A microelectronic DNA chip detects the V617F JAK-2 mutation in myeloproliferative disorders.
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DNA Mutational Analysis↗
Biomedical subjects
A S Bell
Publications and source records attributed to A S Bell.
A real-time PCR assay for quantifying Plasmodium falciparum infections in the mosquito vector.
Transmission-blocking vaccines prevent the development of Plasmodium parasite within the mosquito vector, thereby thwarting the spread of malaria through a community. The gold standard for determining the efficacy of a transmission-blocking vaccine is the standard membrane feeding assay. This assay requires the dissection of mosquitoes and microscopic counting of oocysts present on the mosquito mid-gut, typically at 7-10 days p.i. Here we describe a real-time quantitative PCR assay that is rapid, target-specific and robust, with a sensitive detection threshold and which may be employed earlier p.i. than the standard membrane feeding assay and is applicable to preserved material. The real-time PCR assay utilises the LightCycler platform and SYBR Green I detection system to amplify 180 bp of the asexual form of the Plasmodium falciparum rRNA gene. It has a quantitative range of greater than four orders of magnitude and a detection threshold of 10 parasites. Validation experiments using a monoclonal antibody of known blocking activity revealed the real-time PCR assay to give equivalent results to the standard membrane feeding assay. In addition, the PCR assay can establish the effect of such a monoclonal antibody on the parasites' development within the oocyst and on the sporozoite (the transmissible stage) yield, providing a more pertinent assessment of transmission blocking activity than is possible by the standard membrane feeding assay. This assay may also be employed to monitor the sporogonic development of P. falciparum parasites within the mosquito vector.
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Molecular evidence for the synonymy of two species of Apatemon Szidat, 1928, A. gracilis (Rudolphi, 1819) and A. annuligerum (von Nordmann, 1832) (Digenea: Strigeidae) parasitic as metacercariae in British fishes.
The current study examined rDNA (internal transcribed spacer regions, ITS1 and ITS2) and cytochrome c oxidase subunit 1 (CO1) sequence data of Apatemon annuligerum (originating from two geographical locations) and A. gracilis metacercariae (originating from four natural piscine hosts) to determine the systematic status of these two strigeid digeneans. With the exception of short repeat motifs, the ITS1 regions sequenced demonstrated no intra- or inter-specific sequence variation. ITS2 sequences were 292 bp and CO1 sequences 366 bp in length and identical for both nominal Apatemon species. These sequence data provide strong evidence that the two species are con-specific and that A. annuligerum should be regarded as a junior synonym of A. gracilis.
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A molecular phylogeny of the genus Ichthyocotylurus (Digenea, Strigeidae).
Three nucleotide data sets, two nuclear (ribosomal internal transcribed spacer regions 1 and 2, ITS1 and ITS2) and one mitochondrial (cytochrome c oxidase subunit 1, CO1), were analysed using distance matrix and maximum likelihood methods to determine the inter-relationships amongst the four species attributed to the genus Ichthyocotylurus Odening, 1969. Sequence data obtained from all gene loci investigated supported the position of Ichthyocotylurus variegatus as a species discrete from Ichthyocotylurus platycephalus. Phylogenetic analyses yielded congruent trees, with I. variegatus isolates comprising a common clade to which I. platycephalus constitutes a sister taxon. Ichthyocotylurus erraticus and Ichthyocotylurus pileatus were found to demonstrate a similarly close inter-specific relationship. The greatest intra-generic divergence occurred in the CO1 region (16% variability), with resultant disparities in three to eight encoded amino acids. PCR amplification yielded multiple ITS1 products for all Ichthyocotylurus spp. Analyses of equivalent-sized amplicons showed 5.4% intra-generic variation and several point mutations between I. variegatus isolates from different geographical localities and from different piscine hosts. The ITS2 locus was extremely conserved, with less than 1% variation between species. No intra-specific variation was recorded for any CO1 or ITS2 sequences.
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Single and nested polymerase chain reaction assays for the detection of Microsporidium seriolae (Microspora), the causative agent of 'Beko' disease in yellowtail Seriola quinqueradiata.
Single and nested polymerase chain reaction (PCR) assays were developed for the detection of the microsporidian parasite Microsporidium seriolae, which is responsible for emaciation and even death in farmed Japanese yellowtail. Extremely high rDNA identities exist between this parasite and other members of the as yet unclassified genus, necessitating the design of generic, rather than species-specific primer sets. The nested PCR was several orders of magnitude more sensitive than the standard single PCRs, with visible target product amplified from as little as 0.01 pg of parasite DNA (equivalent to that extracted from a single spore). The specificity of the assays was tested against a range of potential host fishes and 6 other microsporidians infecting either fish or the musculature of their hosts. Single PCRs were found to be specific to the target genus, but the nested PCR replicated rDNA from several different microsporidian genera, limiting its utility. This study highlights problems associated with the use of the rRNA gene for PCR assays of certain microsporidians, but nevertheless provides a rapid and sensitive means for the detection of pre-spore forms not possible by current staining methods. Consequently, these assays may be employed for further studies on the portals of entry, migration to the musculature and transmission of this economically important pathogen.
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Cercarial emergence of Ichthyocotylurus erraticus (Rudolphi, 1809), I. variegatus (Creplin, 1825) and Apatemon gracilis (Rudolphi, 1819) (Digenea: Strigeidae): contrasting responses to light:dark cycling.
Many digenean cercariae have been shown to emerge from their molluscan hosts with distinct shedding patterns that have enabled the discrimination of morphologically similar species, or even strains. In this study the cercarial emission patterns of three strigeid species, Ichthyocotylurus erraticus, I. variegatus and Apatemon gracilis, from experimentally infected natural hosts were found to exhibit rhythms that correlated with the light:dark cycle. Both Ichthyocotylurus spp. exhibited a diurnal pattern of release in which cercariae emerged during the light period. Each demonstrated a latent period before the liberation of large numbers of cercariae and yielded similar numbers of cercariae daily. These rhythms offered no means for the discrimination of these two morphologically similar species. A. gracilis cercariae demonstrated a very different circadian rhythm in which the majority emerged at the onset of darkness with no latent period, whereas the cercarial numbers released daily were far greater. Differences could be related to piscine host behaviour.
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Chaetotaxy and armature of Ichthyocotylurus erraticus (Rudolphi, 1809) and I. variegatus (Creplin, 1825) cercariae (Digenea, Strigeidae).
The armature and chaetotaxy of Ichthyocotylurus erraticus (Rudolphi 1809) and I. variegatus (Creplin 1825) cercariae were studied using scanning electron microscopy (SEM) and silver staining of the argentophilic sensilla. This represents the first detailed investigation of the surface structures of cercariae belonging to this genus. Both species exhibited a similar armature, although differences were recorded in the number of spines comprising the pre-oral tuft and the number of rows of spines in the post-oral collar. The number and the distribution of sensilla were found to be identical for both species of cercariae. Four types of sensilla, common to both species, were identified that differed in cilia length and in the structure of the surrounding collar. The distribution of particular sensillary forms was found to be consistent in both species.
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2(1H)-quinolinones with cardiac stimulant activity. 2. Synthesis and biological activities of 6-(N-linked, five-membered heteroaryl) derivatives.
A series of 6-(N-linked, five-membered heteroaryl)-2(1H)-quinolinone derivatives was synthesized and evaluated for cardiotonic activity. Most compounds were prepared by sulfuric acid catalyzed cyclization of an N-(4-heteroarylphenyl)-3-ethoxypropenamide or by condensation of a 2-amino-5-heteroarylbenzaldehyde or -acetophenone derivative with the ylide derived from triethyl phosphonoacetate. In anesthetized dogs, 6-imidazol-1-yl-8-methyl-2(1H)-quinolinone (3; 25 micrograms/kg) produced a greater increase in cardiac contractility (percentage increase in dP/dt max) than alternative 6-(five-membered heteroaryl)-substituted analogues (4-8). Introduction of 4-methyl (10) or 2,4-dimethyl (13) substituents into the imidazole ring of 3 produced a marked increase in inotropic activity, and these compounds were some 10 and 5 times more potent than milrinone. Most of these quinolinones also displayed positive inotropic effects (decrease in QA interval) in conscious dogs after oral administration (0.0625-1 mg/kg) and in many cases (3, 5-7, 9, 11, 13, 16) there was little difference in activities at both the 1- and 3-h time points. Compound 13 (62.5, 125, 250 micrograms/kg po) demonstrated dose-related cardiac stimulant activity which, in contrast to milrinone, was maintained over the whole 7-h test period. No changes in heart rate were detected at any dose level and compounds 3, 9, 10, and 13 also displayed high selectivity for the stimulation of cardiac contractile force rather than heart rate in the Starling dog heart-lung preparation. Increases in dP/dt max of approximately 50% were accompanied by heart rate changes of less than 10 beats/minute. Physicochemical measurements gave a log P of 1.64 for 13 with pKa values of 7.13 +/- 0.04 and 11.5 +/- 0.2 for the imidazole and quinolinone moieties, respectively. X-ray structural analysis of 13 showed the imidazole and quinolinone rings at 52 degrees to one another in close agreement with the minimum-energy conformation (30 degrees) suggested by PCILO calculations. 6-(2,4-Dimethylimidazol-1-yl)-8-methyl-2-(1H)-quinolinone (13, UK-61,260) is currently undergoing phase II clinical evaluation in congestive heart failure patients.
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2(1H)-quinolinones with cardiac stimulant activity. 3. Synthesis and biological properties of 6-imidazol-1-yl derivatives.
A series of 6-imidazol-1-yl-8-methyl-2(1H)-quinolinones was synthesized and evaluated for cardiac stimulant activity in dogs. The majority of compounds were prepared from an appropriate 6-imidazol-1-yl-2(1H)-quinolinone precursor or by sulfuric acid catalyzed cyclization of an N-(4-heteroarylphenyl)-3-ethoxypropenamide. Introduction of a range of 5-substituents into 6-(2,4-dimethylimidazol-1-yl)-8-methyl-2(1H)-quinolinone (1) reduced inotropic activity in anesthetized dogs (percentage increase in dP/dtmax) although replacement of the 2-methyl group by iodo (10) or cyano (11) substituents was well tolerated. The 2-methyl-4-chloro (15) and 2-methyl-4-(methylthio) (22) derivatives displayed similar potency to 1 (40-50% increase in dP/dtmax, 10-12.5 micrograms/kg) and these compounds were 3-5 times more potent than milrinone. Introduction of iodo (14), cyano (16), or acetyl (17) substituents into the 4-position approximately halved inotropic activity. In conscious dogs, 11 (0.25 mg/kg) and 16 and 17 (0.125 mg/kg) produced similar increases in cardiac contractility (decrease in the QA interval) to 1 (0.125 mg/kg) and maximum responses were maintained for at least 3 h. Dose-related (25, 125, 250 micrograms/kg) cardiac stimulant activity was demonstrated by 17 and after the higher doses a marked response (approximately 30% increase in dP/dtmax) was still observed after 7 h, in contrast to milrinone. The substantial increases in cardiac contractility observed with 16 and 17 in the conscious dog were not accompanied by any tachycardia. These compounds also displayed an overwhelming selectivity for increasing the force of cardiac contraction (greater than 120% increase in dP/dtmax) rather than heart rate (5-10 beats/min decrease) in the Starling heart-lung preparation. As a result of this beneficial pharmacological profile, 6-(4-acetyl-2-methylimidazol-1-yl)-8-methyl-2(1H)-quinolinone (17, UK-66,838) was selected for preclinical development studies.
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7-Heteroaryl-1,2,3,5-tetrahydroimidazo[2,1-b]quinazolin -2(1H)-one derivatives with cardiac stimulant activity.
A series of 7-heteroaryl-1,2,3,5-tetrahydroimidazo[2,1-b]quinazolin++ +-2 (1H)-ones was synthesized and evaluated in dogs for cardiac stimulant activity. Compounds were obtained by a palladium-catalyzed cross-coupling reaction between a heteroarylzinc chloride and a 7-iodo-1,2,3,5-tetrahydroimidazo [2,1-b]quinazolin-2(1H)-one or by cyclization of an N-[(2-aminophenyl)methyl]glycinate with cyanogen bromide. Compared to the parent ring system (3), introduction of a 2,6-dimethylpyridin-3-yl (6), 2,4-dimethylimidazol-1-yl (7), or 1,2,4-triazol-1-yl (8) moiety at the 7-position led to a 13-17-fold increase in positive inotropic activity (percentage increase in dP/dtmax) in anesthetized dogs. Potency could be further enhanced with a 9-methyl substituent (10-12). The most potent member of the series, 7-(2,4-dimethylimidazol-1-yl)-9-methyl-1,2,3,5-tetrahydroimidaz o [2,1-b]quinazolin-2(1H)-one (11) (23% increase in dP/dtmax, 2 micrograms/kg), was 80 times more active than 3 and displayed a 5-fold advantage over milrinone. In conscious dogs, 6 elicited marked and sustained positive inotropic activity (decrease in QA interval) after oral administration (1 mg/kg), whereas 10-12 were 10 times more potent. 11 produced an obvious increase in cardiac contractility (20% increase in dP/dtmax) at low dose levels (25 micrograms/kg) while, after 100 micrograms/kg, the marked response (50% increase in dP/dtmax) was maintained for the whole 7-h test period. In these experiments, 11 had no effect on heart rate, and the compound also displayed exceptional selectivity for increasing the force rather than the rate of cardiac contraction (greater than 150% increase in dP/dtmax) in the Starling heart-lung preparation. These studies demonstrate that the tetrahydroimidazoquinazolinone nucleus is an effective bioisostere for the 2(1H)-quinolinone system and that 11 displays improved cardiac stimulant activity and duration of action when compared to milrinone.
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2(1H)-quinolinones with cardiac stimulant activity. 1. Synthesis and biological activities of (six-membered heteroaryl)-substituted derivatives.
A series of (six-membered heteroaryl)-substituted 2(1H)-quinolinones (1) was synthesized, and structure-activity relationships for cardiac stimulant activity were determined. Most compounds were prepared by acidic hydrolysis of a heteroaryl-2-methoxyquinoline obtained by palladium-catalyzed cross-coupling methodology. Direct reaction of a pyridinylzinc reagent with a 6-haloquinolinone also proved successful. In anesthetized dogs, 6-pyridin-3-yl-2(1H)-quinolinone (3; 50 micrograms/kg) displayed greater inotropic activity (percentage increase in dP/dt max) than positional isomers (2, 4-6), and potency was maintained with either mono- (13, 15) or di- (16) alkylpyridinyl substituents. Introduction of a 4- (24) or 7- (25) methyl group into 3 reduced inotropic activity, whereas the 8-isomer (26) proved to be the most potent member of the series. Compound 26 and the 2,6-dimethylpyridinyl analogue (27) were approximately 6 and 3 times more potent than milrinone. Several quinolinones displayed positive inotropic activity (decrease in QA interval) in conscious dogs after oral administration (1 mg/kg), and 26, 27 were again the most potent members of the series. Compound 27 (0.25, 0.5, 1.0 mg/kg po) demonstrated dose-related cardiac stimulant activity, which was maintained for at least 4 h. No changes in heart rate were observed. Compounds 3, 4, 26, and 27 also selectively stimulated the force of contraction, rather than heart rate, in the dog heart-lung preparation. For a 50% increase in dP/dt max with 27, heart rate changed by less than 10 beats/min. In norepinephrine contracted rabbit femoral artery and saphenous vein, 27 produced dose related (5 X 10(-7) to 5 X 10(-4) M) vasorelaxant activity. The combined cardiac stimulant and vasodilator properties displayed by 27, coupled with a lack of effect on heart rate, should be beneficial for the treatment of congestive heart failure.
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A hyperparasitic microsporidian infecting the salmon louse, Lepeophtheirus salmonis: an rDNA-based molecular phylogenetic study.
The sea louse, Lepeophtheirus salmonis, is an obligate ectoparasitic copepod that lives on the external surface of salmonid fish. It is the most common ectoparasite of marine cage-reared salmonids, causing major economic loss to the aquaculture industry. During a sea louse monitoring programme, samples of L. salmonis were found to harbour an unreported microsporidian parasite. The microsporidian was observed in pre-adult and adult stages of both male and female copepods, with a prevalence of up to 5%. Unfixed spores were slightly pyriform in shape measuring 2.34 microm by 1.83 microm (+/- 0.01 microm) and were not observed to be enclosed by a sporophorous vesicle. The microsporidian infection was observed in all areas of the copepods' body, xenoma-like cysts forming directly under the cuticle in the epidermal tissue layer. In the present study, rDNA (530f-580r) sequence data gathered from the unidentified microsporidian parasite isolated from infected sea lice were compared with equivalents available in the databases in an attempt to identify its systematic position. The microsporidian was found to group within the phylogenetic clade containing the family Enterocytozoonidae, being most similar to members of the intranuclear genus Nucleospora. This is the first report of a hyperparasitic microsporidian infecting a caligid copepod.
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Phylogenetic relationships among microsporidia based on rDNA sequence data, with particular reference to fish-infecting Microsporidium balbiani 1884 species.
Recently, large discrepancies have been identified between microsporidian systematics based on molecular and traditional characteristics. In the current study the 530f-580r region of the rRNA gene of eight microsporidian species was cloned and sequenced. Included were two unclassified species of Microsporidium Balbiani, 1884 and an unidentified microsporidian that infects the musculature of different sea bream species. Sequence identities in excess of 98% indicated that these three species almost certainly are members of the same genus. Phylogenetic analyses of all microsporidian sequence data available for this region of the gene (20 species) and for partial small subunit sequences (51 species of 21 genera) revealed these species to be distinct from the family Pleistophoridae Doflein, 1901 and closely related them to the genus Sproguea Weissenberg, 1976. This clade was found to comprise a sister taxon to that containing the vast majority of fish-infecting species. Broad cladistic divisions were found between terrestrial insect-infecting and fish-infecting species, which together are distant from the aquatic insect-infecting microsporidia. The rRNA gene of certain fish-infecting genera was found to be more highly conserved than previously reported. This has implications for its utility in diagnostic assays and phylogenetic studies at, or close to, the species level.
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