Reduced enzymatic activity of glucokinase after affinity labeling: results from spectrophotometry and electrospray ionization mass spectrometry

Glucokinase catalyzes phosphoryl group transfer from ATP to glucose to form glucose-6-phosphate in the first step of cellular metabolism. While the location of the ATP-binding site of glucokinase was proposed recently, limited information exists on its conformation or the key amino acids involved in substrate binding. Affinity labeling with phenylglyoxal is used to probe possible Arg residues involved in ATP binding. Electrospray ionization mass spectrometry indicates that reaction of purified glucokinase with phenylglyoxal results in as many as six or seven sites of modification, suggesting nonspecific modification. However, preincubation of glucokinase with glucose followed by reaction with phenylglyoxal reveals only two sites of modification. Glucokinase activity assays show that enzyme preincubated with glucose possesses residual activity corresponding to the fraction of unmodified enzyme observed by mass spectrometry, strongly suggesting that glucokinase preincubated with glucose is specifically labeled and inactivated upon modification by phenylglyoxal. The data support the existing conformational model of glucokinase.     

An unexpectedly large working stroke from chymotryptic fragments of myosin II

Recent structural evidence indicates that the light chain domain of the myosin head (LCD) bends on the motor domain (MD) to move actin. Structural models usually assume that the actin-MD interface remains static and the possibility that part of the myosin working stroke might be produced by rotation about the acto-myosin interface has been neglected. We have used an optical trap to measure the movement produced by proteolytically shortened single rabbit skeletal muscle myosin heads (S-1(A1) and S-1(A2)). The working stroke produced by these shortened heads was more than that which the MD-LCD bend mechanism predicts from the full-length (papain) S-1’s working stroke obtained under similar conditions. This result indicates that part of the working stroke may be caused by motor action at the actin-MD interface.     

Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells

To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured. A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam < or = 100 nm) and to measure its local diffusion by high resolution single particle tracking. Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further. Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-stabilized complexes of 26 +/- 13 nm in size diffusing as one entity for minutes.     

Bypassing the kinetic trap of serpin protein folding by loop extension

The native form of some proteins such as strained plasma serpins (serine protease inhibitors) and the spring-loaded viral membrane fusion proteins are in a metastable state. The metastable native form is thought to be a folding intermediate in which conversion into the most stable state is blocked by a very high kinetic barrier. In an effort to understand how the spontaneous conversion of the metastable native form into the most stable state is prevented, we designed mutations of alpha1-antitrypsin, a prototype serpin, which can bypass the folding barrier. Extending the reactive center loop of alpha1-antitrypsin converts the molecule into a more stable state. Remarkably, a 30-residue loop extension allows conversion into an extremely stable state, which is comparable to the relaxed cleaved form. Biochemical data strongly suggest that the strain release is due to the insertion of the reactive center loop into the major beta-sheet, A sheet, as in the known stable conformations of serpins. Our results clearly show that extending the reactive center loop is sufficient to bypass the folding barrier of alpha1-antitrypsin and suggest that the constrain held by polypeptide connection prevents the conversion of the native form into the lowest energy state.     

用性信息素加农药诱杀器防治梨小食心虫的田间试验

在设计出性信息素加农药诱杀器后,以60、135、240、480个／km2诱杀器田间处理,看出以135个／km2诱杀器处理即有明显的防治效果。经在不同的虫口密度果园试验,以诱蛾量下降率、为害率和雌蛾交配率3项指标考察,该诱杀器1次挂出,在田间有效期达70～80天,取得了较理想防治效果。     

Restricted β-galactosidase expression of a hygromycin-lacZ gene targeted to the β- actin locus and embryonic lethality of β-actin mutant mice

Transgenic Research -     

Environmental change and the phenology of European aphids

Aphids, because of their short generation time and low developmental threshold temperatures, are an insect group expected to respond particularly strongly to environmental changes. Forty years of standardized, daily data on the abundance of flying aphids have been brought together from countries throughout Europe, through the EU Thematic Network 'EXAMINE'. Relationships between phenology, represented by date of first appearance in a year in a suction trap, of 29 aphid species and environmental data have been quantified using the residual maximum likelihood (REML) methodology. These relationships have been used with climate change scenario data to suggest plausible changes in aphid phenology. In general, the date of first record of aphid species in suction traps is expected to advance, the rate of advance varying with location and species, but averaging 8 days over the next 50 years. Strong relationships between aphid phenology and environmental variables have been found for many species, but they are notably weaker in species living all year on trees. Canonical variate analysis and principal coordinate analysis were used to determine ordinations of the 29 species on the basis of the presence/absence of explanatory variables in the REML models. There was strong discrimination between species with different life cycle strategies and between species feeding on herbs and trees, suggesting the possible value of trait-based groupings in predicting responses to environmental changes.     

Measuring trap efficiency for bark beetles (Col., Scolytidae)

Abstract:  The relative efficiency of cylindrical, linear and cross-barrier traps for trapping bark beetles was investigated based on a theoretical model. Using this model, the effective trap interception area of each trap type was calculated and trap efficiency was defined as the ratio of the effective interception area to the trap surface area. The relative efficiencies of the three trap types were calculated as the ratios of their respective effective interception areas. Based on this approach, assuming random directional movement of dispersing beetles, the order of efficiency of the three trap types, from highest to lowest, was linear, cross-barrier and cylindrical. The expected ratios of trap catches based on the relative efficiencies of the three trap types were fitted to data from trapping experiments with the mountain pine beetle ( Dendroctonus ponderosae Hopkins). In general, there was large variation in trap catches among traps of the same type but the ratios of mean catches per trap conformed to the expected ratios. The results indicate that the model of trap efficiency could be used for designing efficient traps. The methods presented are amenable for assessing the efficiency of other trap designs.     

硫酸镁微量气泵吸入治疗毛细支气管炎患儿的疗效及对肺功能的影响

目的:探讨硫酸镁微量气泵吸入治疗毛细支气管炎患儿的疗效及对肺功能的影响。方法:选择2012年1月-2014年8月我院收治的毛细支气管炎患儿120例,随机分为研究组和对照组各60例。两组均给予吸氧、吸痰、镇静、止咳、抗感染和支气管扩张剂治疗,研究组再加用25%硫酸镁溶液0.1~0.2ml/(kg.次)气泵吸入治疗,比较两组临床疗效及患儿肺功能变化。结果:研究组总有效率为98.3%,显著高于对照组的88.3%(P0.05);研究组患儿咳嗽、气促、喘息、肺部哮鸣音/啰音消失时间及住院时间显著短于对照组(P0.05);治疗后两组患儿潮气量(VT)、达峰时间比(t PTEF/t E)和v PTEF/v E均明显升高,吸呼比(Ti/Te)明显降低(P0.05),研究组VT、t PTEF/t E和v PTEF/v E显著高于对照组,Ti/Te显著低于对照组(P0.05)。结论:硫酸镁微量气泵吸入治疗小儿毛细支气管炎可以改善患儿喘憋、气促、呼吸困难等症状以及肺功能,临床效果较好。     

诱捕条件对红棕象甲聚集信息素田间效果的影响

红棕象甲Rhynchophorus ferrugineus Oliver是世界著名的严重危害棕榈植物的毁灭性入侵害虫,现已传入我国长江以南广大地区,对我国棕榈植物的安全生产造成严重威胁。本文比较了聚集信息素来源、诱捕器设置高度、颜色和类型对红棕象甲成虫诱捕效果的影响。结果表明:2种不同来源的红棕象甲聚集信息素诱捕效果无显著差异;诱捕器置于地面诱捕效果最好;红色和黑色诱捕器的诱虫量显著优于白色和黄色诱捕器;诱捕器类型对诱虫量无显著影响。本研究结果可为红棕象甲聚集信息素的监测技术提供指导。