A common class of nematode glutathione S-transferase (GST) revealed by the theoretical proteome of the model organism Caenorhabditis elegans.

The genome verified C. elegans free-living nematode model is a new tool for investigating gene expression in human and animal nematode parasites. There is limited information on designating glutathione S-transferase (GST) to specific classes in lower invertebrates such as nematodes. Following cloning, amino acid sequence alignment, recombinant expression and Western blotting we provide evidence of a new GST class in nematodes or lower invertebrates.

Proteomic identification of glutathione S-transferases from the model nematode Caenorhabditis elegans.

Glutathione affinity chromatography and two-dimensional electrophoresis (2-DE) were used to purify glutathione binding proteins from Caenorhabditis elegans. All proteins identified after peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight were found to belong to the glutathione S-transferase (GST) superfamily. From the 26 individual spots identified, 12 different GSTs were isolated. Of these, five were found on the gel only once, whilst the remaining seven were represented by 21 separate spots. Most of the GSTs identified were of the nematode specific class, however, three Alpha class GSTs, a Pi and a Sigma class GST were also isolated.

Proteomic analysis of Fasciola hepatica excretory-secretory products.

This paper describes a global investigation of the components of Fasciola hepatica excretory-secretory (ES) products by a proteomic approach. Despite the absence of a F. hepatica genome sequencing project we have shown that it was possible to identify 29 of the 60 prominent proteins found using two-dimensional gel electrophoresis. As well as cathepsin L proteases, a number of enzymes implicated in parasite protection from the host immune system were also found to be present in relatively large abundance. These included superoxide dismutase, thioredoxin peroxidase, glutathione S-transferases and fatty acid binding proteins, all of which may play a part in the detoxification of reactive oxygen intermediates. Interestingly, ovine superoxide dismutase was the only protein from the host identified on the gel. We suggest that the relative abundance and protective nature of the components of the ES products of this organism play an important role in its survival within the host. The precise identification, to individual NCBI database entries, of a number of glutathione S-transferases and cathepsin Ls from F. hepatica, by peptide mass fingerprinting, was hampered by multi-database submissions of the two protein superfamilies from this organism.

Spo0A directly controls the switch from acid to solvent production in solvent-forming clostridia.

The spo0A genes of Clostridium beijerinckii NCIMB 8052 and Clostridium cellulolyticum ATCC 35319 were isolated and characterized. The C-terminal DNA-binding domains of the predicted products of spo0A from these two organisms, as well as 16 other taxonomically diverse species of Bacillus and Clostridium, show extensive amino acid sequence conservation (56% identity, 65% similarity over 104 residues). A 12-amino-acid motif (SRVERAIRHAIE) that forms the putative DNA recognition helix is particularly highly conserved, suggesting a common DNA target. Insertional inactivation of spo0A in C. beijerinckii blocked the formation of solvents (as well as spores and granulose). Sequences resembling Spo0A-binding motifs (TGNCGAA) are found in the promoter regions of several of the genes whose expression is modulated at the onset of solventogenesis in Clostridium acetobutylicum and C. beijerinckii. These include the upregulated adc gene, encoding acetoacetate decarboxylase (EC 4.1.1. 4), and the downregulated ptb gene, encoding phosphotransbutyrylase (EC 2.3.1.c). In vitro gel retardation experiments using C. acetobutylicum adc and C. beijerinckii ptb promoter fragments and recombinant Bacillus subtilis and C. beijerinckii Spo0A suggested that adc and ptb are directly controlled by Spo0A. The binding affinity was reduced when the 0A boxes were destroyed, and enhanced when they were modified to conform precisely to the consensus sequence. In vivo analysis of wild-type and mutagenized promoters transcriptionally fused to the gusA reporter gene in C. beijerinckii validated this hypothesis. Post-exponential phase expression from the mutagenized adc promoter was substantially reduced, whereas expression from the mutagenized ptb promoter was not shut down at the end of exponential growth.

Parasite proteomics.

Proteomics offers a new set of tools for investigating parasites and parasite-associated disease. In this article, John Barrett, Jim Jefferies and Peter Brophy describe the key technologies involved, including two-dimensional gel electrophoresis, image analysis, biological mass spectroscopy and database searching. The potential applications of proteomics in drug and vaccine discovery are reviewed, as are possible future developments.

Investigation of Fasciola hepatica sample preparation for two-dimensional electrophoresis.

This paper investigates the preparation of Fasciola hepatica samples for two-dimensional electrophoresis (2-DE). Whole samples were prepared by both hot sodium dodecyl sulfate (SDS) solubilisation and precipitation using trichloroacetic acid (TCA) to remove nonprotein contaminants and to inactivate endogenous proteases. Sample preparation had a marked influence on the 2-DE gel profile. TCA precipitation resulted in no measurable improvement in the profile observed, compared to the untreated control. Solubilisation of sample with hot SDS increased the number of protein spots, as did TCA precipitation with the addition of phosphotungstic acid. The preparation of excretory-secretory (ES) products poses problems due to both high salt concentrations and low protein concentration. All precipitation methods used to overcome this gave similar profiles, except acetone alone, which caused depletion of the larger proteins. TCA in acetone gave the best result, similar to that obtained by centrifugal filtration of the sample. Overcrowding of spots in some regions of the 2-DE gel occurred in the whole Fasciola hepatica sample. This problem was alleviated by differential solubilisation, which also resulted in the enrichment of some proteins.

Effect of Fasciola hepatica excretory-secretory products on the metabolic burst of sheep and human neutrophils.

F. hepatica excretory-secretory (ES) products were found to inhibit superoxide output in phorbol myristate acetate (PMA)-stimulated sheep and human neutrophils as measured by spectrophotometry. Luminol-enhanced chemiluminescence by PMA-stimulated neutrophils from both species was again inhibited. However, nitric oxide output by PMA-stimulated human neutrophils was significantly increased in the presence of ES products, whilst in sheep it was inhibited. No major effects were noted using resting neutrophils. The results are discussed in relation to the evolution of parasite defence mechanisms.

Immunomodulation of sheep and human lymphocytes by Fasciola hepatica excretory-secretory products.

Fasciola hepatica excretory-secretory (ES) products (30 micrograms ml-1 total ES protein upwards) significantly decreased tritiated thymidine (3HTh) uptake in phytolectin-stimulated sheep lymphocytes. Lower doses of ES products, however, caused an increase in 3HTh uptake in sheep cells stimulated with phytohaemagglutinin (PHA) and Concanavalin A (Con A) and, indeed, ES products were mitogenic on their own. In contrast, 3HTh uptake by human lymphocyte cultures in the presence of phytolectins was progressively inhibited by increasing doses of ES products, and ES products were not mitogenic on their own. The observed inhibitory effects on 3HTh incorporation were not due to toxicity of ES products.

Polarization and chemokinesis of ovine and human neutrophils in response to Fasciola hepatica excretory-secretory products.

The effect of Fasciola hepatica excretory-secretory products (ES products) on the polarization and chemokinesis of sheep and human neutrophils was investigated. Flukes were cultured overnight in RPMI-1640 medium, and the resulting ES products concentrated by dialysis (cut-off 1.2 kDa) before use. At the concentrations used toxicity tests showed no effect on neutrophil viability, but ES products were capable of causing morphological changes and chemokinesis in both sheep and human neutrophils. The polarizing effect of the ES products was found to be greater in the case of sheep neutrophils with 81% of cells polarizing at 80 micrograms total ES protein/ml as opposed to 36% of human neutrophils. Chemokinesis in response to 80 micrograms total ES protein/ml was significant (P < 0.01) in both species with sheep producing the stronger response. Delipidation of the ES products with Lipidex caused no loss of polarizing activity, nor did the purified lipid fraction show any polarizing effect. ES products lost polarizing activity after heat treatment or trypsin digestion. Preliminary fractionation on an S-300 Sephacryl column suggested the presence of at least four fractions capable of causing polarization in sheep neutrophils.

The minimal width of the periodontal space.

Radiographs of horizontal serial sections of dentate human mandible disclose a picture of the periodontal space, the bony margin of which is highly irregular. The minimal width of this space was measured at four points roughly 90 degrees apart and the average minimal width obtained. It is suggested that the usual clinical radiographic picture of the periodontal space of a particular tooth or teeth is such that a judgment of the width of the space cannot be made with any certainty.