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P M Brophy

Publications and source records attributed to P M Brophy.

At least 19 recordsLinked to original sources
PubMed2001

Biochemical characterisation of a hydrophobic ligand binding protein from the tapeworm Hymenolepis diminuta.

The cestode Hymenolepis diminuta contains an abundant, cytoplasmic, hydrophobic ligand, binding protein (H-HLBP). Studies with polarity sensitive probes suggest a single hydrophobic binding site, the results also indicate that the single tryptophan in the molecule (Trp41) is involved in ligand binding. Of the possible physiological ligands tested, only haematin and retinoids (retinol and retinoic acid) show appreciable binding in addition to fatty acids. H-HLBP also binds a range of anthelmintics, again with K(D) values in the nM range. The interaction of anthelmintics with hydrophobic binding proteins may be important in determining drug specificity and site of action and could have a role in the development of drug resistance.

Animals↗
PubMed2001

A stress-responsive glyoxalase I from the parasitic nematode Onchocerca volvulus.

Glyoxal, methylglyoxal and other physiological alpha-oxoaldehydes are formed by the lipid peroxidation, glycation and degradation of glycolytic intermediates. They are detoxified enzymically by the glyoxalase system. To investigate the physiological function of glyoxalase I in parasitic organisms, the cDNA for glyoxalase I from the filarial nematode Onchocerca volvulus (designated Ov-GloI) has been cloned and characterized. The isolated cDNA contains an open reading frame of 579 bp encoding a protein with a calculated molecular mass of 21930 Da. Owing to the high degree of sequence identity (60%) with human glyoxalase I, for which the X-ray structure is available, it has been possible to build a three-dimensional model of Ov-GloI. The modelled core of Ov-GloI is conserved compared with the human glyoxalase I; however, there are critical differences in the residues lining the hydrophobic substrate-binding pocket of Ov-GloI. A 22 kDa protein was obtained by heterologous expression in Escherichia coli. A homogeneous enzyme preparation was obtained by affinity purification and functional characterization of the recombinant enzyme included the determination of kinetic constants for methylglyoxal and phenylglyoxal as well as inhibition studies. Gel filtration demonstrated a dimeric structure. To assess the role of Ov-GloI as a potential vaccine candidate or serodiagnostic tool, the serological reactivity of the recombinant Ov-GloI was analysed with sera from microfilaria carriers and specific IgG1 antibodies were detected. The effects of oxidative insult, namely plumbagin and xanthine/xanthine oxidase, on the gene transcript level of Ov-GloI were investigated. By using a semi-quantitative PCR ELISA it was shown that Ov-GloI is expressed at elevated levels under conditions of oxidative stress.

Amino Acid Sequence↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed2001

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.

Animals↗
PubMed2001

Gene structure of the extracellular glutathione S-transferase from Onchocerca volvulus and its overexpression and promoter analysis in transgenic Caenorhabditis elegans.

Two highly similar genes encoding unique extracellular, glycosylated glutathione S-transferases (GSTs) of the human-pathogenic nematode, Onchocerca volvulus (Ov-GST1a and Ov-GST1b), have been isolated and characterised. The genes are approximately 3 kb in length and consist of seven exons interrupted by introns of approximately 100 bp in length, with the exception of intron II, which is approximately 1.6 kb in length. Interestingly, exon I and II encode a signal peptide and an N-terminal extension before sequence homology to other GSTs begins. The 5' flanking region was sequenced and analysed for transcription factor binding sites. Consistent with the lack of a TATA box, analysis of the mRNAs by primer extension showed multiple transcription start sites spread over a 60 bp region. To examine the activity and specificity of the Ov-GST1a gene promoter, we have exploited Caenorhabditis elegans as a heterologous transformation system. To analyse whether transgenic C. elegans are able to carry out processing and post-transcriptional modifications of the Ov-GST1a correctly, the protein was ectopically overexpressed in C. elegans. The parasite-derived Ov-GST1a gene product was correctly processed in transgenic C. elegans and posttranslational modifications, such as signal peptide cleavage and N-glycosylation, were performed successfully. This further demonstrates the potential of C. elegans as a host for expression of candidate vaccine antigens from O. volvulus and affirms the role of C. elegans as a model for parasitic nematodes.

Amino Acid Sequence↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed2000

A new diagnostic tool for neurocysticercosis is a member of a cestode specific hydrophobic ligand binding protein family.

A protein of unknown function has been identified as a key serological tool for diagnosis of human tapeworm neurocysticercosis, a major worldwide neurological disease. Our own sequence analysis predicts that this protein is a member of a newly identified cestode specific oligomeric hydrophobic ligand binding protein family. In this report, using a rat cestode model, we confirm that homologues of this protein can bind fatty acids and their derivatives, and thus suggest a biological function for this key diagnostic tool.

Acyl Coenzyme A↗
PubMed2000

Beta-carbonyl substituted glutathione conjugates as inhibitors Of O. volvulus GST2.

A series of beta-carbonyl substituted glutathione conjugates were prepared and evaluated as inhibitors of OvGST2. Their specificity for the parasite derived protein was assessed through comparison with their inhibition of human piGST. Inhibition of OvGST2 has been demonstrated at low micromolar concentrations for these conjugates and selectivity for OvGST2 over human pi-GST of greater than 10-fold has been achieved.

Animals↗
PubMed2000

Purification and characterisation of a novel cysteine conjugate beta-lyase from the tapeworm Moniezia expansa.

The paper presents the first report of the purification of an invertebrate cysteine conjugate beta-lyase (CCBL). CCBL activity was shown to predominate within the cytosolic fraction of tissue from the tapeworm Moniezia expansa. The monomeric cytosolic enzyme was isolated with a M(r) of 72 kDa and co-purified with transaminase activity towards L-aspartate. The substrate profile for M. expansa CCBL is different from that of mammalian CCBLs. Exploiting the differences in mammalian and parasite substrate profiles will facilitate the development of helminth targeted conjugates which will not be activated by host (mammalian) CCBLs.

Animals↗
PubMed2000

Inhibition of glutathione S-transferases (GSTs) from parasitic nematodes by extracts from traditional Nigerian medicinal plants.

Piliostigma thonningii, Ocimum gratissimum, Nauclea latifolia and Alstonia boonei are used in Nigerian traditional medicines against gastrointestinal helminths of animals and man. Proanthocyanidins were detected in Piliostigma and Nauclea, but not Alstonia or Ocimum. Extracts of these plants killed 50% of brine shrimp nauplii at <10 ppm (Nauclea), 100 ppm (Piliostigma) and <1000 ppm (Ocimum and Alstonia), the Nauclea LD50 being similar to the anthelmintic drug piperazine. Extracts were also toxic to the parasitic nematode Haemonchus infective L3 stage. Nematode glutathione-S-transferases (GSTs) are potential drug targets. Apart from Alstonia all the medicinal plants contained heat-stable inhibitory activities against recombinant Ascaris and Onchocerca GSTs in vitro. Piliostigma, Ocimum and Nauclea had IC50s of 2, 10 and 15 microg/mL respectively for Ascaris GST and 4, 8, 28 microg/mL respectively for Onchocerca GST. We suggest that the inhibitory properties of some of these Nigerian plant extracts against GST may contribute to the pharmacological basis of their efficacy against helminths in traditional herbal use.

Animals↗
PubMed2000

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.

Animals↗
PubMed2000

Comparative metal content profiling of parasitic helminths by electron paramagnetic resonance spectrometry: significance for metalloprotein content.

Variation in co-ordination geometries of metal ions bound to proteins imposes electronic states different from free (hydrated) ions in solution. Electron paramagnetic resonance spectroscopy has been used to analyse a selection of parasitic helminths for metal content as an initial step to determination of metallo-enzymes in their ES products under immune stress conditions. Characteristic paramagnetic resonance spectroscopy spectra show clear evidence for the presence of iron, copper, and manganese centres and in the selected parasites. The metals ions are identified as protein-bound as distinct from free metal ions present in aqueous solution, and distinguishable from parasite dietary components derived from host sources. Indication is given that superoxide dismutases may, in part, account for the metal ions observed. The use of electron paramagnetic resonance spectroscopy to identify specific protein-bound metals without prior isolation of the suspected protein is here applied.

Animals↗
PubMed2000

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.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1999

Cysteine conjugate beta-lyase activity in three species of parasitic helminth.

Living organisms employ a variety of metabolic pathways when detoxifying xenobiotic compounds, including the formation of cysteine S-conjugates via glutathione conjugation. However, cysteine conjugate beta-lyase (CCBL) catalysed beta-cleavage, of certain cysteine conjugates, is known to cause cytotoxicity. This study represents the first investigation into the expression of CCBL and other associated enzymes in helminth species. A survey of the three major groups of parasitic helminths [cestodes (Moniezia expansa), digeneans (Fasciola hepatica) and nematodes (Necator americanus, Heligmosomoides polygyrus)] has been made. The presence of CCBL enzymes within Moniezia expansa, Necator americanus and Heligmosomoides polygyrus has been established. Each species was screened for gamma-glutamyl transpeptidase activity and transaminase activity towards L-aspartate, L-alanine, L-albizziin and L-phenylalanine. Aspartate and alanine aminotransferase activity were detected in all four species tested. Gamma-glutamyl transpeptidase activity was only detected in Moniezia expansa and Necator americanus.

Alanine↗
PubMed1999

Effect of Fasciola gigantica somatic extracts and excretory/secretory products on superoxide production by activated neutrophils.

Somatic extracts and excretory/secretory (ES) products from Fasciola gigantica suppress the release of toxic oxygen intermediates by activated sheep neutrophils. At the concentrations used both the somatic extracts and ES products had no effect on neutrophil viability and in both cases the inhibitory effect was reversible. In the somatic extracts, heating or treatment with trypsin destroyed the inhibitory effect. However, the ES products contained at least two immunomodulatory factors. One was less than 10 kDa, the other greater than 50 kDa and both factors were heat and trypsin stable. By acting locally and only causing temporary inhibition of neutrophil function F. gigantica can protect itself from immune mediated oxidative damage without compromising the host's response elsewhere.

Animals↗
PubMed1999

Cu/Zn superoxide dismutase in excretory-secretory products of the human hookworm Necator americanus. An electron paramagnetic spectrometry study.

EPR spectrometry was used to investigate the effect of excretory/secretory product from Necator americanus on superoxide radical anions generated by xanthine/xanthine oxidase as a measure of excretory/secretory product superoxide dismutase activity. Using 1,1',5,5'-dimethylpyrollidine-N-oxide (DMPO) as a superoxide spin-trapping agent a 12-line EPR spectrum characteristic of the DMPO-OOH adduct was observed to decrease in the presence of excretory/secretory product. Superoxide dismutase activity was proportional to excretory/secretory protein concentration, was inhibited with cyanide treatment and was progressively destroyed with increasing time of heat denaturation of excretory/secretory product. Using a purpose-built chamber the superoxide dismutase activity of excretory/secretory product from live worms in culture was shown to accumulate with time to a maximum at 4 h. The electron paramagnetic resonance spectrum obtained for the frozen excretory/secretory product of N. americanus recorded at 77 K is typical of Cu(II) in a protein matrix. The results are consistent with the presence of an active Cu/Zn superoxide dismutase in excretory/secretory product from N. americanus and demonstrate a method for the unequivocal determination of the fate of superoxide anions in the presence of live worms.

Animals↗
PubMed1997

Characterisation and properties of an intracellular lipid-binding protein from the tapeworm Moniezia expansa.

The tapeworm Moniezia expansa contains an extremely abundant cytoplasmic lipid-binding protein (LBP). It is a small protein consisting of 66 amino acids with a molecular mass of 7943 +/- 1.5 Da. The amino acid sequence has been established by Edman degradation and confirmed by PCR analysis. The Moniezia LBP shows no sequence similarity with any previously described binding protein, but does show similarity with antigen B from Echinococcus glanulosus and Echinococcus multilocularis and with Taenia crassiceps antigen. The predicted structure for Moniezia LBP shows four helices and a putative tyrosine kinase site on the loop between helix 1 and 2. Each of the four helices has a well defined hydrophobic face. Studies with fluorescent probes suggest a single hydrophobic binding site. Results indicate that the single tryptophan residue in the molecule (Trp41) is involved in ligand binding, and calculation of the Stern-Volmer quenching constant shows that Trp41 is in a relatively hydrophobic environment.

Amino Acid Sequence↗