Vitellogenin cleavage products as indicators for toxic stress in zebra fish embryos: a proteomic approach

Vitellogenins (Vtgs) are the major yolk proteins in all oviparous animals. Systematic and regulated processing of these during embryogenesis is crucial for embryonic development. In the present study, toxicant-induced disturbance of Vtg degradation processes during Danio rerio (DR) embryogenesis was analysed to establish a sensitive tool for monitoring toxic stress at the molecular level. A 2-DE-based proteomic approach for whole DR embryos was established to study Vtg cleavage products (lipovitellin (Lv) derivatives). Ethanol was chosen as a positive control for a toxicity related change in the proteome of whole zebra fish embryos. Protein extracts from embryos treated with two ethanol concentrations, 0.5 and 2% v/v, showing either no or very strong visible effects, like absent heartbeat and blood circulation, were examined. Significant changes in the Lv pattern were detected for both conditions. The results are interpreted as scope for the use of the high abundant Lv derivatives as sensitive stress indicators in zebra fish embryos reflecting the overall fitness of the intact organisms.     

High Performance Proteomics: 7th HUPO Brain Proteome Project Workshop March 7-9, 2007 Wellcome Trust Conference Centre, Hinxton, UK

The Wellcome Trust Conference Centre at Hinxton, UK, was the meeting place of the 7th HUPO Brain Proteome Project Workshop entitled "High Performance Proteomics". It started on Wednesday, March 7, 2007 with a steering committee meeting followed by a two days series of talks dealing with the standardization and handling of tissues, body fluids as well as of proteomics data. The presentation and accompanying vivid discussions created a picture of actual strategies and standards in recent proteomics.     

Identification of novel bacterial plasminogen-binding proteins in the human pathogen Mycobacterium tuberculosis

Binding and activation of human plasminogen (Plg) to generate the proteolytic enzyme plasmin (Plm) have been associated with the invasive potential of certain bacteria. In this work, proteomic analysis together with ligand blotting assays identified several major Plg-binding spots in Mycobacterium tuberculosis soluble extracts (SEs) and culture filtrate proteins. The identity of 15 different proteins was deduced by N-terminal and/or MS and corresponded to DnaK, GroES, GlnA1, Ag85 complex, Mpt51, Mpt64, PrcB, MetK, SahH, Lpd, Icl, Fba, and EF-Tu. Binding of Plg to recombinant M. tuberculosis DnaK, GlnA1, and Ag85B was further confirmed by ELISA and ligand blotting assays. The binding was inhibited by epsilon-aminocaproic acid, indicating that the interaction involved lysine residues. Plg bound to recombinant mycobacterial proteins was activated to Plm by tissue-type Plg activator. In contrast with recombinant proteins, M. tuberculosis SE enhanced several times the Plg activation mediated by the activator. Interestingly, GlnA1 was able to bind the extracellular matrix (ECM) protein fibronectin. Together these results show that M. tuberculosis posses several Plg receptors suggesting that bound Plg to bacteria surface, can be activated to Plm, endowing bacteria with the ability to break down ECM and basal membranes proteins contributing to tissue injury in tuberculosis.     

Chemical and genetic differentiation of Ligularia tsangchanensis in Yunnan and Sichuan provinces of China

The intraspecific diversity in L. tsangchanensis collected in the Chinese Provinces Yunnan and southwestern Sichuan was studied by chemical and genetic approaches. The samples collected in Yunnan were found to contain cacalol (1) as the sole major component, while samples from Sichuan contained 7alpha- and 7beta-eremophila-9,11-dien-8-one (5 and 6) as well as the 3alpha-angeloyloxy derivative 7 as major components. In addition, the sequences of the internal transcribed spacers (ITSs) of the ribosomal RNA gene indicated that the Yunnan and the Sichuan samples constitute separate clades. These results demonstrate that L. tsangchanensis in Yunnan and Sichuan are distinct.     

Chemical and genetic diversity of Ligularia latihastata and Ligularia villosa in Yunnan Province of China

The chemical constituents of the root extracts and the nucleotide sequences of the atpB-rbcL intergenic region of Ligularia latihastata and L. villosa, collected in northwestern Yunnan Province, were studied. In the twelve collected samples of L. latihastata, two major benzofurans, 5,6-dimethoxy-2-(1-methylethenyl)-1-benzofuran (1) and euparin (2) were detected as major components. The minor compound (2R*,3S*)-5-acetyl-2,3-dihydro-6-hydroxy-2-(1-methylethenyl)-1-benzofuran-3-yl (2Z)-2-[(acetoxy)methyl]but-2-enoate (4) was found to be susceptible to artifact formation upon extraction with EtOH. The intra-specific diversity in chemical composition of the samples was small, but the diversity in the atpB-rbcL sequence was fairly large. Compounds 1 and 2 were also found in the three collected samples of L. villosa, indicating that the two species are chemically close to each other, in agreement with morphological taxonomy.     

Frequently made mistakes in electrophoresis

In spite of text books, instrument manuals, product instructions, and web tutorials there are a number of erroneous protocols around, which lead repeatedly to issues during electrophoresis runs and to inadequate results. The relatively low resolution and short running time of miniformat systems often conceals these issues. However, in high-resolution 2-D electrophoresis in large format gels, one of the most important separation methods in Proteomics, the consequences of these mistakes become more obvious.     

A fundamental regulatory role of formate on thuringiensin production by resting cell of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> YBT-032

In this work, a fundamental regulatory role of formate on thuringiensin production by resting cell of Bacillus thuringiensis YBT-032 was investigated. Nicotinamide adenine dinucleotide (NADH) production and formate dehydrogenase activity increased with formate addition from 0.5 to 2.0 g/L, respectively. However, with the formate addition of 1.5 g/L, the activities of pyruvate kinase and glucose 6-phosphate dehydrogenase reached a peak and increased by 316 and 150% relative to those of the control, respectively. In addition, intracellular production of pyruvate, aspartate, citrate and adenine were significantly enhanced by 75, 66, 32 and 78% as well. An improvement (90%) of thuringiensin production was also successfully obtained. Interestingly to point out, thuringiensin yield was closely correlative with adenine production, and the linear relationship was also observed. The results suggest that appropriate formate addition did act as a modulator and facilitate carbon flux in glycolysis and pentose phosphate pathway to synthesize adenine and thuringiensin via intracellular NADH availability.     

Membrane subproteomic analysis of Mycobacterium bovis bacillus Calmette-Guérin

Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been known for a long time to prevent tuberculosis (TB) worldwide since 1921. Nonetheless, we know little about BCG membrane proteome. In the present study, we utilized alkaline incubation and Triton X-114-based methods to enrich BCG membrane proteins and subsequently digested them using proteolytic enzyme. The recovered peptides were further separated by 2-D LC and identified by ESI-MS/MS. As a result, total 474 proteins were identified, including 78 integral membrane proteins (IMPs). Notably, 18 BCG IMPs were described for the first time in mycobacterium. Further analysis of the 78 IMPs indicated that the theoretical molecular mass distribution of them ranged from 8.06 to 167.86 kDa and pI scores ranged from 4.40 to 11.60. Functional classification revealed that a large proportion of the identified IMPs (67.9%, 53 out of 78) were involved in cell wall and cell processes functional group. In conclusion, here we reported a comprehensive profile of the BCG membrane subproteome. The present investigation may allow the identification of some valuable vaccine and drug target candidates and thus provide basement for future designing of preventive, diagnostic, and therapeutic strategies against TB.