Characteristics of Novel Insect Defensin-Based Membrane-Disrupting Trypanocidal Peptides

Synthetic D- and L-amino acid type cationic 9-mer peptides (all sequences were synthesized as D- or L-amino acids) derived from the active sites of insect defensins were tested for their ability to modify the growth of blood-stream form African trypanosomes in vitro. One of them, the D-type peptide A (RLYLRIGRR-NH₂), irreversibly suppressed proliferation of the Trypanosoma brucei brucei GUTat3.1 parasite. The presence of negatively charged phosphatidylserine on the surface of the parasites was demonstrated, suggesting electrostatic interaction between the peptide and the phospholipids. Furthermore, this peptide was found to alter trypanosome membrane-potentials significantly, an effect apparently due to the removal of the parasite’s plasma membrane. The potential toxic effects of D-peptide A on mammalian cells was assessed using human brain microvascular endothelial cells. Only minor effects were found when the endothelial cells were exposed for 16 h to peptide concentrations of less than 200 μM. These findings suggest that insect defensin-based peptides represent a potentially new class of membrane-disrupting trypanocidal drugs.     

Cape buffalo mitogenomics reveals a Holocene shift in the African human-megafauna dynamics

Africa is unique among the continents in having maintained an extraordinarily diverse and prolific megafauna spanning the Pleistocene-Holocene epochs. Little is known about the historical dynamics of this community and even less about the reasons for its unique persistence to modern times. We sequenced complete mitochondrial genomes from 43 Cape buffalo (Syncerus caffer caffer) to infer the demographic history of this large mammal. A combination of Bayesian skyline plots, simulations and Approximate Bayesian Computation (ABC) were used to distinguish population size dynamics from the confounding effect of population structure and identify the most probable demographic scenario. Our analyses revealed a late Pleistocene expansion phase concurrent with the human expansion between 80 000 and 10 000 years ago, refuting an adverse ecological effect of Palaeolithic humans on this quarry species, but also showed that the buffalo subsequently declined during the Holocene. The distinct two-phased dynamic inferred here suggests that a major ecological transition occurred in the Holocene. The timing of this transition coincides with the onset of drier conditions throughout tropical Africa following the Holocene Optimum (～9000-5000 years ago), but also with the explosive growth in human population size associated with the transition from the Palaeolithic to the Neolithic cultural stage. We evaluate each of these possible causal factors and their potential impact on the African megafauna, providing the first systematic assessment of megafauna dynamics on the only continent where large mammals remain abundant.     

Genetic diversity and isolation in African Buffalo (Syncerus caffer)

We studied genetic diversity in 58 buffalo from the Kruger National Park (KNP) and Willem Pretorius Nature Reserve (WPNR). Thirty-three protein-encoding loci were resolved; three were polymorphic. Average heterozygosity (H) values did not differ substantially between adult and sub-adult animals from the KNP (2.65 and 2.89%, respectively), but were lower in animals from the isolated WPNR herd (H = 1.48% and only 3% polymorphic loci compared to 9.1%). Representative levels of genetic diversity exist in the large but disease-carrying herd, whereas the smaller disease-free herds available for translocations appear less polymorphic.     

Protein crystallography and infectious diseases.

The current rapid growth in the number of known 3-dimensional protein structures is producing a database of structures that is increasingly useful as a starting point for the development of new medically relevant molecules such as drugs, therapeutic proteins, and vaccines. This development is beautifully illustrated in the recent book, Protein structure: New approaches to disease and therapy (Perutz, 1992). There is a great and growing promise for the design of molecules for the treatment or prevention of a wide variety of diseases, an endeavor made possible by the insights derived from the structure and function of crucial proteins from pathogenic organisms and from man. We present here 2 illustrations of structure-based drug design. The first is the prospect of developing antitrypanosomal drugs based on crystallographic, ligand-binding, and molecular modeling studies of glycolytic glycosomal enzymes from Trypanosomatidae. These unicellular organisms are responsible for several tropical diseases, including African and American trypanosomiases, as well as various forms of leishmaniasis. Because the target enzymes are also present in the human host, this project is a pioneering study in selective design. The second illustrative case is the prospect of designing anti-cholera drugs based on detailed analysis of the structure of cholera toxin and the closely related Escherichia coli heat-labile enterotoxin. Such potential drugs can be targeted either at inhibiting the toxin's receptor binding site or at blocking the toxin's intracellular catalytic activity. Study of the Vibrio cholerae and E. coli toxins serves at the same time as an example of a general approach to structure-based vaccine design. These toxins exhibit a remarkable ability to stimulate the mucosal immune system, and early results have suggested that this property can be maintained by engineered fusion proteins based on the native toxin structure. The challenge is thus to incorporate selected epitopes from foreign pathogens into the native framework of the toxin such that crucial features of both the epitope and the toxin are maintained. That is, the modified toxin must continue to evoke a strong mucosal immune response, and this response must be directed against an epitope conformation characteristic of the original pathogen.     

Myelin Proteolipid Protein Contains Thioester-Linked Fatty Acids

Myelin proteolipid protein (PLP) is known to contain long-chain, covalently bound fatty acids. Previous studies, including our own, have suggested the occurrence of an oxyester type of linkage between fatty acids and PLP. However, we found that protein-SH groups are required in the acylation reaction, suggesting the possible presence of thioesters. In the present study, we have examined the nature of the acyl-PLP linkages by determining whether free thiol groups are generated on removal of fatty acids. Incubation of reduced and carboxyamidomethylated proteolipid apoprotein (RCM-APL) with 0.2 M hydroxylamine and [14C]iodoacetamide at pH 7.5 and 37 degrees C resulted in the release of fatty acids and the concomitant labeling of newly formed thiol groups. Incubation with Tris or methylamine at pH 7.5 failed to remove fatty acids and generate free -SH groups. The possibility that on treatment buried thiol groups became exposed was essentially excluded because (1) similar results were obtained in 2-chloroethanol, a solvent in which acylated and deacylated PLP have the same conformation, and (2) small PLP peptides were labeled only in the presence of hydroxylamine. On incubation with [14C]methylamine at pH 9.0, RCM-APL was not labeled, thus excluding the occurrence of intramolecular thiol esters. On the other hand, fatty acids were released as radioactive N-methyl fatty acylamide, indicating the presence of intermolecular thioesters between fatty acids and protein. These results demonstrate that a large proportion of fatty acids covalently bound to PLP are liked to -SH groups.     

Cobra Venom Contains a Protein of the CRISP Family

Amino acid sequences of several fragments of the 25k protein (molecular mass 24 953 Da) previously isolated from cobra Naja kaouthia(Kukhtina et al., Bioorg. Khim., 2000, vol. 26, pp. 803–807) were determined. Their comparison with the primary structures of known proteins showed that the 25k protein belongs to the CRISP family and is the first protein of this type identified in cobra venoms.     

基于亲和素-生物素双夹心体系测定外源性蛋白血清动态浓度的非抗体依赖新方法

建立一种非抗体依赖的检测外源性蛋白多肽分子代谢动力学血清浓度及动态变化的新方法．链亲和素为捕获分子,加入待测生物素标记蛋白或合成肽,辣根过氧化物酶标记链亲和素为检测分子,构成链亲和素-生物素标记大分子-酶链亲和素的双夹心体系,并进行特异性、敏感性、准确性及稳定性评价．本检测体系灵敏度高,可达0.3125 μg/L,且可通过改变亲和素包被浓度调整检出敏感度与检测范围．准确性回收率为97.82%～107.92%,批内、批间变异系数分别< 5.76% 和< 8.42%,并成功应用在生物素标记人血清白蛋白(biotin-HSA)与生物素标记鸡卵黏蛋白(biotin-OVM)的小鼠血清浓度动态检测．本方法不依赖抗体与放射性核素,可望在外源性蛋白多肽大分子及其药物的体内定量检测和代谢动力学研究中广泛应用．     

Effects of Chemical Immobilization on Survival of African Buffalo in the Kruger National Park

ABSTRACT Capturing, immobilizing, and fitting radiocollars are common practices in studies of large mammals, but success is based on the assumptions that captured animals are representative of the rest of the population and that the capture procedure has negligible effects. We estimated effects of chemical immobilization on mortality rates of African buffalo (Syncerus caffer) in the Kruger National Park, South Africa. We used a Cox proportional hazards approach to test for differences in mortality among age, sex, and capture classes of repeatedly captured radiocollared buffalo. Capture variables did not improve model fit and the Cox regression did not indicate increased risk of death for captured individuals up to 90 days postcapture [exp (β) = 1.07]. Estimated confidence intervals, however, span from a halving to a doubling of the mortality rate (95% CI = 0.56–2.02). Therefore, capture did not influence survival of captured individuals using data on 875 captures over a 5-year period. Consequently, long-term research projects on African buffalo involving immobilization, such as associated with research on bovine tuberculosis, should result in minimal capture mortality, but monitoring of possible effects should continue.     

利用噬菌体抗体库筛选U251细胞血清应答基因蛋白特异抗体

利用噬菌体表面展示抗体库对不同血清处理U251细胞吸附的抗体进行差异筛选,筛选获得血清饥饿细胞吸附的阳性噬菌体克隆96个和血清饥饿后恢复血清培养细胞吸附的阳性噬菌体克隆82个。细胞免疫组化检测发现应答反应差异较大的抗体2个,即血清饥饿培养细胞特异反应的抗体1个（11号抗体）和血清饥饿后恢复血清培养细胞特异反应的抗体1个（2号抗体）,其中2号抗体在恢复血清培养细胞中的应答反应强于血清饥饿培养细胞,是一个血清应答基因蛋白特异抗体,且在血清饥饿后恢复血清培养不同时间的U251细胞中具有一定的特异性反应。该研究为寻找与细胞周期调控有关的因子奠定了基础,同时对肿瘤的诊断和治疗研究也有重要意义。     

蛋白质相互作用研究的新技术与新方法

目前,蛋白质相互作用已成为蛋白质组学研究的热点. 新方法的建立及对已有技术的改进标志着蛋白质相互作用研究的不断发展和完善．在技术改进方面,本文介绍了弥补酵母双杂交的蛋白定位受限等缺陷的细菌双杂交系统;根据目标蛋白特性设计和修饰TAP标签来满足复合体研究要求的串联亲和纯化技术,以及在双分子荧光互补基础上发展的动态检测多个蛋白质间瞬时、弱相互作用的多分子荧光互补技术．还综述了近两年建立的新方法：与免疫共沉淀相比,寡沉淀技术直接研究具有活性的蛋白质复合体;减量式定量免疫沉淀方法排除了蛋白质复合体中非特异性相互作用的干扰;原位操作的多表位－配基绘图法避免了样品间差异的影响,以及利用多点吸附和交联加固研究弱蛋白质相互作用的固相蛋白质组学方法.     

Serum antibody profiles in individuals with latent Mycobacterium tuberculosis infection

One‐third of the world's humans has latent tuberculosis infection (LTBI), representing a large pool of potentially active TB. Recent LTBI carries a higher risk of disease progression than remote LTBI. Recent studies suggest important roles of antibodies in TB pathology, prompting us to investigate serum antibody profiles in a cohort with LTBI. In this single‐center prospective observational study, we analyzed IgG‐antibody concentrations against five major Mycobacterium tuberculosis (Mtb) antigens (including 6 kDa early secretory antigenic target (ESAT6), CFP10, and antigen 85A, which are expressed mainly in the growth phase; and mycobacterial DNA‐binding protein 1 (MDP1) and alpha‐crystallin like protein (Acr), which are expressed in the dormant phases) in individuals with recent (n=13) or remote (n=12) LTBI, no Mtb infection (n=19), or active TB (n=15). Antibody titers against ESAT6 and MDP1 were significantly higher in individuals with recent LTBI than in those with no Mtb infection or remote LTBI. All pairwise antibody titers against these five major antigens were significantly correlated throughout the stages of Mtb infection. Five individuals with recent LTBI had significantly higher antibody titers against ESAT6 (P = 0.03), Ag85A (P = 0.048), Acr (P = 0.057), and MDP1 (P = 0.0001) than in individuals with remote LTBI; they were also outside the normal range (+2 SDs). One of these individuals was diagnosed with active pulmonary TB at 18‐month follow‐up examination. These findings indicated that concentrations of antibodies against both multiplying and dormant Mtb are higher in recent LTBI and that individuals with markedly higher antibody titers may be appropriate candidates for prophylactic therapy.     

电针对大鼠胃黏膜损伤修复的血清蛋白质差异表达研究

目的：观察电针胃经穴对大鼠胃黏膜损伤修复的血清蛋白质差异表达,为进一步阐明针刺效应的体液机理提供理论依据。方法：用表面增强激光解吸离子化飞行时间质谱（SELDI—TOF—MS）技术和WCX2（弱阳离子交换芯片）对正常大鼠血清和电针大鼠血清进行蛋白质指纹图谱检测分析,通过Biomarker Wizard和Biomarker Patterns System软件判别分析处理数据并结合生物信息学方法筛选差异表达蛋白质。结果：与正常大鼠血清比较,电针大鼠血清蛋白质在质荷比为2000-50000有25个蛋白质峰差异有显著意义,其中有19个标志蛋白在电针胃经大鼠血清中呈现高表达,6个标志蛋白在电针胃经大鼠血清中呈现低表达。结论：电针可促进胃黏膜损伤大鼠血清蛋白质差异表达,这种差异蛋白质可能与电针促进胃黏膜损伤修复效应密切相关。     

糖化血红蛋白、糖化血清蛋白与急性脑梗死预后的相关性

目的:分析急性脑梗死患者临床预后与糖化血红蛋白(HbA1c)水平及糖化血清蛋白(GSP)水平的相关性。方法:以80例健康体检者为对照组,以80例急性脑梗死患者为观察组,对比两组入组时HbA1c水平及GSP水平差异。并根据改良Ran Kin评分将观察组分为轻症组(50例)及重症组(30例),对比两组间入组时HbA1c水平及GSP水平差异。并对观察组入组时HbA1c水平及GSP水平与治疗前后改良Ran Kin评分差值的相关性。结果:观察组HbA1c水平及GSP水平均明显高于对照组(P0.05),而重症组HbA1c水平及GSP水平同样明显高于轻症组(P0.05),同时观察组入组时HbA1c水平及GSP水平与治疗前后改良Ran Kin评分差值存在显著的负向直线相关性(P0.05)。结论:急性脑梗死患者入组时HbA1c水平及GSP水平与患者预后存在显著的相关性,可用于与评价患者的临床预后。     

疟疾免疫血清对红内期疟原虫作用的研究进展

疟原虫是人体疟疾的病原体,其在人体内发育可以分为肝内期和红内期。对红内期疟原虫免疫机制的研究表明,疟原虫作为“抗原”侵入人体后,人血清中就会产生能够对抗该疟原虫的免疫活性物质,其中抗体和细胞因子发挥主要的免疫保护作用。抗体具有降低疟疾发病率的作用,然而不同抗原诱导产生的抗体的保护作用强度不同。 Th1和Th2型细胞因子在控制原虫血症、建立抗体介导的抗疟免疫机制、以及多种病理损伤的发生中发挥重要作用。着重就免疫血清中的抗体和细胞因子对红内期疟原虫的免疫作用做一概述。     

重组标签蛋白在蛋白质纯化中的研究进展

重组蛋白的表达纯化是研究蛋白质结构与功能的重要环节之一,表达重组蛋白宿主细胞的监测筛选和重组蛋白低水平可溶性表达、低回收率以及不稳定易水解等问题一直是蛋白质高通量纯化工艺中的难题。近年来,将多肽或蛋白质作为标签,与目标蛋白共表达的方法已经很大程度地解决了这一问题。因此,针对不同特性的蛋白质选择合适的标签纯化策略在重组蛋白纯化研究中是相当关键的环节。本文回顾了几种传统标签蛋白的研究概况(His-tag,Arg-tag,Flag-tag,GST-tag,MBP-tag等),着重对近年来新开发的了几种标签蛋白(Si-tag,Halo-tag,Intein-CBD-tag,ELPs-tag等)进行了深入探讨,并对新标签蛋白在蛋白质纯化中的应用前景作以展望。     

血清蛋白与4,5-二溴荧光素相互作用及其分析应用的研究

在 0 .10mol/mL的醋酸溶液中 ,4,5 二溴荧光素能与血清蛋白形成稳定的复合物 ,最大吸收波长 482nm ,与试剂比较 ,红移了 12nm。据此建立了测定血清蛋白的方法 ,用于BSA和HSA的测定 ,分别在 2～ 14mg·L-1有线性关系 ,表观摩尔吸光系数分别为 3.12× 10 5L·mol-1·cm-1和 3.2 7× 10 5L·mol-1·cm-1。应用该法测定了人血清样品总蛋白含量 ,结果令人满意。     

Serum Protein Profiling Reveals a Landscape of Inflammation and Immune Signaling in Early-stage COVID-19 Infection

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Highlights

• •In-depth profiling of the serum proteome in early-stage COVID-19 patients.
• •A landscape of inflammation and immune signaling related to the SARS-CoV-2 infection.
• •CCL2 and CXCL10 medicated cytokine signaling pathways may correlate with neutrophil and lymphocyte respectively.

     

沙丘芦苇特有内含物与水芦叶绿体及其蛋白质的相互作用特征

以水生芦苇为材料,通过吸附试验、亲和试验和荧光光谱试验探讨了沙丘芦苇(Phragmites communis Trin)的特有内含物PAAC与水芦叶绿体吸附特性及其作用位点,并在固定化的条件下研究其与水芦叶片可溶性蛋白的相互作用特点,以阐明PAAC对叶绿体的保护作用机理.结果显示:(1)水芦叶绿体对PAAC的吸附符合Freundlich吸附模型,吸附量随pH的增加而增大并于pH 7.6时达到最大值(0.86 μg/μg),吸附量虽随离子强度的提高而降低,但降幅较小.(2)PAAC可与水芦可溶性蛋白结合,0.26 mol/L的NaCl溶液能够洗脱与PAAC结合的蛋白质,两者间的相互作用以盐键为主,且结合蛋白质的种类特异性不明显.(3)PAAC与水芦蛋白质之间存在着相互结合作用并可能存在变构效应,它对蛋白质的荧光猝灭作用是生成PAAC-蛋白质复合物的静态猝灭,它与蛋白质的结合常数在1.0×10~5左右及结合位点数为1∶1.     

蛋白质微技术及其在医学领域中的应用

蛋白质微阵列是生物芯片的一种,其主要优势在于应用平面上的有序排列的许多管、腔（孔）或各自独立的点来进行样本检测,使大量样本的平行分析成为可能。应用此技术可同时分析诸多蛋白质的生物化学活性、蛋白质与蛋白质间、蛋白质与DNA间、蛋白质与RNA间,以及蛋白质与配体间的相互作用,从而在临床诊断、药物研究、环境监测、食品卫生等方面显示出其广阔的应用前景。     

植物生物反应器生产药用蛋白

以转基因植物作为生物反应器规模化生产药用蛋白已经成为国际上植物基因工程一个新的发展趋势。本文综述了这一领域的现状、优势、存在的问题及发展前景。