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Biomedical subjects

E M Hoey

Publications and source records attributed to E M Hoey.

At least 19 recordsLinked to original sources
PubMed2007

Evidence for multiple mitochondrial lineages of Fasciola hepatica (liver fluke) within infrapopulations from cattle and sheep.

The economic, veterinary, and medical impact of the parasite Fasciola hepatica, liver fluke, is difficult to alleviate due to increasing incidences of resistance to the principal anthelmintic drugs. These have occurred in widely separated regions. The rate of response to selection imposed by such drugs will be dependent on the genetic variation present in the F. hepatica gene pool, but this is at present unknown. We have assessed the genetic diversity of mitochondrial haplotypes found in the infrapopulation of flukes recovered from a calf of known provenance and from six other cattle and sheep hosts located in Ireland and four from elsewhere. Our results revealed that at least ten different mitochondrial composite PCR-restriction fragment length polymorphism haplotypes had been acquired by a single animal in 1 year, and there was comparable diversity in six other definitive hosts carrying field-acquired infections. The extent of divergence between these fluke lineages suggests that they predate the last ice age and, thus, cannot have developed in Northern Europe. A consequence of this high level of diversity is that there will be frequent selection for anthelmintic resistance and rapid responses to climatic changes.

Animals↗
PubMed2004

Response of two isolates of Fasciola hepatica to treatment with triclabendazole in vivo and in vitro.

In this study, the susceptibility of two isolates of Fasciola hepatica--the Fairhurst and Oberon isolates--to treatment with triclabendazole was investigated, both in vivo and in vitro. The Fairhurst isolate originated in England, but has since been maintained in Australia; the Oberon isolate originated in Australia. Triclabendazole had a very high efficacy against the Fairhurst isolate. In sheep (dose: 10 mg/kg), the efficacy ranged from 78.4% at 2 weeks post-infection to 98.5% at 6 weeks post-infection. In cattle (dose: 12 mg/kg) efficacy was 89% at 2 weeks post-infection and 100% at 12 weeks. In contrast, against the Oberon isolate, triclabendazole had 0% efficacy against 2-week-old flukes in sheep (dose: 10 mg/kg) and 5% against 4-week-old flukes. Surface changes to flukes of the two isolates were assessed by scanning electron microscopy following treatment in vitro for 24 h in triclabendazole sulphoxide (15 and 50 microg/ml). Disruption took the form of blebbing, swelling and furrowing of the tegument and was greater in the Fairhurst than the Oberon isolate. Surface changes generally were more severe in the anterior than posterior region of the fluke and the dorsal surface was also consistently more severely affected than the ventral surface. Disruption was more severe at the higher drug concentration for both isolates. The morphological data is consistent with the efficacy data, which indicates that the Fairhurst isolate of F. hepatica is susceptible to triclabendazole treatment, whilst the Oberon isolate is refractory.

Animals↗
PubMed2004

The occurrence and significance of triploidy in the liver fluke, Fasciola hepatica.

Karyotyping of Fasciola hepatica samples from Britain and Ireland has identified a triploid isolate which is effectively aspermic, rendering it necessarily asexually reproducing. Considering the extensive presence of asexually reproducing diploid and triploid Fasciola in Asia it is suggested that facultative gynogenesis is widespread in this parasite. This has important implications for the population genetics and evolution of Fasciola, especially in relation to the development and spread of drug resistance, and must be considered in the mathematical modelling of this process.

Animals↗
PubMed2003

Fasciola hepatica: surface and internal tegumental changes induced by treatment in vitro with the sulphoxide metabolite of albendazole ('Valbazen').

A morphological study has been carried out to determine the effect of the active sulphoxide metabolite of the benzimidazole anthelmintic, albendazole (ABZ-SO) on the adult liver fluke, Fasciola hepatica. Whole flukes were treated with ABZ-SO for 12 and 24 h at a concentration of 10 microg/ml. The changes in response to drug treatment were examined by scanning electron microscopy (SENI), transmission electron microscopy (TEM) and tubulin immunocytochemistry (ICC). No surface changes were apparent following 12 h ABZ-SO treatment, but localized blebbing was observed after 24 h, which became more extensive towards the posterior region of both surfaces. TEM of sections from the posterior midbody region revealed that ABZ-SO caused the accumulation of secretory bodies in the tegumental cells and in their cytoplasmic connections and, after 24 h, just above the basal plasma membrane. Localized blebbing of the apical membrane also occurred. The morphology of the Golgi complexes within the tegumental cells began to change after 12 h treatment with ABZ-SO and, by 24 h, few complexes were observed. A distinct increase in tubulin immunoreactivity occurred after 12 h treatment, but this decreased after 24 h. The results obtained are consistent with those expected for microtubule inhibition. They are discussed in relation to the action of established microtubule inhibitors, as well as the benzimidazole derivative, triclabendazole.

Albendazole↗
PubMed2002

Triclabendazole-resistant Fasciola hepatica: beta-tubulin and response to in vitro treatment with triclabendazole.

Resistance in Fasciola hepatica to triclabendazole ('Fasinex') has emerged in several countries. Benzimidazole resistance in parasitic nematodes has been linked to a single amino acid substitution (phenylalanine to tyrosine) at position 200 on the beta-tubulin molecule. Sequencing of beta-tubulin cDNAs from triclabendazole-susceptible and triclabendazole-resistant flukes revealed no amino acid differences between their respective primary amino acid sequences. In order to investigate the mechanism of triclabendazole resistance, triclabendazole-susceptible and triclabendazole-resistant flukes were incubated in vitro with triclabendazole sulphoxide (50 microg/ml). Scanning and transmission electron microscopy revealed extensive damage to the tegument of triclabendazole-susceptible F. hepatica, whereas triclabendazole-resistant flukes showed only localized and relatively minor disruption of the tegument covering the spines. Immunocytochemical studies, using an anti-tubulin antibody, showed that tubulin organization was disrupted in the tegument of triclabendazole-susceptible flukes. No such disruption was evident in triclabendazole-resistant F. hepatica. The significance of these findings is discussed with regard to the mechanism of triclabendazole resistance in F. hepatica.

Animals↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed2001

Partial nucleotide sequences, and routine typing by polymerase chain reaction-restriction fragment length polymorphism, of the brown trout (Salmo trutta) lactate dehydrogenase, LDH-C1*90 and *100 alleles.

The cDNA nucleotide sequences of the lactate dehydrogenase alleles LDH-C1*90 and *100 of brown trout (Salmo trutta) were found to differ at position 308 where an A is present in the *100 allele but a G is present in the *90 allele. This base substitution results in an amino acid change from aspartic acid at position 82 in the LDH-C1 100 allozyme to a glycine in the 90 allozyme. Since aspartic acid has a net negative charge whilst glycine is uncharged, this is consistent with the electrophoretic observation that the LDH-C1 100 allozyme has a more anodal mobility relative to the LDH-C1 90 allozyme. Based on alignment of the cDNA sequence with the mouse genomic sequence, a local primer set was designed, incorporating the variable position, and was found to give very good amplification with brown trout genomic DNA. Sequencing of this fragment confirmed the difference in both homozygous and heterozygous individuals. Digestion of the polymerase chain reaction products with BslI, a restriction enzyme specific for the site difference, gave one, two and three fragments for the two homozygotes and the heterozygote, respectively, following electrophoretic separation. This provides a DNA-based means of routine screening of the highly informative LDH-C1* polymorphism in brown trout population genetic studies. Primer sets presented could be used to sequence cDNA of other LDH* genes of brown trout and other species.

Alleles↗
PubMed2000

Conformational changes during proteolytic processing of a picornavirus capsid proteins.

We have used synthetic peptide antibodies to probe conformational changes that occur during the cleavage cascade which generates the capsid proteins of a picornavirus. The initial translation product of 97 kDa, the precursor of all four structural proteins, is cleaved to form a 63 kDa fragment which, we show, has significantly different folding characteristics to both its larger parent and its products. We demonstrate that proteolytic cleavages as distant as 520 residues from epitopes confer sufficiently large conformational changes as to render them unrecognisable. To our knowledge, this is the first demonstration of this phenomenon in the picornavirus system.

Animals↗
PubMed2000

The major tegumental antigen of Fasciola hepatica contains repeated elements.

In order to provide a better understanding of the interaction between the liver fluke (Fasciola hepatica) and the immune system of its mammalian host immunoreactive lambda bacteriophage clones containing F. hepatica cDNA have been isolated. Plasmids from these clones were sequenced and found to encode a family of proteins containing certain common elementst. All the clones contained a coding repeating sequence (RRRXCA) which is conserved at the nucleic acid level followed by a non-repeating element coding for the C terminal used by the proteins which shows conservation of amino acids at certain positions. Antisera raised against a beta-galactosidase fusion protein with one of these sequences as a terminal extension was used to localize the immunoreactive antigens. Binding was predominantly in the tegument of the juvenile fluke but was reduced in the adult tegument. The wall of the uterus showed strong reactivity in the adult. Rats immunized with the beta-galactosidase fusion protein showed enhanced resistance to challenge infections. The role of these antigens in the host response to infection by F. hepatica is discussed.

Amino Acid Sequence↗
PubMed2000

Species-specific oligonucleotide probes for macroalgae: molecular discrimination of two marine fouling species of Enteromorpha (Ulvophyceae).

The green seaweeds Enteromorpha intestinalis and E. compressa are important fouling organisms commonly found in polluted and nutrient-enriched marine and brackish water habitats, where they are used in environmental monitoring. Discrimination of the two species is extremely difficult because of overlapping morphological characters. In this study a quick molecular method for species identification was developed based on the nuclear rDNA ITS2 sequence data of 54 E. intestinalis samples and 20 E. compressa samples from a wide geographical range. Oligonucleotide probes were designed for species-specific hybridization to dot-blots of the PCR-amplified ITS1, 5.8S gene and ITS2 fragment of both E. intestinalis and E. compressa. Specificity of the oligonucleotide probes was confirmed by tests with taxonomically diverse species that could morphologically be confused with E. intestinalis or E. compressa. This is the first use of species-specific probes for macroalgae. The restriction endonuclease NruI digested specifically the amplified PCR product from E. compressa into two fragments detectable on agarose gels, but no suitable restriction sites were identifiable in the PCR product of E. intestinalis.

Base Sequence↗
PubMed1995

Nitric oxide synthase activity and expression in retinal capillary endothelial cells and pericytes.

The purpose of this study was to examine nitric oxide synthase (NOS) expression in the retinal vasculature in vivo and to study nitric oxide (NO) synthesis in vitro in retinal microvascular endothelial cells and pericytes. Immunoreactivity was examined using a polyclonal antibody raised against porcine cerebellar nitric oxide synthase on frozen sections cut from postmortem human retina and trypsin digests of rat retinal vasculature. The synthesis of nitrite, a stable end product from the interaction of NO with molecular oxygen, was measured in culture supernatants of retinal microvascular cells under basal and stimulated conditions. Expression of constitutive NOS (cNOS) in these cells was examined using the polymerase chain reaction (PCR). Strong NOS immunoreactivity was seen in the endothelium of choroidal and retinal vessels. Nitrite synthesis was documented in supernatants from cultured microvascular endothelial cells which increased significantly following exposure to A23187 and cytokines. Nitrite synthesis by pericytes was not detectable under basal conditions or following stimulation with A23187. Bacterial lipopolysaccharide (LPS), a potent inducer of NOS, caused an increase in nitrite concentrations in pericyte supernatants 24 h after stimulation suggesting the presence of inducible NOS (iNOS). PCR amplification confirmed the presence of the cNOS gene in endothelial cells but not in pericytes. Retinal vascular endothelial cells express significant amounts of NOS constitutively in vivo and in vitro which is activated by Ca++. Also, endothelial cells can be stimulated to synthesize iNOS by cytokines. Retinal pericytes too show iNOS activity following exposure to bacterial LPS. These results suggest that the nitric oxide synthase/nitric oxide pathway may be involved in the regulation of microcirculatory haemodynamics in the retina.

Amino Acid Oxidoreductases↗
PubMed1994

The nucleotide sequence of the 5'non-coding and capsid coding genome regions of two bovine enterovirus strains.

The sequence of cDNA clones representing the 5' non-coding regions (NCR) and capsid regions of two bovine enteroviruses (strains PS-87 and RM-2; serotype two viruses) have been determined and compared with that obtained from a serotype one strain (VG-5-27). All three strains showed a longer 5' NCR compared to human enteroviruses and rhinoviruses due in part to a hundred residue insertion approximately at a hundred residues in from the 5' end. However, another domain occurring at nucleotide 187-222 in poliovirus is absent in each bovine enterovirus. Comparisons of the predicted structural protein amino acid sequences indicate that PS-87 shares most sequence identity with RM-2 and then with VG-5-27 in that order. The VP1 protein of PS-87 and RM-2 are shorter than the equivalent VP1 of VG-5-27 due in part to a truncation at their C-terminii. VP3 is only slightly smaller than VP2 in each virus.

Amino Acid Sequence↗
PubMed1993

A nucleotide sequence comparison of coxsackievirus B4 isolates from aquatic samples and clinical specimens.

Ten coxsackievirus B4 (CVB4) strains isolated from clinical and environmental sources in Northern Ireland in 1985-7, were compared at the nucleotide sequence level. Dideoxynucleotide sequencing of a polymerase chain reaction (PCR) amplified fragment, spanning the VP1/P2A genomic region, classified the isolates into two distinct groups or genotypes as defined by Rico-Hesse and colleagues for poliovirus type 1. Isolates within each group shared approximately 99% sequence identity at the nucleotide level whereas < or = 86% sequence identity was shared between groups. One isolate derived from a clinical specimen in 1987 was grouped with six CVB4 isolates recovered from the aquatic environment in 1986-7. The second group comprised CVB4 isolates from clinical specimens in 1985-6. Both groups were different at the nucleotide level from the prototype strain isolated in 1950. It was concluded that the method could be used to sub-type CVB4 isolates and would be of value in epidemiological studies of CVB4. Predicted amino acid sequences revealed non-conservation of the tyrosine residue at the VP1/P2A cleavage site but were of little value in distinguishing CVB4 variants.

Base Sequence↗
PubMed1992

Characterization of neutralizing antibodies to bovine enterovirus elicited by synthetic peptides.

Six synthetic peptides corresponding to regions of bovine enterovirus (BEV), strain VG-5-27, elicited antibodies in mice which reacted with the virus in various assays. These antibodies have been characterised on the basis of their ability to (1) neutralize the virus, (2) bind to the intact virus particle in an immunoprecipitation test, (3) react with the denatured viral proteins, and (4) give immunofluorescent staining of virus infected cells. We have also determined the proportion of antipeptide antibody which binds to the virus in each case. All of the sera immunoprecipitated the virus and neutralized its activity to varying extents. Two of the sera specific for VP 1 sequences failed to react with denatured VP 1 whereas all the other antisera reacted with their respective parental proteins. All of the sera reacted with VG-5-27 infected cells in an immunofluorescence test. The proportion of antibodies to each peptide recognizing intact virus was variable and did not appear to correlate with neutralizing activity. In addition, the ability of each of the sera to react with and neutralize three other strains of the virus was analysed. With one of these strains significant cross-neutralization was observed.

Amino Acid Sequence↗
PubMed1992

Characterization of viral DNAs from cells infected with chicken anaemia agent: sequence analysis of the cloned replicative form and transfection capabilities of cloned genome fragments.

The viral DNAs induced by the unclassified animal virus, chicken anaemia agent (CAA), during replication in MDCC-MSB 1 cells have been investigated. Analyses after S1 nuclease, restriction endonuclease and denaturation treatments indicated that infected cell extracts contained genome-size, single-stranded DNA (M(r) 2.3 kb), closed and open circular, double-stranded replicative form (RF) DNAs (M(r) 2.3 kbp) and a population of smaller double-stranded DNAs (M(r) 0.8 kbp). Recombinant plasmids containing 2.3 kbp CAA RF fragments cloned at the PstI, BamHI and EcoRI sites failed to transfect MDCC-MSB 1 cells. However, one plasmid, which contained two 2.3 kbp CAA RF fragments ligated in tandem at the PstI site, and cloned 2.3 kbp PstI, BamHI and EcoRI fragments, excised from their respective plasmids by restriction endonuclease digestion, were capable of transfection. The nucleotide sequence of the circular genome (2298 bp) of the Cux-1 isolate of CAA has indicated the presence of three overlapping open reading frames (ORFs) of 52 kDa, 24 kDa and 13 kDa on one strand. The existence of these ORFs was corroborated by analyses of partial sequences from three other isolates. The non-coding region of the CAA genome contained sequences with putative regulatory function. These results are discussed in relation to the "rolling circle" model of DNA replication.

Amino Acid Sequence↗
PubMed1990

Chemically synthesized peptides elicit neutralizing antibody to bovine enterovirus.

Synthetic peptides representing 14 regions of the bovine enterovirus structural proteins were used to raise antibodies in mice. The peptides were predicted using amino acid sequence alignments with the position of antigenic sites on other picornaviruses. Five of the anti-peptide antibodies reacted with the virus in an immunoprecipitation test. Furthermore, each of these anti-peptide antibodies neutralized virus infectivity; those directed against peptides of VP2 and VP3 neutralized to a greater extent than those directed against peptides of VP1. The positions of these epitopes in the viral structural proteins are discussed in relation to corresponding positions in other picornaviruses.

Amino Acid Sequence↗
PubMed1988

The complete nucleotide sequence of a bovine enterovirus.

The complete nucleotide sequence of the genome of a bovine enterovirus (strain VG-5-27) has been determined using molecular cloning and DNA sequencing techniques. Excluding the poly(A) tract the genome was 7414 nucleotides long and contained a 5' non-coding region which at 818 nucleotides was longer than that of most picornaviruses. The single large open reading frame encoded a potential polyprotein of 2175 amino acids which showed considerable homology with other enteroviruses. This homology has allowed us to predict the possible cleavage sites of the polyprotein and to identify other features of structural and functional significance which might help us to understand the constraints involved in the evolutionary divergence of these viruses.

Amino Acid Sequence↗