蝗虫微孢子虫对蝗虫脂肪含量的影响

用东亚飞蝗Locusta migratoria manilensis作为活体寄主,将4龄蝗蝻接种蝗虫微孢子虫Nosema locustae后,对虫体总脂含量和血淋巴中的甘油脂含量、脂肪酶活力进行了测定,结果表明：蝗虫微孢子虫的寄生可导致东亚飞蝗虫体总脂含量和血淋巴甘油酯含量大幅度下降及血淋巴脂肪酶活力大幅度上升。根据病虫生理指标提出了一种新的病级鉴定方法。     

蝗虫微孢子虫对东亚飞蝗卵黄原蛋白含量的影响

采用免疫学方法,对东亚飞蝗Locusta migratoria manilensis感染蝗虫微孢子虫Nosema locustae后体内卵黄蛋白含量的变化进行了研究。结果表明,感病蝗虫与对照健虫相比,卵黄发生有严重障碍,脂肪体和卵巢中卵黄原蛋白或卵黄蛋白含量极低,导致感病雌虫丧失产卵能力。脂肪体中卵黄原蛋白含量最高峰健虫为18.7 mg/mL,而病虫只有4.7 mg/mL;血淋巴中卵黄原蛋白含量最高峰健虫为7.6 mg/mL,而病虫只有2.6 mg/mL;卵巢中卵黄蛋白含量最高峰健虫为73.4 mg/mL,而病虫只有4.9 mg/mL。     

黄粉虫β-1,3-葡聚糖识别蛋白的分离纯化及部分生物学功能

采用中性盐沉淀、凝胶层析等常规方法纯化黄粉虫Tenebrio molitor血淋巴中的β-1,3-葡聚糖识别蛋白,并对其在酚氧化酶原激活系统中的作用进行了初步的研究。结果表明:黄粉虫血淋巴的β-1,3-葡聚糖识别蛋白的分子量约为70 kDa,主要分布于血浆中。纯化的β-1,3-葡聚糖识别蛋白只能特异性地识别β-1,3-葡聚糖而不能识别肽聚糖。在β-1,3- 葡聚糖所诱导的酚氧化酶原的激活过程中,随着酚氧化酶原激活程度的提高,内源性β-1,3-葡聚糖识别蛋白的含量逐渐减少。抗β-1,3-葡聚糖识别蛋白多克隆抗体对黄粉虫血淋巴中由β-1,3-葡聚糖所诱导的酚氧化酶活性起抑制作用,且该抑制作用呈现一种剂量依赖性的趋势。上述结果有助于深入了解β-1,3-葡聚糖对黄粉虫血淋巴酚氧化酶原激活系统的激活作用。     

柞蚕感染微孢子虫后血淋巴免疫应答蛋白质的分离与鉴定

了解柞蚕Antheraea pernyi感染微孢子虫初期血淋巴内免疫系统及刺激应答相关蛋白质种类, 本研究以柞蚕5龄雌幼虫的起蚕（结束4眠, 刚完成蜕皮的幼虫）添食柞蚕微孢子虫Nosema pernyi为材料, 对感染后血淋巴利用SDS-PAGE进行分离后, 利用LC-MS/MS质谱技术和蛋白质组学分析对差异蛋白质条带进行鉴定。结果显示： 感染微孢子虫144 h后, 血淋巴中分子量约为44 kD （AP44）和28 kD （AP28）的蛋白质条带表达量增高。质谱分析AP28和AP44蛋白质条带样品, 共鉴定117个不重复蛋白质, 其中2个样品共有蛋白质12个, AP28独有蛋白质52个, AP44独有蛋白质53个。对质谱数据利用COG数据库进行搜寻鉴定, 显示AP28和AP44的鉴定蛋白质中涉及柞蚕免疫系统及刺激应答生物过程的蛋白质共有29个, 其中AP28中包括热激蛋白、 泛素样蛋白、 泛素结合酶E2、 保幼激素环氧水解酶、 微管结合蛋白、 溶菌酶、 ADP-核糖基化因子、 防御蛋白、 肽聚糖识别蛋白等15个, AP44中包括DRK、 酚氧化酶原、 类免疫球蛋白等10个; 二者共有热激蛋白hsp21.4、 酚氧化酶原、 抗菌肽等4个。本研究结果可以为今后研究柞蚕对微孢子虫的免疫应答及防御机制提供参考。     

棉铃虫血淋巴酚氧化酶活性的微量测定

昆虫血淋巴黑化的形成由激活酚氧化酶原的级联系统所引发 ,酚氧化酶在昆虫体液免疫中起着重要作用。用抗凝剂从棉铃虫血淋巴中分离获得了血浆及完整的血细胞 ,以L DOPA为底物 ,牛胰蛋白酶为激活剂 ,测定了血浆及血细胞裂解液中酚氧化酶及酚氧化酶原的活性。结果表明 ,血浆及血细胞中两者都有一定量的分布。这一昆虫血淋巴酚氧化酶的微量测定方法 ,所需样品量少 ,耗时短 ,简便易行。     

家蝇幼虫血淋巴蛋白组及血淋巴中热稳定蛋白研究

目的 初步研究家蝇幼虫血淋巴蛋白组学特征及其中的热稳定蛋白.方法 使用破壁法提取三龄幼虫血淋巴原液,设置全血淋巴组、沸水浴处理组,采用不同pH范围双向电泳胶条,分析血淋巴中蛋白的pH、分子量分布特点,采用二级质谱对蛋白点进行分子鉴定.结果 家蝇幼虫血淋巴中蛋白的分子量主要分布在10 kDa～66kDa之间,采用pH3～10的IPG胶,检测到286个蛋白点,采用pH4～7的IPG胶,测到601个蛋白点;血淋巴经沸水浴处理后,检测到41个蛋白点,分子量低于30 kDa,比未处理组减少了93.1％,对其中11个蛋白点进行质谱分析,鉴定出4个功能蛋白,包括存储蛋白、气味结合蛋白、铁蛋白重链样蛋白和铁蛋白2轻链蛋白同系物.结论 双向电泳能很好地分析家蝇幼虫血淋巴中的蛋白,血淋巴中的蛋白等电点主要分布在pH4～7之间;血淋巴经沸水浴处理后可去掉大量变性蛋白,其上清中可能包含耐热性较好的功能蛋白.     

短时高温胁迫对瓜实蝇生长发育及繁殖的影响

为明确瓜实蝇对短时高温胁迫的耐受性。利用人工气候箱模拟短时高温胁迫,测定了不同高温处理(34、36、38、40、42、44、46、48℃)12 h,对不同发育阶段瓜实蝇的存活率和生长发育的影响。结果表明短时高温显著影响瓜实蝇的存活,随温度升高,瓜实蝇各虫态的存活率逐渐降低;高温处理12 h后瓜实蝇卵、幼虫、蛹、雌成虫、雄成虫的致死中温度LT 50分别为35.48、37.55、41.85、43.62、43.32℃;34~42℃短时高温胁迫对瓜实蝇各虫态的发育历期无明显影响,44℃时其发育历期均显著增长;46℃、48℃处理下各虫态死亡率较高,不能正常发育;随着处理温度的升高,雌成虫产卵前期不断增长,单雌产卵量呈下降趋势,成虫寿命不断缩短,后代雌性比增大。44℃及以上的短时高温胁迫不利于瓜实蝇的生长发育,40℃及以上的短时高温胁迫不利于瓜实蝇的繁殖,雌性瓜实蝇对短时高温的胁迫的适应性强于雄性,随着处理温度的升高,后代雌性比例增大。     

外源保幼激素类似物对七星瓢虫血淋巴中卵黄原蛋白含量的影响

本文以不同剂量的保幼激素(JH)类似物点滴七星瓢虫雌虫,测定了生殖过程中卵母细胞的长度及血淋巴中卵黄原蛋白(Vg)及总蛋白的含量.结果表朋:1.点滴不同剂量ZR-512于虫体后,皆可促进卵母细胞生长,尤以点滴100μg效果最为明显.2.点滴ZR-512后可以促进Vg合成,血淋巴中Vg含量皆明显提高,以点滴100μg效果最好.3.点滴不同剂量ZR-512后,雌虫血淋巴中总蛋白含量变化与Vg含量变化的规律相似.4.血淋巴中的Vg含量与卵母细胞长度有一定相关性.另外,讨论了不同剂量外源JH类似物对CA活性、内源JH水平、Vg含量以及卵母细胞生长的影响.     

短时高温对意大利蝗存活和生殖的影响

【目的】明确短时高温对意大利蝗Calliptamus italicus(L.)存活和生殖的影响,为气候变暖趋势下意大利蝗灾害的预测预报及综合防治提供理论依据。【方法】采集初羽化24h内的意大利蝗雌雄成虫,于30℃光照培养箱中饲养。设置4个温度处理组,分别为33、36、39、42℃,每日处理4h,之后放回30℃光照培养箱中继续饲养。未经过短时高温处理的意大利蝗为对照组。观察记录各组意大利蝗存活率、寿命、产卵前期、产卵期、单雌产卵量、卵巢发育情况及卵巢中卵黄蛋白含量,并分析变化规律。【结果】短时高温对意大利蝗成虫存活率无显著影响,但对其寿命有显著影响(P0.01),其中33℃处理4h,意大利蝗雌雄成虫寿命显著延长(P0.01),而36~42℃范围内处理4h,意大利蝗雌雄成虫寿命显著缩短(P0.01)。短时高温对意大利蝗产卵前期有显著影响(P0.05),随温度升高,产卵前期缩短,但对产卵期没有显著影响(P0.05)。短时高温对意大利蝗单雌产卵量有显著影响(P0.01),其中33℃处理组单雌产卵量为(57.6±2.4)粒,显著高于对照组(P0.01),36~42℃范围内,随温度升高单雌产卵量显著降低(P0.01)。短时高温对意大利蝗卵巢长度、鲜重及发育历期有显著影响(P0.01),但对卵巢宽度没有显著影响(P0.05),且随温度升高,卵巢长度、宽度、鲜重及发育历期均呈下降趋势。短时高温可使卵黄蛋白在卵巢中提前沉积,并对其含量(峰值)有显著影响(P0.01),其中33℃处理组卵黄蛋白含量(峰值)为(49.795±6.253)mg/mL,显著高于对照组(P0.01),36~42℃范围内处理4h,卵黄蛋白含量(峰值)显著减少(P0.01)。【结论】33℃处理4h,意大利蝗存活及生殖特性显著提高,而36℃及以上高温对意大利蝗生长繁殖产生不利影响。     

高温对家蚕三品系血淋巴中糖水平的影响（英文）

家蚕Bombyx mori的两个二化性品系热耐受型NB4D2和热敏感型CSR2均适合于温带气候,而多化性的PM（Pure Mysore) 品系适合于热带气候,将这3种品系5龄幼虫分别置于32℃和36℃的高温下,观察高温对其5龄幼虫至蛹期血淋巴中糖含量及海藻糖酶活性的影响。结果表明： PM幼虫和蛹的死亡率均小于NB4D2和CSR2。在蜕皮期间血淋巴海藻糖水平较高,而葡萄糖水平及海藻糖酶活性较低。32℃和36℃的高温下,幼虫蜕皮期间血淋巴中糖含量及海藻糖酶活性仅在其各自的水平上表现为小幅度的增加。蜕皮后幼虫血淋巴中海藻糖含量显著下降,而葡萄糖含量和海藻糖酶活性显著上升。在较高温度下,蜕皮后幼虫血淋巴中海藻糖含量下降幅度更大,而葡萄糖含量及海藻糖酶活性上升水平也更加显著。25±1℃下取食幼虫血淋巴中葡萄糖含量显著下降,海藻糖含量显著上升;3℃和36℃下PM 和NB4D2取食幼虫血淋巴葡萄糖和海藻糖含量以及海藻糖酶活性增加,而CSR2均减少或降低。吐丝幼虫血淋巴中葡萄糖含量及海藻糖酶活性显著下降,海藻糖小幅度下降。而在较高温度下,耐热型PM 和NB4D2吐丝家蚕血淋巴糖含量含量和海藻糖酶活性明显增加,而热敏感型CSR2的则明显下降。这3种品系蛹发育期的血淋巴糖含量及海藻糖酶活性均下降。在两较高温度下,PM蛹期血淋巴糖和海藻糖酶活性增加,而NB4D2 36℃时增加幅度小于32℃时。对于CSR2,32℃时观察到其血淋巴葡萄糖含量增加,但当环境温度增加到36℃时其血淋巴葡萄糖含量降至正常水平下。然而,当CSR2的蛹置于32℃和36℃时血淋巴海藻糖含量及其酶活性下降,且36℃时下降幅度更大。因此,桑蚕对高温的适应取决于家蚕的品系及发育阶段,并可通过其血淋巴糖及海藻糖酶活性水平进行验证。     

The heat capacity of proteins

The heat capacity plays a major role in the determination of the energetics of protein folding and molecular recognition. As such, a better understanding of this thermodynamic parameter and its structural origin will provide new insights for the development of better molecular design strategies. In this paper we have analyzed the absolute heat capacity of proteins in different conformations. The results of these studies indicate that three major terms account for the absolute heat capacity of a protein: (1) one term that depends only on the primary or covalent structure of a protein and contains contributions from vibrational frequencies arising from the stretching and bending modes of each valence bond and internal rotations; (2) a term that contains the contributions of noncovalent interactions arising from secondary and tertiary structure; and (3) a term that contains the contributions of hydration. For a typical globular protein in solution the bulk of the heat capacity at 25°C is given by the covalent structure term (close to 85% of the total). The hydration term contributes about 15 and 40% to the total heat capacity of the native and unfolded states, respectively. The contribution of non-covalent structure to the total heat capacity of the native state is positive but very small and does not amount to more than 3% at 25°C. The change in heat capacity upon unfolding is primarily given by the increase in the hydration term (about 95%) and to a much lesser extent by the loss of noncovalent interactions (up to ～5%). It is demonstrated that a single universal mathematical function can be used to represent the partial molar heat capacity of the native and unfolded states of proteins in solution. This function can be experimentally written in terms of the molecular weight, the polar and apolar solvent accessible surface areas, and the total area buried from the solvent. This unique function accurately predicts the different magnitude and temperature dependences of the heat capacity of both the native and unfolded states, and therefore of the heat capacity changes associated with folding/unfolding transitions. © 1995 Wiley-Liss, Inc.     

Characterization of membrane association of Rinderpest virus matrix protein

Paramyxovirus matrix protein is believed to play a crucial role in the assembly and maturation of the virus particle by bringing the major viral components together at the budding site in the host cell. The membrane association capability of many enveloped virus matrix proteins has been characterized to be their intrinsic property. In this work, we have characterized the membrane association of Rinderpest virus matrix (M) protein. The M protein of Rinderpest virus when expressed in the absence of other viral proteins is present both in the cytoplasm and plasma membrane. When expressed as GFP fusion protein, the M protein gets localized into plasma membrane protrusions. High salt and alkaline conditions resulted in partial dissociation of M protein from cell membrane. Thus, M protein behaves like an integral membrane protein although its primary structure suggests it to be a peripheral membrane protein.     

A large scale test of computational protein design: folding and stability of nine completely redesigned globular proteins

A previously developed computer program for protein design, RosettaDesign, was used to predict low free energy sequences for nine naturally occurring protein backbones. RosettaDesign had no knowledge of the naturally occurring sequences and on average 65% of the residues in the designed sequences differ from wild-type. Synthetic genes for ten completely redesigned proteins were generated, and the proteins were expressed, purified, and then characterized using circular dichroism, chemical and temperature denaturation and NMR experiments. Although high-resolution structures have not yet been determined, eight of these proteins appear to be folded and their circular dichroism spectra are similar to those of their wild-type counterparts. Six of the proteins have stabilities equal to or up to 7kcal/mol greater than their wild-type counterparts, and four of the proteins have NMR spectra consistent with a well-packed, rigid structure. These encouraging results indicate that the computational protein design methods can, with significant reliability, identify amino acid sequences compatible with a target protein backbone.     

Characterization of the impact of alternative splicing on protein dynamics: the cases of glutathione S-transferase and ectodysplasin-A isoforms

Recent studies have shown how alternative splicing (AS), the process by which eukaryotic genes express more than one product, affects protein sequence and structure. However, little information is available on the impact of AS on protein dynamics, a property fundamental for protein function. In this work, we have addressed this issue using molecular dynamics simulations of the isoforms of two model proteins: glutathione S-transferase and ectodysplasin-A. We have found that AS does not have a noticeable impact on global or local structure fluctuations. We have also found that, quite interestingly, AS has a significant effect on the coupling between key structural elements such as surface cavities. Our results provide the first atom-level view of the impact of AS on protein dynamics, as far as we know. They can contribute to refine our present view of the relationship between AS and protein disorder and, more importantly, they reveal how AS may modify structural dynamic couplings in proteins.     

A review of visualisations of protein fold networks and their relationship with sequence and function

Proteins form arguably the most significant link between genotype and phenotype. Understanding the relationship between protein sequence and structure, and applying this knowledge to predict function, is difficult. One way to investigate these relationships is by considering the space of protein folds and how one might move from fold to fold through similarity, or potential evolutionary relationships. The many individual characterisations of fold space presented in the literature can tell us a lot about how well the current Protein Data Bank represents protein fold space, how convergence and divergence may affect protein evolution, how proteins affect the whole of which they are part, and how proteins themselves function. A synthesis of these different approaches and viewpoints seems the most likely way to further our knowledge of protein structure evolution and thus, facilitate improved protein structure design and prediction.     

Improvement of protein stability in protein microarrays

Protein stability in microarrays was improved using protein stabilizers. PEG 200 at 30% (w/v) was the most efficient stabilizer giving over 4-fold improvement in protein stability compared to without the stabilizer. PEG 200 above 10% (w/v) in the array solution prevented the evaporation of water in the sample and thereby improved protein stability in the microarray. When the streptavidin-biotin binding reaction was performed under optimized conditions, biotin-BSA-fluorescein isothiocyanate (FITC) was detected from 1 ng ml^–1 to 5 g ml^–1 by fluorescence analysis.     

Differential effects of five types of antipathogenic plant peptides on model membranes

The effects of five antipathogenic plant peptides, wheat α-thionin, potato PTH1 defensin, barley LTP2 lipid transfer protein, and potato tuber DL1 and DL2 defensins, have been tested against phospholipid vesicles (liposomes). Wheat thionin very actively induces aggregation and leakage of negatively charged vesicles. LTP2 displays the same activities, although to a limited extent. Under certain conditions PTH1 and DL2 induce vesicle aggregation, but not leakage. Potato defensin DL1 failed to show any effect on liposomes. The same peptides have been assayed against a plant pathogenic bacterium, both the membrane-active and -inactive compounds having efficient antibacterial action.     

Rapid cooperative two-state folding of a miniature alpha-beta protein and design of a thermostable variant

There is a great deal of interest in developing small stably folded miniature proteins. A limited number of these molecules have been described, however they typically have not been characterized in depth. In particular, almost no detailed studies of the thermodynamics and folding kinetics of these proteins have been reported. Here we describe detailed studies of the thermodynamics and kinetics of folding of a 39 residue mixed alpha-beta protein (NTL9(1-39)) derived from the N-terminal domain of the ribosomal protein L9. The protein folds cooperatively and rapidly in a two-state fashion to a native state typical of those found for normal globular proteins. At pH 5.4 in 20mM sodium acetate, 100mM NaCl the temperature of maximum stability is 6 degrees C, the t(m) is 65.3 degrees C, deltaH degrees (t(m)) is between 24.6 kcalmol(-1) and 26.3 kcalmol(-1), and deltaC(p) degrees is 0.38 kcalmol(-1)deg(-1). The thermodynamic parameters are in the range expected on the basis of per residue values determined from databases of globular proteins. H/2H exchange measurements reveal a set of amides that exchange via global unfolding, exactly as expected for a normal cooperatively folded globular protein. Kinetic measurements show that folding is two-state folding. The folding rate is 640 s(-1) and the value of deltaG degrees calculated from the folding and unfolding rates is in excellent agreement with the equilibrium value. A designed thermostable variant, generated by mutating K12 to M, was characterized and found to have a t(m) of 82 degrees C. Equilibrium and kinetic measurements demonstrate that its folding is cooperative and two-state.