Recombinant human glucose-6-phosphate dehydrogenase. Evidence for a rapid-equilibrium random-order mechanism.

Cloning and over-expression of human glucose 6-phosphate dehydrogenase (Glc6P dehydrogenase) has for the first time allowed a detailed kinetic study of a preparation that is genetically homogeneous and in which all the protein molecules are of identical age. The steady-state kinetics of the recombinant enzyme, studied by fluorimetric initial-rate measurements, gave converging linear Lineweaver-Burk plots as expected for a ternary-complex mechanism. Patterns of product and dead-end inhibition indicated that the enzyme can bind NADP+ and Glc6P separately to form binary complexes, suggesting a random-order mechanism. The Kd value for the binding of NADP+ measured by titration of protein fluorescence is 8.0 microm, close to the value of 6.8 microm calculated from the kinetic data on the assumption of a rapid-equilibrium random-order mechanism. Strong evidence for this mechanism and against either of the compulsory-order possibilities is provided by repeating the kinetic analysis with each of the natural substrates replaced in turn by structural analogues. A full kinetic analysis was carried out with deaminoNADP+ and with deoxyglucose 6-phosphate as the alternative substrates. In each case the calculated dissociation constant upon switching a substrate in a random-order mechanism (e.g. that for NADP+ upon changing the sugar phosphate) was indeed constant within experimental error as expected. The calculated rate constants for binding of the leading substrate in a compulsory-order mechanism, however, did not remain constant when the putative second substrate was changed. Previous workers, using enzyme from pooled blood, have variously proposed either compulsory-order or random-order mechanisms. Our study appears to provide unambiguous evidence for the latter pattern of substrate binding.     

切鳍标记对唐鱼游泳能力的影响

本研究通过测定完全切除背鳍、臀鳍、尾鳍或双侧胸鳍后唐鱼（Tanichthys albonubes）临界游泳速度来评价切鳍标记对游泳能力的影响。研究结果表明,在速度增量（ΔU）为36 mm/s,持续时间（Δt）分别为5 min、10 min、15 min、20 min、25 min、30 min的条件下,唐鱼临界游泳速度会随着持续时间的延长而降低。鳍组织完整的唐鱼〔体长（24 ± 1）mm〕,在不同持续时间（Δt）条件下,其绝对临界游泳速度（Uacrit）为（251.98 ± 11.04）mm/s ~（333.78±12.44）mm/s;同等条件下,切除唐鱼的背鳍或臀鳍均不会对唐鱼的绝对临界游泳速度造成显著影响（P＞0.05）,但切除唐鱼的尾鳍或胸鳍后其临界游泳速度与对照组相比极显著降低（P＜0.01）,其中切除尾鳍后绝对临界游泳速度平均下降47.20%,切除胸鳍后平均下降22.98%,两者间存在极显著的差异（P＜0.01）(图2)。研究表明背鳍切除可作为野外唐鱼短期标记-重捕的手段之一。     

白银寿的组织培养与快速繁殖

1 植物名称Haworthia emelyae,car.emelyae V.Poelln,中文惯称白银寿. 2 材料类别成年的白银寿春生花茎子房,实验材料来源于日本奈良多肉植物研究会. 3 培养条件(1)启动培养基:MS 6-BA2mg·L-1(单位下同) KT1 NAA 0.2;(2)叶基分化培养基:MS 6-BA0.5 KT2;(3)继代与增殖培养基:MS 6-BA 0.5 KT1 NAA 0.05;(4)复壮与生根培养基:1/2MS DPU(3,31'-二苯基脲)1 NAA 0.2.以上培养基均加入3%蔗糖和1.6%聚合胶(polygel),pH 6.0.培养温度为(25±2)℃,光照时间8 h·d-1,光照强度约为60μmol·m-2·s-1.     

Hierarchical responses of plant stoichiometry to nitrogen deposition and mowing in a temperate steppe

Background and aims

Stoichiometric relations drive powerful constraints on many ecosystem processes. However, our understanding of the hierarchical responses of plant C:N:P stoichiometry at different levels of biological organization to global change factors remains limited.

Methods

we examined the plant C:N:P stoichiometric responses to N deposition and mowing (hay making) at both species- and community-level by carrying out a 4-year field experiment in the temperate steppe of northern China.

Results

Our results showed that N addition and mowing resulted in higher plant N concentrations, lower C:N, and higher N:P at both species- and community-level. Mowing had a limited negative influence on the effects of N addition. We observed divergent responses of both plant P concentrations and C:P to N addition at species-level and community-level: N addition led to higher plant P and lower C:P at species-level, but this effect was not observed at the community-level.

Conclusions

Our results indicate that stoichiometric responses at community-level to N addition and mowing diverge from more traditionally examined species-specific responses. Our results suggest that the hierarchical responses of plant stoichiometry to anthropogenic disturbance deserves more attention when we model the interactions of terrestrial ecosystem C, N, and P cycling under scenarios of increasing N availability concomitantly occurring with active land management.     

Diversity and composition of understory vegetation in the tropical seasonal rain forest of Xishuangbanna, SW China

Tropical forests vegetation and community research have tended to focus on the tree component, and limited attention has been paid to understory vegetation. Species diversity and composition of the understory of tropical seasonal rain forest were inventoried in a 625 m2 area (for sapling layer) and a 100 m2 area (for herb/seedling layer) in three 1 ha plots. We found 3068 individuals belonging to 309 species, 192 genera and 89 families. The most important family as determined by the Family Importance Value (FIV) was Rubiaceae in both sapling and herb/seedling layers. In terms of Importance Value Index (IVI), the shrub Mycetia gracilis (Rubiaceae) was the most important species in the sapling layer and the pteridophyte Selaginella delicatula (Selaginellaceae) was the most ecological significant species in the herb/seedling layer. Much more vascular plant species were registered in the understory than in the tree layer totaled among the three plots. The species diversity did not differ significantly among the tree layer, sapling layer and herb/seedling layer. Given that we still know little about the understory plant community for growth forms other than trees, the results from the present study indicate that more attention should be paid to the understory vegetation during the decision-making process for biodiversity conservation in the tropical forests.     

卵形鲳鯵对刺激隐核虫的免疫应答和免疫保护研究

用刺激隐核虫(Cryptocaryon irritans)的幼虫对卵形鲳鯵(Trachinotus ovatus)进行腹腔注射和体表感染,然后每隔一周用阻动试验(Immobilization assay)检测免疫鱼的抗血清和皮肤培养液对刺激隐核虫幼虫的阻动效价,在第14周中,分别用亚致死剂量和致死剂量的刺激隐核虫幼虫对免疫鱼攻毒以检测所产生的免疫保护力。实验结果显示:两种免疫方法都能让卵形鲳鯵的血清和皮肤生成阻动刺激隐核虫幼虫的特异性抗体,并能使被免疫鱼获得明显的免疫保护,但是体表感染免疫组的血清和皮肤培养液的阻动效价都要比腹腔注射免疫组高,所获得的免疫保护力也更强。同时还发现,免疫鱼血清和皮肤培养液中的抗体存在明显的差异:两者的最初生成时间、达到峰值的时间、变化规律以及阻动效价等都不一致。因此,我们推测鱼类的系统免疫应答和皮肤黏膜免疫应答有可能是相互独立的,或者是不同步的。鱼类的体液免疫应答,特别是黏膜免疫应答对抵御刺激隐核虫的感染起了重要的作用,采用刺激隐核虫虫体疫苗可能成为预防海水鱼类白点病的一种选择。       

卵形鲳鲹对刺激隐虫的免疫应答和免疫保护研究

用刺激隐核虫(Cryptocaryon irritans)的幼虫对卵形鲳NFDA8(Trachinotus ovatus)进行腹腔注射和体表感染,然后每隔一周用阻动试验(Immobilization assay)检测免疫鱼的抗血清和皮肤培养液对刺激隐核虫幼虫的阻动效价,在第14周中,分别用亚致死剂量和致死剂量的刺激隐核虫幼虫对免疫鱼攻毒以检测所产生的免疫保护力.实验结果显示:两种免疫方法都能让卵形鲳鲹的血清和皮肤生成阻动刺激隐核虫幼虫的特异性抗体,并能使被免疫鱼获得明显的免疫保护,但是体表感染免疫组的血清和皮肤培养液的阻动效价都要比腹腔注射免疫组高,所获得的免疫保护力也更强.同时还发现,免疫鱼血清和皮肤培养液中的抗体存在明显的差异:两者的最初生成时间、达到峰值的时间、变化规律以及阻动效价等都不一致.因此,我们推测鱼类的系统免疫应答和皮肤黏膜免疫应答有可能是相互独立的,或者是不同步的.鱼类的体液免疫应答,特别是黏膜免疫应答对抵御刺激隐核虫的感染起了重要的作用,采用刺激隐核虫虫体疫苗可能成为预防海水鱼类白点病的一种选择.     

猪链球菌2型全基因组DNA芯片的研制及基因表达谱技术平台的建立

目的：研制猪链球菌2型（SS2）全基因组DNA芯片,建立SS2基因表达谱技术平台。方法：利用SS2全基因组序列,挑选出2194条基因,经PCR扩增出2156条基因并将产物纯化,点样制备芯片;将芯片用于表达谱研究,采用实时定量PCR验证表达谱结果,对芯片进行可靠性分析。结果：芯片杂交数据与实时定量PCR验证显示了较高的相关性,二者相关系数r=0.87。结论：研制了一批SS2全基因组DNA芯片,并建立了基于DNA芯片的表达谱技术平台。     

基于Avi-tag技术的人胚肾细胞内增强型绿色荧光蛋白的生物素化、纯化和检测方法

目的:建立并优化基于Avi-tag标签技术的人胚肾细胞增强型绿色荧光蛋白(eGFP)的定点生物素化标记、纯化和检测方法。方法:分别构建具有Avi-tag标签的eGFP真核表达载体plenti-Avi-eGFP和BirA酶真核过表达载体pQCXIH-BirA,将plenti-Avi-eGFP和pQCXIH-BirA共转染人胚肾293T细胞,12 h后观察Avi-tag标签对eGFP蛋白在细胞内定位的影响;48 h后裂解细胞,用链霉亲和素珠子纯化生物素标记的eGFP,SDS-PAGE观察eGFP纯化和富集情况,并优化基于Western印迹的生物素化eGFP检测方法。结果:Avi-tag标签对eGFP在细胞内的定位无影响,同时BirA酶在293T细胞内可将带Avi-tag标签的eGFP标记上生物素;生物素化的eGFP可特异性地被链霉亲和素珠子纯化和富集,纯度可达95%;Western印迹检测生物素化蛋白的最终条件为5%的BSA作为封闭液和终浓度为100 ng/mL的链霉亲和素-HRP。结论:建立了基于Avi-tag技术的人胚肾细胞内增强型绿色荧光蛋白的生物素化标记、纯化与检测方法,为该方法的广泛应用奠定了前期技术基础。     

非泛素依赖地降解蛋白质研究进展

如何识别和选择性降解蛋白质是细胞生命过程中非常重要的环节,泛素-蛋白酶体需能降解途径的发现,揭示了蛋白质在细胞内选择性降解的普遍方式,成为研究焦点.然而,很少关注蛋白酶体以非泛素依赖方式降解蛋白质的可能性.近年来,已发现不少蛋白质被蛋白酶体以非泛素依赖方式降解.该途径涉及降解某些短寿命的调节蛋白、错误折叠蛋白、衰老蛋白和氧化蛋白,以及新合成蛋白的"质量控制",并涉及病理过程如癌症、神经退行性疾病,所以具有非常重要的生理和病理作用.总结了近一二十年来发现的一些具有代表性的被蛋白酶体以非泛素依赖方式降解的蛋白质,并重点论述了其作用的分子机制,以期以点带面地展示这一领域的研究概况.