藏药赛北紫堇总生物碱镇痛药效学研究

本文采用小鼠热板法和醋酸扭体法观察藏药赛北紫堇总生物碱的镇痛作用;藏药赛北紫堇总生物碱灌胃给药,在小鼠热板法和扭体法实验中能提高小鼠热刺激引起的痛阈值(P＜0.05),显著减少醋酸所致的小鼠扭体反应(P＜0.05).结果表明藏药赛北紫堇总生物碱具有明显的镇痛作用.     

比色法结合HPLC指纹图谱法用于诃子质量控制研究

采用比色法与HPLC指纹图谱法对诃子进行质量控制,建立了诃子中的主要药效成分总鞣质含量测定方法以及诃子的HPLC指纹图谱质量控制方法。结果诃子的总鞣质含量达到20.6%,生成的对照指纹图谱与各图谱相似度≥0.94。本实验建立的评价方法能够快速、准确、简便、全面地评价中药诃子质量,在一定程度上弥补药典中对诃子的质量控制不足之处。     

我国不同基因型水痘-带状疱疹病毒流行毒株gE基因序列分析

为进一步了解我国不同地区流行的水痘—带状疱疹病毒(VZV)病毒株的基因型分布和分析代表性病毒株gE基因序列。于2010年12月至2011年6月期间,收集来自北京市、长春市、拉萨市和乌鲁木齐市4个地区水痘和带状疱疹患者的疱疹液拭子和皮肤痂片,共计18份。用单个核苷酸多态性(SNP)谱的方法确定病毒株的基因型。然后采用聚合酶链反应(PCR)方法扩增gE基因的全长片段,并进行测序分析。SNP分析显示18个病毒株的基因型共为4种,其中有7株属于clade2遗传支,1株属于clade3遗传支,4株为clade5遗传支。另有6株病毒兼有不同遗传支的特征,按照目前国际通用的分型方法不能归属于任何明确的遗传支。对不同病毒株gE基因序列分析,除了发现3个国外已有报道的1个同义突变(T660C)和2个反义突变(C119T、C1606A),还发现了3个新的反义突变(C56T、C1109T和C917A)和4个同义突变(C54T、T1075C、T816C和G279A)。首次在我国新疆自治区发现了clade5遗传支的VZV,在长春市还发现了目前尚未能分型的6株病毒。对部分病毒株gE基因序列分析,在gE的e1和c1抗原表位的编码区内中检出1个新型反义突变(C917A),需要进一步研究该突变对该病毒免疫原性和致病性的影响。     

An improved dual-tube megaprimer approach for multi-site saturation mutagenesis

Saturation mutagenesis is a powerful tool in protein engineering. Even though QuikChange site-directed mutagenesis method is dominantly used in laboratories, it could not be successfully applied to the generation of a focused mutant library of human glutathione transferase A2-2. In the present study, we further developed an improved versatile dual-tube approach of randomizing difficult-to-amplify targets, exhibiting significant improvement towards equal distribution of nucleotides at randomized sites compared to other published methods.     

Purification and characterization of a dehalogenase from Pseudomonas stutzeri DEH130 isolated from the marine sponge Hymeniacidon perlevis

2-haloacid dehalogenases are enzymes that are capable of degrading 2-haloacid compounds. These enzymes are produced by bacteria, but so far they have only been purified and characterized from terrestrial bacteria. The present study describes the purification and characterization of 2-haloacid dehalogenase from the marine bacterium Pseudomonas stutzeri DEH130. P. Stutzeri DEH130 contained two kinds of 2-haloacid dehalogenase (designated as Dehalogenase I and Dehalogenase II) as detected in the crude cell extract after ammonium sulfate fractionation. Both enzymes appeared to exhibit stereo-specificity with respect to substrate. Dehalogenase I was a 109.9-kDa enzyme that preferentially utilized D-2-chloropropropionate and had optimum activity at pH 7.5. Dehalogenase II, which preferentially utilized L-2-chloropropionate, was further purified by ion-exchange chromatography and gel filtration. Purified Dehalogenase II appeared to be a dimeric enzyme with a subunit of 26.0-kDa. It had maximum activity at pH 10.0 and a temperature of 40 °C. Its activity was not inhibited by DTT and EDTA, but strongly inhibited by Cu²⁺, Zn²⁺, and Co²⁺. The K _m and V _max for L-2-chloropropionate were 0.3 mM and 23.8 μmol/min/mg, respectively. Its substrate specificity was limited to short chain mono-substituted 2-halocarboxylic acids, with no activity detected toward fluoropropionate and monoiodoacetate. This is the first report on the purification and characterization of 2-haloacid dehalogenase from a marine bacterium.     

Proteomic and physiological analyses reveal detoxification and antioxidation induced by Cd stress in Kandelia candel roots

The heavy metal Cadmium (Cd), added to the water bodies through weathering of rocks and human activities, constitutes one of the major environmental pollutants toxic to plants. This study examines the proteome changes in roots of actively growing Kandelia candel (L.) Druce when challenged with Cd. This mangrove-like species proliferates in estuaries and bays and is a potential choice for phytoremediation of Cd. A total of 53 proteins were up- or down-regulated following a short-term Cd treatment. The identities of the differentially expressed proteins were determined by MALDI-TOF/TOF. Approximately half of the up-regulated proteins are involved in oxidative response, including antioxidant enzymes, enzymes required for glutathione biosynthesis, enzymes in TCA and PPP cycles for generating ATP, NADH and NADPH. These results support the prediction that a prompt antioxidative response is necessary for the reduction of the oxidative stress caused by Cd and set the stage for further investigating of Cd up-regulated proteins in Kandelia candel.     

Effects of learning experience on behaviour of the generalist parasitoid Sclerodermus pupariae to novel hosts

Massicus raddei Blessig (Coleoptera: Cerambycidae), also referred to as the oak long‐horned beetle (OLB), is a non‐natural host for the generalist parasitoid Sclerodermus pupariae Yang et Yao (Hymenoptera: Bethylidae). To determine whether this generalist parasitoid might be a suitable agent for the control of OLB, the adaptive learning experience of adult female parasitoids to OLB larvae was investigated in the laboratory. A Y‐tube olfactometer bioassay was used to examine the effects of adaptive learning experience on the foraging ability of parasitoids for OLB larvae. The results indicated that parasitoids were significantly attracted by the volatiles of ash bark, Fraxinus velutina, with emerald ash borer (EAB), Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) larvae and larval frass, after exposure to ash bark mixed with EAB larval frass (learning condition A). In contrast, after exposure to oak bark, Quercus liaotungensis, mixed with OLB larval frass (learning condition C), parasitoids showed significant preference for the volatiles of oak bark with OLB larvae and larval frass. On the basis of the results of no‐choice tests, we found that parasitoids exposed to learning condition C had greater paralysis efficiency and higher OLB larvae parasitism rates than those exposed to learning condition A or no experience. Furthermore, parasitoids fed on OLB larvae in learning condition C had significantly greater paralysis efficiency and higher OLB larvae parasitism rates than other parasitoids tested. Parasitoids fed on EAB larvae in learning condition A had the lowest paralysis efficiency and OLB larvae parasitism rates among the parasitoids tested. These findings suggested that adaptive learning significantly enhanced the ability of a generalist parasitoid to utilize a novel host. This may provide a new approach to controlling non‐natural hosts using generalist parasitoids.     

Comparative proteomic and physiological analysis of diurnal changes in Nostoc flagelliforme

Nostoc flagelliforme is a terrestrial cyanobacterium species whose metabolism follows an obvious diurnal pattern. Diurnal changes at physiological and proteomic levels of N. flagelliforme were obtained. In the morning (7:00 H), net photosynthesis, dark respiration, as well as the activities of total Rubisco, nitrogenase, glutamine synthetase, SOD and CAT were comparatively high. All these physiological activities significantly decreased in the afternoon (13:00 H), and then slowly increased in the evening (19:00 H). Thirty-one differentially expressed proteins with a variety of important functions were reproducibly detected and identified over a diurnal cycle. These proteins were categorized according to their predicted functions into secretion and regulation (15.79 %), antioxidative processes (21.05 %), nitrogen metabolism (10.53 %), carbohydrate and energy metabolism (10.53 %), as well as cell division (2.63 %). The remaining proteins had unclassified/unknown functions (21.05 %) or were unidentified (18.42 %). The results suggested a metabolic shift from active (7:00 H) to quiescent (13:00 H) and then to active (19:00 H) over the diurnal cycle. The differential expression level of ferritin, Mn-CAT, SOD and Fe-SOD may serve as molecular markers for the diurnal metabolism in N. flagelliforme.