Isolation and partial sequencing of a pancreatic polypeptide-like neuropeptide from the liver fluke, Fasciola hepatica.

1. A pancreatic polypeptide (PP)-immunoreactive neuropeptide has been isolated and partially sequenced from the liver fluke, Fasciola hepatica. 2. Gel permeation chromatography of an acid ethanol extract of cattle flukes showed that the peptide is similar in size to mammalian (bovine) PP. 3. The Fasciola peptide was purified to homogeneity by means of reverse-phase HPLC, employing different column chemistries. 4. The purified peptide was sequenced using automated gas-phase Edman degradation and the first 24 amino acid residues determined.     

Characterisation of a beta-tubulin gene from the liver fluke, Fasciola hepatica

This study represents the first beta-tubulin sequence from a trematode parasite, namely, the liver fluke, Fasciola hepatica. PCR of genomic DNA showed that at least one beta-tubulin gene from F. hepatica contains no introns. A number of amino acids in the primary sequence of fluke tubulin are different from those described previously in various nematode species and the cestode, Echinococcus multilocularis. beta-Tubulin is an important target for benzimidazole anthelmintics, although (with the exception of triclabendazole) they show limited activity against F. hepatica. The amino acid differences in fluke beta-tubulin are discussed in relation to the selective toxicity of benzimidazoles against helminths and the mechanism of drug resistance.     

Presclerotized eggshell protein from the liver fluke Fasciola hepatica

Trematode parasites protect their eggs with a tough tanned eggshell. Eggshell precursor proteins are synthesized and stockpiled within the extensive vitellaria of the animal. A major eggshell precursor protein with an apparent molecular weight of 31,000 and pI of 7.4 was isolated from the vitellaria of Fasciola hepatica. This protein, which represents 6-7% of the total protein in mature Fasciola, is unique in containing rather high levels of the amino acid 3,4-dihydroxyphenylalanine (DOPA), i.e., 110 residues per 1000. Other prominent amino acids are glycine, aspartic acid, and lysine. A prominent DOPA-containing tryptic peptide derived from eggshell precursor protein has the sequence Gly-Gly-Gly-DOPA-Gly-Gly-DOPA-Gly-Lys. DOPA residues disappear during the maturation of the eggshell and by treatment in vitro with mushroom polyphenol oxidase. This disappearance may be related to the formation of cross-links in the eggshell protein.     

Characterisation of two calmodulin-like proteins from the liver fluke, Fasciola hepatica

Calmodulin is a calcium ion-sensing signalling protein found in eukaryotics. Two calmodulin-like gene sequences were identified in an EST library from adult liver flukes. One codes for a protein (FhCaM1) homologous to mammalian calmodulins (98% identity), whereas the other protein (FhCaM2) has only 41% identity. These genes were cloned into expression vectors and the recombinant proteins were expressed in Escherichia coli. Gel shift assays showed that both proteins bind to calcium, magnesium and zinc ions. Homology models have been built for both proteins. As expected, FhCaM1 has a highly similar structure to other calmodulins. Although FhCaM2 has a similar fold, its surface charge is higher than FhCaM1. One of the potential metal ion-binding sites has lower metal-ion co-ordination capability, while another has an adjacent lysine residue, both of which may decrease the metal-binding affinity. These differences may reflect a specialised role for FhCaM2 in the liver fluke.     

Proteomic analysis of glutathione transferases from the liver fluke parasite, Fasciola hepatica

The parasite Fasciola hepatica causes major global disease of livestock, with increasing reports of human infection. Vaccine candidates with varying protection rates have been identified by pre-genomic approaches. As many candidates are part of protein superfamilies, sub-proteomics offers new possibilities to systematically reveal the relative importance of individual family proteins to vaccine formulations within populations. The superfamily glutathione transferase (GST) from liver fluke has phase II detoxification and housekeeping roles, and has been shown to contain protective vaccine candidates. GST were purified from cytosolic fractions of adult flukes using glutathione- and S-hexylglutathione-agarose, separated by 2-DE, and identified by MS/MS, with the support of a liver fluke EST database. All previously described F. hepatica GST isoforms were identified in 2-DE. Amongst the isoforms mapped by 2-DE, a new GST, closely related to the Sigma class enzymes is described for the first time in the liver fluke. We also describe cDNA encoding putative Omega class GST in F. hepatica.     

Isolation and characterization of microsatellite markers in the liver fluke (Fasciola hepatica)

Six microsatellite markers were isolated from Fasciola hepatica, a re‐emerging parasite that causes important veterinary and public health problems. In a sample of 52 liver flukes from a region of hyperendemicity (Bolivian Altiplano), five microsatellite were polymorphic. Our results showed that liver flukes present important genetic variability, suggesting a preferential outcrossing reproduction mode for this hermaphroditic parasite.     

Understanding genetic diversity of the liver fluke Fasciola hepatica

Economical breeding is important to obtain maximum gain from the breeding in the animal sector. The economic loss has to be eliminated or should be minimized. The liver fluke, Fasciola hepatica, present mostly in sheep and dairy cattle affect the yield of animals and even cause their death. To eliminate or minimize the impact of these parasites on the animals, it is important to understand the genetic diversity of the liver fluke populations and the relationship between parasite and host at regional bases. This research was carried out to determine diversity by sequence analysis of the mitochondrial ND1 gene and ribosomal ITS1 region.     

A histidine-rich protein from the vitellaria of the liver fluke Fasciola hepatica

The vitellaria are an extensive network of glandular cells and ducts distributed throughout the peripheral tissues of the liver fluke Fasciola hepatica. Eggshell precursor proteins are produced and stockpiled in the vitelline cells of mature flukes. Vitelline protein C has an extraordinary composition: the amino acid 3,4-dihydroxyphenyl-L-alanine (DOPA) and histidine each comprise about 20% of the residues, while glycine represents 41-42% in all variants of what appears to be a microheterogeneous protein family. Protein C has an apparent molecular weight of 16,000-17,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although the protein appears homogeneous following polyacrylamide gel electrophoresis in Tris-glycine with SDS and a acetic acid-urea, electrophoresis in borate, however, suggests that the vitelline protein consists of four or more closely related proteins weighing from 16,000 to 18,500. Isoelectric focusing of the protein family in the presence of 8 M urea resolved only two species having pI values of 6.89 and 6.99. A single N-terminus having the sequence H-H-W-D-G-DOPA-G-DOPA-G was detected. The primary structure of vitelline protein C is characterized by a repeated motif consisting of (G-X)n, where X is Ser, DOPA, or His. Most of the His occurs as G-H repeats in a pepsin-resistant fragment of the protein. Previously, a 31-kDa protein, representing up to 6% of the total protein in the fluke, was reported [Waite, J. H., & Rice-Ficht, A (1987) Biochemistry 26, 7819-7825] to contain significant levels of DOPA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantification and partial characterisation of regulatory peptides in the liver fluke, Fasciola hepatica, from different mammalian hosts.

1. Extracts of the liver fluke, Fasciola hepatica from three different hosts (cow, sheep, rat) have been subjected to radioimmunoassay using antisera to 6 mammalian regulatory peptides. 2. Immunoreactivity was measured to pancreatic polypeptide, substance P, peptide histidine isoleucine and gastrin-releasing peptide. Levels of each peptide varied considerably in flukes from different hosts. 3. Reverse-phase HPLC of rat and sheep fluke extracts revealed three molecular forms of tachykinin immunoreactivity and single peaks of pancreatic polypeptide and peptide histidine isoleucine immunoreactivity. No GRP-immunoreactivity was detected by RIA of HPLC fractions.     

Physiological and morphological effects of genistein against the liver fluke, Fasciola hepatica

A study has been carried out to determine the activity of genistein against adult liver fluke, Fasciola hepatica. Flukes were incubated in vitro in genistein at a concentration of 0.27 mg/ml (=1 mM). They ceased to move after 3 h, at which point the experiment was terminated and the specimens prepared for examination by scanning and transmission electron microscopy. Surface changes to the flukes comprised swelling and blebbing, especially in the posterior region of the flukes, and there was particular disruption to the spines, accompanied by some spine loss. Fine structural changes to the tegumental syncytium indicated an accelerated release of secretory bodies at the surface, but a reduction in their production within the cell bodies. Autophagic activity was evident in the tegumental cells, a phenomenon that was also observed in the gastrodermal cells. Disruption to the testis and vitelline follicles was severe, with an apparent block in the normal developmental sequence of the spermatogenic and vitelline cells, respectively. Shell protein production by the vitelline cells was also disrupted. In separate experiments, somatic muscle strips were exposed to concentrations of genistein ranging from 1 microm to 1 mm. There were statistically significant increases in the frequency and/or amplitude of muscle contractions at concentrations of 10 microm, 100 microm and 1 mm. The results suggest that genistein is capable of causing severe morphological and neuromuscular disruption to adult flukes in vitro over a short time-span.     

A native 13-kDa fatty acid binding protein from the liver fluke Fasciola hepatica

A 13-kDa fatty acid binding protein (FABP) (Fh13) has been isolated from the cytosol of adult Fasciola hepatica and its physicochemical and binding characteristics determined. Fh13 appears to exist as a dimer in native solution. Binding of the fluorescent fatty acid analogue 11-((5-dimethyl aminonaphthalene-1-sulfonyl) amino) undecanoic acid (DAUDA) to Fh13 results in changes in the emission spectrum, which are reversed by oleic acid. The binding activity for DAUDA determined from titration experiments revealed a single binding site per monomeric unit with Kd of 1.5 microM. The displacement of DAUDA by competitive nonfluorescent ligands allowed Kd values for oleic (2.5 microM), retinoic (2.8 microM), palmitic (4.1 microM) and arachidonic acid (6.1 microM) to be calculated. Ten commonly used anthelmintics were evaluated for binding to Fh13, but only bithionol showed binding activity commensurate with those of the putative natural ligands (Kd 6.8 microM).     

Biochemical characterisation of the recombinant peroxiredoxin (FhePrx) of the liver fluke, Fasciola hepatica

The parasitic helminth Fasciola hepatica secretes a 2-Cys peroxiredoxin (Prx) that may play important functions in host-parasite interaction. Recombinant peroxiredoxin (FhePrx) prevented metal-catalyzed oxidative nicking of plasmid DNA and detoxified hydrogen peroxide when coupled with Escherichia coli thioredoxin and thioredoxin reductase (k(cat)/K(m)=5.2 x 10(5)M(-1)s(-1)). Enzyme kinetic analysis revealed that the catalytic efficiency of FhePrx is similar to other 2-Cys peroxiredoxins; the enzyme displayed saturable enzyme Michaelis-Menten type kinetics with hydrogen peroxide, cumene hydroperoxide and t-butyl hydroperoxide, and is sensitive to concentrations of hydrogen peroxide above 0.5 mM. Like the 2-Cys peroxiredoxins from a related helminth, Schistosoma mansoni, steady-state kinetics indicate that FhePrx exhibits a saturable, single displacement-like reaction mechanism rather than non-saturable double displacement (ping-pong) enzyme substitution mechanism common to other peroxiredoxins. However, unlike the schistosome Prxs, FhePrx could not utilise reducing equivalents supplied by glutathione or glutathione reductase.