The use of limited proteolysis to probe interdomain and active site regions of beta-galactosidase (Escherichia coli)

Limited proteolysis by pancreatic elastase (EC 3.4.21.36) and chymotrypsin (EC 3.4.21.1) was used to study the domain structure and active site of beta-galactosidase (EC 3.2.1.23) (Escherichia coli). Treatment with elastase resulted in a rapid cleavage between residues Ala-732 and Ala-733. No inactivation accompanied this cleavage suggesting that this bond is in a hinge region of the protein. Some slow cleavages beyond the initial one were observed to occur and were accompanied by inactivation. Treatment of beta-galactosidase with chymotrypsin resulted in cleavages first between Trp-585 and Ser-586 and then between Phe-601 and Cys-602. The first of these cleavages resulted in total inactivation of beta-galactosidase. The presence of monovalent ions or isopropyl-beta-D-thiogalactopyranoside protected against the cleavages but when Mg2+ or Mn2+ was present in the reaction mixture, the bond between Trp-585 and Ser-586 was more susceptible to the action of chymotrypsin. These data demonstrate that the conformation of beta-galactosidase around Trp-585 and Ser-586 is dramatically affected by the binding of ions and isopropyl-beta-D-thiogalactopyranoside. The mutant M15 beta-galactosidase, which is missing residues 11 through 41 and is an inactive dimer rather than an active tetramer, was found to be much more labile to proteases than native beta-galactosidase, but the same initial cleavages were found to occur. In addition, trypsin cleaved the M15 protein between Arg-431 and Trp-432 while native beta-galactosidase was stable to trypsin.     

大劣按蚊吸血活动与交配受精的关系

大劣按蚊Anophelos dirus是我国及东南亚地区的重要传疟媒介。研究表明成蚊羽化后,在未喂血的情况下,可有较高的交配受精率。在同一蚊笼,吸血机会完全相同的条件下,大蚊笼受精雌蚊吸血率为59.88%,未受精雌蚊的吸血率为35.38%;小蚊笼受精雌蚊的吸血率为74.13%,未受精雌蚊吸血率仅为43.56%。正常交配雌蚊的吸血率为49.71%,未进行交配的处女雌蚊吸血率为28.42%。同为正常交配蚊群,喂血组雌蚊受精率55.85%,不喂血组的受精率为44.51%。初步认为大劣按蚊的吸血活动与交配受精有一定的关系。     

糙叶败酱挥发油镇静作用的研究

本文观察了败酱科植物糙叶败酱(Patrinia scabra Bunge)根和根茎中制得的挥发油的镇静作用,井与黄花败酱挥发油做了比较。结果表明,此油灌胃给予数组小鼠,剂量0.45ml/kg,显示如下的作用:[1]能显著延长由于腹腔注射戊巴比妥钠(50mg/kg)引起的小鼠睡眠时间,但其作用强度弱于黄花败酱挥发油。(2)一次灌胃给予小鼠大剂量10.46g/kg的糙叶败酱挥发油,连续观察10天,动物外观正常,无一死亡,体重增加与对照组相似。     

鲢、鳙在天然条件下的摄食强度(Ⅱ)武汉东湖鲢、鳙周年摄食强度的研究

武汉东湖的鲢(Hypophthalmichthy molitrix)鳙(Aristichthys nobilis)在天然条件下摄食强度具季节性变化。摄食强度高峰处于夏季,低谷处于冬季。在实验条件下,按周年采样期间水温变化范围,测定鱼的肠管排空率。食物通过鱼肠管时间(Y_p—h)与水温(X_t—℃)的关系为: 鲢Y_p=270.63 X_t~(0.6408) 鳙Y_p=280.46 X_t~(0.6642) 根据修正后Bajkov公式(D=C (24.A)/n),估算鱼的日粮。鱼日粮(Y_D)与水温(X_t)关系为: 鲢Y_D=0.2683e~(0.1503X_t) 鳙Y_D=0.0075X_t~(2.2715) 计算鱼在天然条件下周年月粮及年粮。鲢、鳙对天然饵料年消耗量分别为18.924公斤及17.39公斤,饵料系数分别为18.02及13.38。     

“引黄济青”输水沿线水生生物分布与蓄水库富营养化关系的研究

“引黄济青”是国家“七五”重点工程,由黄河下游博兴县境内打渔张闸引水,在渠首沉沙后,再经惠民、东营、潍坊和青岛的10个县区约253km的明渠输送,到达唠山县棘洪滩蓄水库蓄存,实现向青岛市日供水3.0×10~5m~3。明渠输水过程中,来白沿线的营养物质会污染水体,加之输水在库中滞留时间长,会不会发生富营养化,这是社会普遍关注的问题。     

自养黄杆菌合成羟基丁酸和羟基戊酸共聚体的发酵研究

采用本实验室从土壤中分离到的一株自养黄杆菌进行了羟基丁酸和羟基戊酸共聚体〔P(HB-co-HV)〕的发酵试验。实验结果表明,该菌株是自养黄杆菌葡萄糖运输突变株,可以葡萄糖、果糖、蔗糖、麦芽糖、乙酸盐、乳酸盐和苹果酸盐作为唯一碳源,尤以葡萄糖和果糖效果最佳。硫酸铵、氯化铵和蛋白胨等不同氮源不影响其生长,却影响细胞中P(HB-co-HV)的含量和P(HB-co-HV)中HV/HB的比例。应用两阶段控制方式,经42h的补料分批发酵,细胞浓度达34.9g·L~(-1),P(HB-co-HV)浓度达25.28g·L~(-1)。细胞和P(HB-co-HV)生产速率系数分别为0.83g·L~(-1)”·h~(-1)和0.61g·L~(-1)·h~(-1)。以基质为基准的细胞得率系数(Yx/s)、产物得率系数(Yp/s)和以干细胞为基准的产物得率系数(Yp/x)分别为0.283(g/g)、0.174(g/g)和0.73(g/g)。改变培养基中碳氮源组分可将P(HB-co-HV)中HB的含量调节在24％～78％之间。     

湿地松粉蚧夏季数量凋落的原因分析

利用生命表技术及其相应的控制指数分析方法,对新侵入害虫湿地松粉蚧Ｏｒａｏｅｌｌａａ－ｃｕｔａ（Ｌｏｂｄｅｌｌ）夏季数量凋落的某些因子进行了量化分析。结果表明：在广东南部的新侵入区,夏季高温引起的松梢迅速老化,上代为害以后引起的营养质量的变化、拥挤以及煤污病的严重发生等,均对湿地松粉蚧夏季种群数量的凋落有着明显的影响,其总的排除作用控制指数ＥＩＰＣ为４６．８９,如果排除这几个因子的作用,下代种群的数量将为当代的４６．８９倍。     

脑桥呼吸调整中枢向中缝大核下行投射的研究

实验在23只苯巴比妥钠麻醉(i.p.30mg/kg)的成年猫上进行。在脑桥呼吸调整中枢(NPBM-KF)共记录到67个单位放电可被电刺激中缝大核(NRM)所逆行兴奋。其中有7个单位为呼吸相关性单位(吸气性6、呼气性1),占脑桥87个呼吸相关性单位总数的8％。逆行兴奋潜伏期在0.4—2.5ms之间,平均1.2ms。中缝大核内微量注入麦角辣根过氧化酶(WGA-HRP)后在NPBM-KF区观察到大量HRP标记神经元。本实验结果表明,发自脑桥呼吸调整中枢神经元的轴突可投射到中缝大核。这一投射通路可能与呼吸及痛觉调节有关。     

褪黑素调节烟草丁香假单胞菌抗性的功能研究

为分析褪黑素(N-乙酰-5-甲氧基色胺)在植物先天免疫中的功能及调控机理,研究以病原菌丁香假单胞杆菌(Pseudomonas syringae pv.tomato DC3000,Pst DC3000)—烟草互作系统为模型,检测了病原菌侵染对烟草褪黑素相关基因表达的影响,并探讨了褪黑素对植物叶片病原菌生长以及气孔开度和活性氧自由基(reactive oxygen species,ROS)含量的影响以及调控机理。结果表明:(1)Pst DC3000处理提高了烟草褪黑素合成(NtSNAT1)和受体(NtPMTR1)基因表达,且外源褪黑素处理降低了叶片中的病原菌含量。(2)与野生型植物相比,过表达大豆GmSNAT1基因显著提高了转基因烟草中内源褪黑素含量和NtPMTR1的表达,且转基因烟草叶片中的Pst DC3000菌落数显著下降。(3)外源褪黑素和细菌鞭毛蛋白多肽flg22处理诱导了野生型和转基因烟草保卫细胞中ROS产生和气孔关闭,且转基因植物对褪黑素和flg22诱导的气孔关闭和ROS产生比野生型烟草更加敏感。综上所述,研究表明褪黑素可能通过受体NtPMTR1介导的信号途径促进保卫细胞ROS产生,诱导气孔关闭,从而降低病原菌Pst DC3000的入侵。     

Poplar (Populus nigra L.) plants transformed with aBacillus thuringiensis toxin gene: insecticidal activity and genomic analysis

Insect-resistant poplar (Populus nigra L.) plants have been produced by infecting leaves withAgrobacterium tumefaciens strains carrying a binary vector containing different truncated forms of aBacillus thuringiensis (B.t.) toxin gene under a duplicated CaMV 35S promoter. Putative transgenic plants were propagated by cuttings at two experimental farms (in Beijing and Xinjiang, China). At 2–3 years after transformation, 17 of them were selected on the bases of insect-tolerance and good silvicultural traits, and evaluated for insect resistance, for the presence of theB.t. toxin DNA fragment (Southern blots and PCR) and for the expression of the transgene (western and northern blots). Somaclonal variation, as suggested by the appearance of permanent changes in the shape of the leaves, was also investigated with molecular tools (RFLP (restriction fragment length polymorphism), RAPD (random amplified polymorphic DNA) and microsatellite DNA).Bioassays withApochemia cineraius andLymantria dispar on the leaves of the selected clones showed different and, in some cases, high levels of insecticidal activity. The molecular analysis demonstrated integration and expression of the foreign gene. Somatic changes were correlated to extensive genomic changes and were quantified in dendrograms, in terms of genomic similarity. The analysis of control plants suggested that genomic changes were correlated to thein vitro culture step necessary forA. tumefaciens-mediated gene transfer, rather than to the integration of the foreign genes.Three transgenic clones (12, 153 and 192), selected for insect resistance, reduced morphological changes and promising silvicultural traits, are now under large-scale field evaluation in six different provinces in China.