一种蛋白质B细胞表位展示系统的构建及验证

目的:建立一种能够有效展示蛋白质B细胞表位的载体系统,以用于表位特异性抗体的制备和表位疫苗的设计。方法:选用抗体的可变区作为蛋白质B细胞表位展示的骨架蛋白,B细胞表位的展示部位为CDR3区。全基因合成骨架蛋白基因,通过重叠PCR将B细胞表位编码序列插入到骨架蛋白基因中,原核表达目的蛋白,利用Sephacryl S-100层析柱进行蛋白纯化,用纯化的蛋白按常规方法免疫小鼠,采用ELISA法检测免疫血清的滴度及特异性,通过Western blot和间接免疫荧光技术进一步验证免疫血清对目的蛋白的识别。结果:成功构建了3种表位展示蛋白,均表现出了很好的抗原性,表位展示蛋白免疫血清具有较好的特异性,能够特异识别所展示的B细胞表位。结论:抗体可变区作为骨架蛋白能够很好地展示B细胞表位,免疫小鼠后获得的免疫血清表现出了较好的特异性。     

大鼠骨骼肌多催化功能蛋白酶的提取、鉴定及其抗血清的制备

为了研究多催化功能蛋白酶（ｍｕｌｔｉｃａｔａｌｙｔｉｃａｌｐｒｏｔｅｉｎａｓｅ,ＭＣＰ）在负氮平衡形成中的作用,以大鼠骨骼肌为原料,提取此酶并制备其抗血清．将大鼠骨骼肌粗提物经４５％～６５％饱和度硫酸铵分级盐析、阴离子交换层析和凝胶过滤,最后从Ｓｅｐｈａｒｏｓｅ４Ｂ层析柱上获得单一活性洗脱峰．酶活性用Ｃａｒｂｏｂｅｎｚｏｘｙ－Ｖａｌ－Ｇｌｙ－Ａｒｇ－４－ｎｉｔｒｉｎｉｌｉｄｅａｃｅｔａｔｅ作底物检测．非变性ＰＡＧＥ银盐染色显示单一区带的骨骼肌多催化功能蛋白酶,ＳＤＳ－ＰＡＧＥ银盐染色显示１０条亚基电泳区带,分子量在２５～３２ｋＤ之间．纯化的酶免疫兔８周后,抗血清效价达１３２,用分级盐析和离子交换层析纯化抗血清,显示单一电泳区带的ＩｇＧ．Ｗｅｓｔｅｒｎ－ｂｌｏｔ分析显示只在２５～３２ｋＤ之间出现多条亚基区带．这些结果提示已获得电泳纯ＭＣＰ及其较高特异性的多克隆抗体．     

Genetic engineering of cytokinin metabolism: Prospective way to improve agricultural traits of crop plants

Cytokinins (CKs) are ubiquitous phytohormones that participate in development, morphogenesis and many physiological processes throughout plant kingdom. In higher plants, mutants and transgenic cells and tissues with altered activity of CK metabolic enzymes or perception machinery, have highlighted their crucial involvement in different agriculturally important traits, such as productivity, increased tolerance to various stresses and overall plant morphology. Furthermore, recent precise metabolomic analyses have elucidated the specific occurrence and distinct functions of different CK types in various plant species. Thus, smooth manipulation of active CK levels in a spatial and temporal way could be a very potent tool for plant biotechnology in the future. This review summarises recent advances in cytokinin research ranging from transgenic alteration of CK biosynthetic, degradation and glucosylation activities and CK perception to detailed elucidation of molecular processes, in which CKs work as a trigger in model plants. The first attempts to improve the quality of crop plants, focused on cereals are discussed, together with proposed mechanism of action of the responses involved.     

SDS‐PAGE‐free protocol for comprehensive identification of cytochrome P450 enzymes and uridine diphosphoglucuronosyl transferases in human liver microsomes

Comprehensive identification of cytochrome P450 enzymes (CYPs) and uridine diphosphoglucuronosyl transferases (UGTs) in human liver microsomes (HLMs) was performed with an SDS‐PAGE‐free protocol. HLMs were solubilized with 5% v/v ionic liquid, 1‐butyl‐3‐methyl imidazolium tetrafluoroborate, followed by tryptic digestion, and 2D‐SCX‐RPLC‐ESI‐MS/MS (LTQ XL) analysis in triplicate. In total, 27 CYPs and 12 UGTs were confidently identified with average sequence coverage as 30.99 and 25.07%, average peptide number as 14 and 13, and average unique peptide number as 7 and 4, respectively. The highly similar isoforms of CYP3A, CYP2C, and CYP4F subfamilies could be unambiguously differentiated from each other, despite the fact that the sequence similarity of CYP2C9 and CYP2C19 is 91%. In addition, protein spectral count was used to approximately evaluate the relative abundance of identified CYPs and UGTs, and the results agreed with previous immunochemistry reports.     

Tissue Response to a Supplement High in Aluminum and Silicon

The objective was to determine the effects of sodium zeolite A (SZA) on mineral metabolism and tissue mineral composition in calves. Twenty calves were placed on study at 3 days of age and were placed into one of two groups: SS, which received 0.05% BW SZA added to their milk replacer, and CO, which received only milk replacer. Blood samples were taken on days 0, 30, and 60 for mineral analysis. Urine and feces were collected on day 30 for mineral metabolism, and on day 60, the calves were euthanized, and samples were taken from numerous organs for mineral analyses. Aluminum retention was increased in the SS calves (p = 0.001). Silicon concentrations were increased in the aorta, spleen, lung, muscle, and kidney of the SS calves, and aluminum was increased in all SS tissues (p < 0.05). Calcium concentrations were increased in aorta, liver, muscle, and tendon; phosphorus concentrations were increased in aorta, but decreased in plasma; magnesium concentrations were increased in aorta, heart, kidney, liver, and pancreas, but decreased in plasma; and iron concentrations were decreased in kidney and liver (p < 0.05). The accumulation of tissue aluminum and therefore potential adverse consequences may preclude any benefits of using SZA as a dietary supplement.     

Induction and floral determination in the terminal bud of Nicotiana tabacum L. cv. Maryland Mammoth,a short-day plant

Floral determination in the terminal bud of the short-day plant Nicotiana tabacum cv. Maryland Mammoth has been investigated. Plants grown continuously in short days flowered after producing 31.4±1.6 (SD) nodes while plants grown continuously in long days did not flower and produced 172.5±9.5 nodes after one year. At various ages, expressed as number of leaves that were at least 1.0 cm in length above the most basal 10-cm leaf, one of three treatments was performed on plants grown from seed in short days: 1) whole plants were shifted from short days to long days, 2) the terminal bud was removed and then rooted and grown in long days, and 3) the terminal bud was removed and then rooted and grown in short days. Whole plants flowered only when shifted from short days to long days at age 15 or later. Only rooted terminal buds from plants at age 15 or older produced plants that flowered when grown in long days. Only terminal buds from plants at age 15 or older that were rooted and grown in short days produced the same number of nodes as they would have produced in their original locations while buds from younger plants produced more nodes than they would have in their original locations. Thus, determination for floral development in the terminal bud, as assayed by rooting, is simultaneous with the commitment to flowering as assayed by shifting whole plants to non-inductive conditions.Abbreviations LD long day(s) - SD short day(s) - DN dayneutral     

Design,synthesis and evaluation of potent and selective inhibitors of mono-(ADP-ribosyl)transferases PARP10 and PARP14

A series of diaryl ethers were designed and synthesized to discern the structure activity relationships against the two closely related mono-(ADP-ribosyl)transferases PARP10 and PARP14. Structure activity studies identified 8b as a sub-micromolar inhibitor of PARP10 with?～15-fold selectivity over PARP14. In addition, 8k and 8m were discovered to have sub-micromolar potency against PARP14 and demonstrated moderate selectivity over PARP10. A crystal structure of the complex of PARP14 and 8b shows binding of the compound in a novel hydrophobic pocket and explains both potency and selectivity over other PARP family members. In addition, 8b, 8k and 8m also demonstrate selectivity over PARP1. Together, this study identified novel, potent and metabolically stable derivatives to use as chemical probes for these biologically interesting therapeutic targets.     

Evaluation of chalcones as inhibitors of glutathione S‐transferase

Glutathione S‐transferases (GSTs) are the superfamily of multifunctional detoxification isoenzymes and play an important role in cellular signaling. In the present study, potential inhibition effects of chalcones were tested against human GST. For this purpose, GST was purified from human erythrocytes with 5.381 EU?mg^?1 specific activity and 51.95% yield using a GSH–agarose affinity chromatographic method. The effects of chalcones on in vitro GST activity were tested at various concentrations. K_i constants of chalcones were found in the range of 7.76–41.93 μM. According to the results, 4‐fluorochalcone showed a better inhibitory effect compared with the other compounds. The inhibition mechanisms of 2'‐hydroxy‐4‐methoxychalcone and 4‐methoxychalcone were noncompetitive, whereas the inhibition mechanisms of 4'‐ hydroxychalcone, 4‐ fluorochalcone, and 4,4'‐ diflurochalcone were competitive.     

Direct gene transfer into Actinidia deliciosa protoplasts: analysis of transient expression of the CAT gene using TLC autoradiography and a GC-MS-based method

Chloramphenicol acetyl transferase (CAT) gene was used as a reporter gene to assess the conditions for polyethylene glycol (PEG)-mediated transfection of kiwifruit protoplasts. The effect of plasmid concentration and the presence of carrier DNA were each assessed by analysing CAT activity in transfected protoplasts using thin-layer chromatography (TLC) autoradiographic detection of acetylated chloramphenicol. A gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) non-radioactive method was developed for monitoring CAT gene activity. This method provides a high speed of analysis (30 min) and precise means of detecting acetylated products at the nanomolar level, enabling quantification at very low transfection rates. Using this method we optimized plasmid and PEG concentration and also assessed the effect of heat shock on transfection. The best CAT activity was obtained using 30% polyethylene glycol 4000 and by submitting protoplasts to heat shock (45 °C, 5 min) prior to transfection.     

Distribution of glutathione transferases in Gram-positive bacteria and Archaea

Glutathione transferases (GSTs) have been widely studied in Gram-negative bacteria and the structure and function of several representatives have been elucidated. Conversely, limited information is available about the occurrence, classification and functional features of GSTs both in Gram-positive bacteria and in Archaea. An analysis of 305 fully-sequenced Gram-positive genomes highlights the presence of 49 putative GST genes in the genera of both Firmicutes and Actinobacteria phyla. We also performed an analysis on 81 complete genomes of the Archaea domain. Eleven hits were found in the Halobacteriaceae family of the Euryarchaeota phylum and only one in the Crenarchaeota phylum. A comparison of the identified sequences with well-characterized GSTs belonging to both Gram-negative and eukaryotic GSTs sheds light on their putative function and the evolutionary relationships within the large GST superfamily. This analysis suggests that the identified sequences mainly cluster in the new Xi class, while Beta class GSTs, widely distributed in Gram-negative bacteria, are under-represented in Gram-positive bacteria and absent in Archaea.