High level expression of recombinant Mycobacterium tuberculosis culture filtrate protein CFP32 in Pichia pastoris

Difficulty in obtaining large quantities of Mycobacterium tuberculosis (MTB) proteins remains a major obstacle in the development of subunit vaccines and diagnostic reagents for tuberculosis. A major reason is because Escherichia coli has not proven to be an optimal host for the expression of MTB genes. In this article, we used the yeast Pichia pastoris to express high levels of CFP32, a culture filtrate protein restricted to the MTB complex and a potential target antigen for serodiagnosis of tuberculosis in patients. Using shaker flasks, we generated a P. pastoris clone expressing CFP32 as a secreted protein fused to the myc-(His)₆ tag, at a yield of 0.5 g of purified protein per liter of culture. Recombinant CFP32 (rCFP32) produced in P. pastoris has a molecular weight of 35 kDa, which is slightly higher than that of the native protein We identified putative acylation and glycosylation sites in the CFP32 amino acid sequence that suggested post-translational modifications may contribute to the size difference. The NH₂-terminal peptide sequencing of rCFP32 showed that the signal peptide alpha factor is correctly excised. In addition, rCFP32 reacted with the sera of patients with tuberculosis. These data are the first to show that P. pastoris is a suitable host for high-yield production of good quality mycobacterium antigens, and especially culture filtrate proteins that have vaccine and diagnostic potential.     

沙打旺组培根对其它作物种的生长干扰

采用正交设计,研究了B5培养基蔗糖和大量元素、微量元素及有机组分含量调节对沙打旺组培根生长的影响;并采用玻璃皿滤纸培养法,进行了培养滤液对萝卜和小麦幼苗生长干扰作用的研究.结果表明,培养滤液的生长干扰作用实际是化感效应;同时,营养胁迫是引发沙打旺化感效应的重要原因.有望为探明沙打旺化感作用营养胁迫机理提供初步的科学依据.     

Induction of Systemic Acquired Resistance in Arachis hypogaea L. by Sclerotium rolfsii Derived Elicitors

Plants evolve a strategy to survive the attacks of potential pathogens by inducing the microbial signal molecules. In this study, plant defence responses were induced in four different varieties of Arachis hypogaea (J‐11, GG‐20, TG‐26 and TPG41) using the fungal components of Sclerotium rolfsii in the form of fungal culture filtrate (FCF) and mycelial cell wall (MCW), and the levels of defence‐related signal molecule salicylic acid (SA), marker enzymes such as peroxidase (POX), phenylalanine ammonia lyase (PAL), β‐1,3‐glucanase and lignin were determined. There was a substantial fold increase in POX, PAL, SA, β‐1,3‐glucanase and lignin content in FCF‐ and MCW‐treated plants of all varieties of groundnut when compared to that of control plants. The enzyme activities were much higher in FCF‐treated plants than in MCW‐treated plants. The increase in fold activity of enzymes and signal molecule varied between different varieties. These results indicate that the use of fungal components (FCF and MCW) had successfully induced systemic resistance in the four different varieties of groundnut plants against Sclerotium rolfsii.     

Comparative proteomic profiling of culture filtrate proteins of less and highly virulent Francisella tularensis strains

The facultative intracellular bacterium Francisella tularensis is the causal agent of the serious infectious disease tularemia. Despite the dynamic progress, which has been made in last few years, important questions regarding Francisella pathogenicity still remain to be answered. Generally, secreted proteins play an important role in pathogenicity of intracellular microbes. In this study, we investigated the protein composition of the culture filtrate proteins of highly virulent F. tularensis subsp. tularensis, strain SCHU S4 and attenuated F. tularensis subsp. holarctica, live vaccine strain using a comparative proteomic analysis. The majority of proteins identified in this study have been implicated in virulence mechanisms of other pathogens, and several have been categorized as having moonlighting properties; those that have more than one unrelated function. This profiling study of secreted proteins resulted in the unique detection of acid phosphatase (precursor) A (AcpA), β-lactamase, and hypothetical protein FTT0484 in the highly virulent strain SCHU S4 secretome. The release of AcpA may be of importance for F. tularensis subsp. tularensis virulence due to the recently described AcpA role in the F. tularensis escape from phagosomes.     

Reaction of common bean callus to culture filtrate of Colletotrichum lindemuthianum: Differences in the composition and toxic activity of fungal culture filtrates

Culture filtrate of Colletotrichum lindemuthianum caused dark brown lesions on the lower surface veins of bean leaves. This phenomenon was used as a bioassay to study the production of toxic fungal metabolites. Calli from anthracnose-susceptible bean cultivars 'Collacia', 'Andecha' and 'Seronda' were sensitive to a 12.5% solution of race 38 filtrate or to a 25% solution of race 7 filtrate. In contrast, calli from anthracnose-resistant bean genotypes A 247, TU, PI 207262, 'Collacia' × 'Tu', 'Collacia' × AB 136 and 'Collacia' × PI 207262 did not develop browning. Culture filtrates were passed through an ionic-exchange resin and a gel filtration resin. Toxic activity of fractions from two races of the fungal pathogen was different, although in both races slight necrosis was produced by the same nine fractions. Pathogenicity could be related with common substances and toxicity could be identified with differential compounds. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro and ex vitro Selection of Potato Plantlets for Resistance to Early Blight

Reaction to the culture filtrate of Alternaria solani (Sorauer) was used as an indicator in an in vitro screening test to select lines with decreased field susceptibility to early blight from a population of 1000 putative mutants. Plantlets of cv. ‘Desirée derived from irradiated callus of potato were inoculated in vitro with a culture filtrate of A. solani (Sorauer). Of the 45 lines selected and subsequently evaluated under conditions of natural infection in the greenhouse six showed lesser degrees of early blight infection than the cv. Desirée control. The six lines selected in the greenhouse retained lower degrees of infection during 2 years of field trials.     

Selenoprotein P—Expression,functions, and roles in mammals

Selenoprotein P (Sepp1) is a secreted protein that is made up of 2 domains. The larger N-terminal domain contains 1 selenocysteine residue in a redox motif and the smaller C-terminal domain contains the other 9 selenocysteines. Sepp1 isoforms of varying lengths occur but quantitation of them has not been achieved. Hepatic synthesis of Sepp1 affects whole-body selenium content and the liver is the source of most plasma Sepp1. ApoER2, a member of the lipoprotein receptor family, binds Sepp1 and facilitates its uptake into the testis and retention of its selenium by the brain. Megalin, another lipoprotein receptor, facilitates uptake of filtered Sepp1 into proximal tubule cells of the kidney. Thus, Sepp1 serves in homeostasis and distribution of selenium. Mice with deletion of Sepp1 suffer greater morbidity and mortality from infection with Trypanosoma congolense than do wild-type mice. Mice that express only the N-terminal domain of Sepp1 have the same severity of illness as wild-type mice, indicating that the protective function of Sepp1 against the infection resides in the N-terminal (redox) domain. Thus, Sepp1 has several functions. In addition, plasma Sepp1 concentration falls in selenium deficiency and, therefore, it can be used as an index of selenium nutritional status.     

乳酸杆菌发酵滤液对人宫颈癌细胞株Hela细胞的细胞周期和细胞凋亡的影响

目的观察乳酸杆菌DM9811发酵滤液对宫颈癌细胞株Hela细胞的细胞周期和细胞凋亡的影响,探索乳酸杆菌发酵滤液对宫颈癌细胞作用的可能机制。方法用光镜、电镜和流式细胞仪分析不同浓度乳酸杆菌DM9811发酵滤液对Hela细胞凋亡的诱导效果;用流式细胞仪分析不同浓度乳酸杆菌DM9811发酵滤液对Hela细胞细胞周期的影响。结果（1）乳酸杆菌DM9811发酵滤液可诱导宫颈癌Hela细胞凋亡。形态学观察处理后的Hela细胞,可见细胞变形,细胞皱缩,体积变小,细胞间隙增大,细胞核固缩。流式细胞仪分析,1%、2%的乳酸杆菌DM9811发酵滤液在48、72h可诱导Hela细胞凋亡;5%的乳酸杆菌发酵滤液在24、48和72h均可诱导Hela细胞凋亡。（2）乳酸杆菌DM9811发酵滤液阻滞宫颈癌Hela细胞于S期,不同浓度的乳酸杆菌发酵滤液作用24、48和72h均可使S期细胞比阴性对照组增多。结论乳酸杆菌DM9811发酵滤液可诱导部分Hela细胞凋亡,其对Hela细胞的生长抑制作用可能通过S期阻滞实现。     

RDOCK: refinement of rigid-body protein docking predictions

We present a simple and effective algorithm RDOCK for refining unbound predictions generated by a rigid-body docking algorithm ZDOCK, which has been developed earlier by our group. The main component of RDOCK is a three-stage energy minimization scheme, followed by the evaluation of electrostatic and desolvation energies. Ionic side chains are kept neutral in the first two stages of minimization, and reverted to their full charge states in the last stage of brief minimization. Without side chain conformational search or filtering/clustering of resulting structures, RDOCK represents the simplest approach toward refining unbound docking predictions. Despite its simplicity, RDOCK makes substantial improvement upon the top predictions by ZDOCK with all three scoring functions and the improvement is observed across all three categories of test cases in a large benchmark of 49 non-redundant unbound test cases. RDOCK makes the most powerful combination with ZDOCK2.1, which uses pairwise shape complementarity as the scoring function. Collectively, they rank a near-native structure as the number-one prediction for 18 test cases (37% of the benchmark), and within the top 4 predictions for 24 test cases (49% of the benchmark). To various degrees, funnel-like energy landscapes are observed for these 24 test cases. To the best of our knowledge, this is the first report of binding funnels starting from global searches for a broad range of test cases. These results are particularly exciting, given that we have not used any biological information that is specific to individual test cases and the whole process is entirely automated. Among three categories of test cases, the best results are seen for enzyme/inhibitor, with a near-native structure ranked as the number-one prediction for 48% test cases, and within the top 10 predictions for 78% test cases. RDOCK is freely available to academic users at http://zlab.bu.edu/ approximately rong/dock.