部是国家重点保护野生植物物种丰富度最高的地区,尤其是四川中部、云南南部和东南部、广西北部、广东北部与海南。(2)热点网格的保护成效分析表明,171个(85.50%)热点网格得到了有效保护(含80.50%的物种),29个(14.50%)热点网格未得到自然保护区的保护(含51.20%物种)。(3)通过比较当前与未来气候变化下国家重点保护野生植物的潜在分布区分布,发现未来潜在分布区将向西藏东南部、广西西南部、广东南部以及福建南部等地扩张,而向环四川盆地、云南南部和贵州南部等地缩减。因此,需要加强这些区域生物多样性的动态监测,持续关注气候变化对该区域国家重点保护野生植物的影响。基于该研究所确定的热点网格、保护成效以及潜在分布区的分析结果,可为国家重点保护野生植物多样性优先保护区的确定和保护政策的制定提供有力的数据支持与参考。