RESUMEN
AIM AND METHODS: Obesity in humans is associated with proteinuria and an increased glomerular filtration, possibly related to an increase in glomerular capillary pressure. We investigated in obese and lean Zucker rats (10-12 weeks old) whether this might be related to alterations in the diameter of preglomerular and postglomerular microvessels and their reactivity to the resistance regulator angiotensin II (AngII), using the hydronephrotic kidney model. RESULTS: The obese rats exhibited a hyperinsulinaemic, euglycaemic state and hypertension. Urinary protein concentration and fluid intake were both increased threefold. Basal diameters of distal interlobular arteries (ILAs) and afferent arterioles (AAs) were larger in the obese rat than in the lean rat (ILA: 25.7 +/- 0.3 vs. 23.0 +/- 0.4 microm and AA: 18.8 +/- 0.3 vs. 16.7 +/- 0.5 microm, respectively; p = 0.01), while diameters of efferent arterioles (EAs) were smaller in obese animals (14.2 +/- 1.1 vs. 18.2 +/- 1.2 microm; p = 0.05). AngII induced a concentration-dependent constriction in ILA, AA and EA with an augmented response in the obese compared with the lean rats. Thus, at higher concentrations, AngII abolished the diameter difference between obese and lean animals in preglomerular microvessels while exaggerating that in postglomerular arterioles. CONCLUSIONS: Our data indicate that in obese rats, a vasodilated state in small preglomerular microvessels and a vasoconstricted state in the postglomerular arterioles exist. Although AngII cancelled the former, the latter remained. Therefore, these data reveal periglomerular vascular changes that may play a role in glomerular dysfunction and renal pathology associated with obesity.
Asunto(s)
Angiotensina II/farmacología , Glomérulos Renales/irrigación sanguínea , Microcirculación/efectos de los fármacos , Obesidad/fisiopatología , Vasoconstrictores/farmacología , Animales , Arteriolas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Hidronefrosis/fisiopatología , Glomérulos Renales/fisiopatología , Masculino , Ratas , Ratas Zucker , Técnicas de Cultivo de Tejidos , Resistencia Vascular/efectos de los fármacosRESUMEN
Nematode nicotinic acetylcholine receptors (nAChRs) are the sites of action for the anthelmintic drug levamisole. Recent findings indicate that the molecular mechanism of levamisole resistance may involve changes in the number and/or functions of target nAChRs. Accordingly, we have used an RT-PCR approach to isolate and characterise partial cDNA clones (tca-1 and tca-2) encoding putative nAChR subunits from the economically important trichostrongyloid, Teladorsagia circumcincta. The predicted tca-1 gene product is a 248 aa fragment (TCA-1) which contains structural motifs typical of ligand-binding (alpha-) subunits, and which shows very high sequence similarities (98.8 and 97.2% amino acid identities) to the alpha-subunits encoded by tar-1 and hca-1 from Trichostrongylus colubriformis and Haemonchus contortus, respectively. Sequence analyses of partial tca-1 cDNAs from one levamisole-resistant and two susceptible populations of T. circumcincta revealed polymorphism at the predicted amino acid level, but there was no apparent association of any particular tca-1 allele with resistance. tca-2 encodes a 67 aa fragment (TCA-2) containing the TM4 transmembrane domain and carboxyl terminus of a putative nAChR structural (non-alpha) subunit. The deduced amino acid sequence of TCA-2 shows highest similarity (75% amino acid identity) to ACR-2, a structural subunit involved in forming levamisole-gated ion channels in Caenorhabditis elegans, but low similarity (43% identity) to the corresponding regions of TAR-1 and HCA-1. tca-2 is the first nAChR subunit gene of this type to be isolated from parasitic nematodes, and it provides a basis for further characterisation of structural subunits in trichostrongyloids.
Asunto(s)
Receptores Nicotínicos/genética , Trichostrongyloidea/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Resistencia a Medicamentos , Datos de Secuencia Molecular , ARN Mensajero/genética , Receptores Nicotínicos/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Trichostrongyloidea/químicaRESUMEN
For the first time we used amplified fragment length polymorphism on individual nematode parasites to analyse the genetic diversity between and within isolates during consecutive stages of increased benzimidazole resistance and of increased levamisole resistance of Haemonchus contortus. The genetic diversity of the H. contortus genome turned out to be unusually high, within and between the isolates. The difference between individuals of an isolate could be as high as between individuals of two different mammalian species that do not interbreed. During benzimidazole selection the genetic constitution of the population was changed, but did not lead to a decrease in the genetic diversity. The selection for levamisole resistance resulted in a limited reduction of the genetic diversity only after the first selection step. The extensive genetic diversity apparently has allowed a fast and flexible response of H. contortus to drug selection as shown by the appearance of drug resistant isolates. This selection however has little or no effect on the extent of the genetic diversity of these resistant isolates. Implications for more sustainable control methods are discussed.
Asunto(s)
Antihelmínticos/farmacología , Haemonchus/efectos de los fármacos , Haemonchus/genética , Animales , Bencimidazoles/farmacología , Análisis por Conglomerados , Dermatoglifia del ADN , ADN de Helmintos/química , ADN de Helmintos/aislamiento & purificación , Resistencia a Medicamentos/genética , Variación Genética/efectos de los fármacos , Variación Genética/genética , Hemoncosis/tratamiento farmacológico , Hemoncosis/parasitología , Haemonchus/aislamiento & purificación , Levamisol/farmacología , Análisis Multivariante , Reacción en Cadena de la Polimerasa/veterinaria , Polimorfismo Genético/efectos de los fármacos , Polimorfismo Genético/genética , Ovinos , Enfermedades de las Ovejas/tratamiento farmacológico , Enfermedades de las Ovejas/parasitologíaRESUMEN
We have evaluated whether sequence polymorphisms in the rRNA intergenic spacer region can be used to study the relatedness of two subspecies of Trypanosoma brucei. Thirteen T. brucei isolates made up of 6 T. b. brucei and 7 T. b. gambiense were analyzed using restriction fragment length polymorphism (RFLP). By PCR-based restriction mapping of the ITS1-5.8S-ITS2 ribosomal repeat unit, we found a fingerprint pattern that separately identifies each of the two subspecies analyzed, with unique restriction fragments observed in all but 1 of the T. b. gambiense "human" isolates. Interestingly, the restriction profile for a virulent group 2 T. b. gambiense human isolate revealed an unusual RFLP pattern different from the profile of other human isolates. Sequencing data from four representatives of each of the two subspecies indicated that the intergenic spacer region had a conserved ITS-1 and a variable 5.8S with unique transversions, insertions, or deletions. The ITS-2 regions contained a single repeated element at similar positions in all isolates examined, but not in 2 of the human isolates. A unique 4-bp [C(3)A] sequence was found within the 5.8S region of human T. b. gambiense isolates. Phylogenetic analysis of the data suggests that their common ancestor was a nonhuman animal pathogen and that human pathogenicity might have evolved secondarily. Our data show that cryptic species within the T. brucei group can be distinguished by differences in the PCR-RFLP profile of the rDNA repeat.
Asunto(s)
Variación Genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei gambiense/genética , Animales , Secuencia de Bases , Análisis por Conglomerados , Secuencia de Consenso , ADN Protozoario/química , ADN Ribosómico/química , Genotipo , Humanos , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , ARN Ribosómico 5.8S/genética , Secuencias Repetitivas de Ácidos Nucleicos , Alineación de Secuencia , Especificidad de la Especie , Trypanosoma brucei brucei/clasificación , Trypanosoma brucei gambiense/clasificaciónRESUMEN
Expressed sequence tags from the parasitic nematode Haemonchus contortus were generated in order to identify anchor loci for comparative mapping between nematode genomes and candidate targets for future control measures. In total, 370 SL1 trans-spliced cDNAs from different developmental stages representing 195 different genes were partially sequenced. From these expressed sequence tags 50% were similar to genes with a known or predicted function and 19% were similar to nematode sequences with no ascribed function. From the first, free-living L1 and L3 stages relatively many cDNAs matched to housekeeping genes, and 11% (L1) or 23% (L3) of the encoded proteins were predicted to contain signal peptides. In contrast, no function could be ascribed to most of the cDNAs from the early L5 and adult parasitic stages, but for 30% (L5) or 55% (adult) of the encoded proteins a signal sequence was predicted. This limited analysis suggests that during the transition from the free-living to parasitic stages gene expression shifts towards the synthesis of less conserved extracellular proteins. These proteins offer the best perspectives for vaccine development and the development of anthelmintic drugs. In contrast, cDNAs from the first larval stages may be most suitable for comparative mapping with the free-living nematode Caenorhabditis elegans.
Asunto(s)
Etiquetas de Secuencia Expresada , Expresión Génica , Hemoncosis/veterinaria , Haemonchus/crecimiento & desarrollo , Haemonchus/genética , Proteínas del Helminto/genética , Animales , Clonación Molecular , ADN Complementario/genética , Femenino , Genes de Helminto , Marcadores Genéticos , Hemoncosis/parasitología , Proteínas del Helminto/biosíntesis , Masculino , Datos de Secuencia Molecular , Señales de Clasificación de Proteína/genética , Análisis de Secuencia de ADN , Ovinos , Enfermedades de las Ovejas/parasitologíaRESUMEN
The alarming development of anthelmintic resistance in important gastrointestinal nematode parasites of man and live-stock is caused by selection for specific genotypes. In order to provide genetic tools to study the nematode populations and the consequences of anthelmintic treatment, we isolated and sequenced 59 microsatellites of the sheep and goat parasite Haemonchus contortus. These microsatellites consist typically of 2-10 tandems CA/GT repeats that are interrupted by sequences of 1-10 bp. A predominant cause of the imperfect structure of the microsatellites appeared mutations of G/C bp in the tandem repeat. About 44% of the microsatellites were associated with the HcREP1 direct repeat, and it was demonstrated that a generic HcREP1 primer could be used to amplify HcREP1-associated microsatellites. Thirty microsatellites could be typed by polymerase chain reaction (PCR) of which 27 were polymorphic. A number of these markers were used to detect genetic contamination of an experimental inbred population. The microsatellites may also contribute to the genetic mapping of drug resistance genes.
Asunto(s)
Repeticiones de Dinucleótido/genética , Variación Genética , Haemonchus/genética , Animales , Antinematodos/farmacología , Bencimidazoles/farmacología , ADN de Helmintos/genética , Resistencia a Medicamentos/genética , Marcadores Genéticos , Genotipo , Haemonchus/clasificación , Haemonchus/crecimiento & desarrollo , Haemonchus/aislamiento & purificación , Datos de Secuencia Molecular , Análisis de Secuencia de ADNAsunto(s)
Marcadores Genéticos , Haemonchus/genética , Intrones/genética , Animales , Secuencia de Bases , Secuencia Conservada , Cartilla de ADN/química , ADN Complementario/química , ADN de Helmintos/química , Resistencia a Medicamentos/genética , Haemonchus/efectos de los fármacos , Mutación , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
We have previously characterized a Tcl-like transposable element Hctcl, from the parasitic nematode Haemonchus contortus. Here we describe the genetic variation of Hctcl insertion sites in H. contortus populations differing in geographical origin, resistance to chemotherapeutics and level of inbreeding. Clear differences between populations were observed on Southern blots with a Hctcl-specific probe. Sequencing the 5'- or 3'-flanks of individual Hctcl integration sites allowed the design of PCR reactions between a Hctcl-specific primer and the flanking regions. This revealed a considerable variation of integration sites of Hctcl both within and between populations, although several integrations were shared by populations of different geographical origin. For four of the eight markers allele frequencies were shifted during selection for resistance to chemotherapeutics and/or inbreeding. For two positions both the 5' and 3' regions flanking Hctcl were isolated and PCR showed that for these two positions the variation of transposon associated markers between populations were indeed caused by variation in integration of Hctcl. For these two positions co-dominant markers were developed. These results indicate that Hctcl insertions may serve as genetic markers for H. contortus.
Asunto(s)
Elementos Transponibles de ADN , Variación Genética , Haemonchus/genética , Animales , Antinematodos/farmacología , Secuencia de Bases , Southern Blotting , ADN de Helmintos/genética , ADN de Helmintos/aislamiento & purificación , Resistencia a Medicamentos/genética , Marcadores Genéticos , Haemonchus/efectos de los fármacos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Análisis de Secuencia de ADNRESUMEN
Hctc1, a member of the Tc1-family of transposable elements was isolated from the parasitic nematode Haemonchus contortus. Hctc1 is 1590 bp long, is flanked by 55 bp inverted repeats and carries a single open reading frame of a 340 amino acid transposase-like protein. Hctc1 is similar to Tc1 of Caenorhabditis elegans and elements Tcb1 and Tcb2 of Caenorhabditis briggsae in the inverted terminal repeats, the open reading frame, as well as the target insertion sequence. Furthermore, the copy number of Hctc1 is comparable with the Tc1 copy number in low copy strains of C. elegans. The sequence of Hctc1 is highly variable in H. contortus due to deletions, insertions and point mutations, with at least five distinct length variants of Hctc1. Most of the Hctc1 variation was within rather than between H. contortus populations. The high level of sequence variation is probably due to variation generally found for members of the Tc1-family, as well as a high background level of genetic variation of H. contortus.
Asunto(s)
Elementos Transponibles de ADN/genética , Haemonchus/genética , Polimorfismo Genético , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN de Helmintos/análisis , Dosificación de Gen , Variación Genética , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADNRESUMEN
Polymorphic molecular markers are being identified to characterize the genomes of parasitic nematodes. The aim is to construct a map with markers evenly spread over the six chromosomes. With such a map, regions can be identified that are under selection pressure when attempts are being made to eradicate worms, be it by drugs, vaccines or genetic resistance in the sheep. Several types of markers have been developed, microsatellites, transposon-associated markers, amplified fragment length polymorphism (AFLP) and expressed sequence tag (EST) markers. Linkage groups can be constructed using several genetic crosses between inbred and drug resistant strains. EST markers will be especially important for comparative mapping with the genome of Caenorhabditis elegans, and therefore localization of the linkage group on a chromosome. It will then be possible to identify functional genes close to markers that have changed allele frequencies under selection pressure and identify the mechanisms of resistance to parasite control.
Asunto(s)
ADN de Helmintos/genética , Marcadores Genéticos , Genoma , Nematodos/genética , Polimorfismo Genético , Animales , Haemonchus/genéticaRESUMEN
A P-glycoprotein gene probe from the sheep parasitic nematode Haemonchus contortus was developed and used to analyse restriction fragment length polymorphisms between susceptible isolates and isolates resistant to either benzimidazole; levamisole and benzimidazole; or benzimidazole, ivermectin and closantel. No polymorphism could be correlated with any of the different resistances. A P-glycoprotein gene probe was also isolated from the human nematode parasite Onchocerca volvulus and an Onchocerca-specific PCR was developed.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Sondas de ADN/genética , Resistencia a Múltiples Medicamentos/genética , Haemonchus/efectos de los fármacos , Haemonchus/genética , Onchocerca volvulus/efectos de los fármacos , Onchocerca volvulus/genética , Secuencia de Aminoácidos , Animales , Southern Blotting , Clonación Molecular , ADN de Helmintos , Electroforesis en Gel de Agar , Filaricidas , Genes de Helminto/genética , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Alineación de Secuencia , Ovinos , Especificidad de la EspecieRESUMEN
To develop tools to analyse parasitic nematode population structures and the effects of selection pressure on the nematode population, we isolated and characterised 13 microsatellite markers of the nematode Haemonchus contortus. The density of CA/GT microsatellites, once in 575 kb, is lower than in mammals. Although the isolated CA/GT repeats were imperfect, the majority of the microsatellites were polymorphic in one or more populations. An extensive genetic diversity both within and between populations could be established. Two-thirds of the CA/GT microsatellites were followed by a variable number of 128 bp direct repeat elements, HcREP1. HcREP1 is a repetitive element in the H. contortus genome, which is homologous to the repetitive TcREP element in the nematode Trichostrongylus colubriformis.
Asunto(s)
Ligamiento Genético , Haemonchus/genética , Repeticiones de Microsatélite , Polimorfismo Genético , Animales , Secuencia de Bases , ADN de Helmintos/aislamiento & purificación , Repeticiones de Dinucleótido , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
The anthelminitic drug levamisole is thought to bind to nicotinic acetylcholine receptors of nematodes. It is possible that resistance to this drug is associated with either a change in binding characteristics or a reduction in the number of nicotinic acetylcholine receptors. Therefore, the molecular mechanism of levamisole resistance in the parasitic nematode Haemonchus contortus was studied by isolating and characterising cDNA clones encoding a putative ligand binding nicotinic acetylcholine receptor subunit, HCAl, of two susceptible and one levamisole resistant population. Hcal is related to unc-38, a nicotinic acetylcholine receptor subunit gene associated with levamisole resistance in Caenorhabditis elegans. Although extensive sequence analyses of hcal sequences revealed polymorphism at amino acid level, no association with levamisole resistance could be detected. Restriction fragment length polymorphism analyses confirmed that, although polymorphism was detected, no selection of a specific allele of hcal has taken place during selection for levamisole resistance in various levamisole resistant populations.
Asunto(s)
Antinematodos/farmacología , Genes de Helminto , Haemonchus/efectos de los fármacos , Haemonchus/genética , Levamisol/farmacología , Receptores Nicotínicos/genética , Secuencia de Aminoácidos , Animales , Antinematodos/metabolismo , Secuencia de Bases , Caenorhabditis elegans/genética , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/genética , ADN de Helmintos/genética , Resistencia a Medicamentos/genética , Haemonchus/metabolismo , Levamisol/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Receptores Nicotínicos/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Parasitic helminths (worms) cause serious infectious diseases in humans and domestic animals. Control of these infections relies mostly on chemotherapeutics (the anthelmintics), but resistance has developed against most of these broad-spectrum drugs in many parasite species. These resistant parasites are being used to elucidate the molecular mechanisms of drug resistance and drug action. This has led to the development of sensitive assays to detect resistant parasites, but this has not delayed the emergence of additional drug resistant parasite populations. Therefore, as development of new drugs by pharmaceutical companies is slow, we may have to be prepared for a time when broad-spectrum drugs are no longer effective, especially against worms of sheep.
Asunto(s)
Antinematodos/uso terapéutico , Infecciones por Nematodos/tratamiento farmacológico , Infecciones por Nematodos/veterinaria , Animales , Antineoplásicos/farmacología , Bencimidazoles/farmacología , Resistencia a Medicamentos , Haemonchus/efectos de los fármacos , Ovinos , Enfermedades de las Ovejas/tratamiento farmacológico , Tubulina (Proteína)/genéticaRESUMEN
To investigate the characteristics of selection for levamisole resistance in Haemonchus contortus, the consecutive nematode generations of an in vivo selection were monitored with a newly developed egg-hatch assay. The in vivo selection was started with a population not previously exposed to any anthelmintics (SHS). At first, the levamisole resistance progressed gradually in successive nematode generations by treating sheep with increasing doses of levamisole, the initial dose being 1 mg kg-1. Treatment with 5 mg kg-1 levamisole resulted, however, in a steep increase of resistance. The selection was ended after six generations, since a level of 30 mg kg-1 levamisole, which is not far from the toxic level for sheep, was reached. The final population, RHS6, was studied in a controlled test. Treatment of RHS6-infected sheep with 30 mg kg-1 levamisole caused an 80% decrease of faecal egg output, and a reduction of 34% in worm numbers. It was remarkable that only the number of female adults was reduced. RHS6 showed a reduced viability, but a fertility similar to the starting population SHS.
Asunto(s)
Antinematodos/farmacología , Haemonchus/efectos de los fármacos , Levamisol/farmacología , Selección Genética , Animales , Bioensayo , Resistencia a Medicamentos/genética , Femenino , Hemoncosis/tratamiento farmacológico , Hemoncosis/parasitología , Óvulo/efectos de los fármacos , Óvulo/crecimiento & desarrollo , OvinosRESUMEN
Many anaerobically functioning eukaryotes have an anaerobic energy metabolism in which fumarate is reduced to succinate. This reduction of fumarate is the opposite reaction to succinate oxidation catalyzed by succinate-ubiquinone oxidoreductase, complex II of the aerobic respiratory chain. Prokaryotes are known to contain two distinct enzyme complexes and distinct quinones, menaquinone and ubiquinone (Q), for the reduction of fumarate and the oxidation of succinate, respectively. Parasitic helminths are also known to contain two different quinones, Q and rhodoquinone (RQ). This report demonstrates that RQ was present in all examined eukaryotes that reduce fumarate during anoxia, not only in parasitic helminths, but also in freshwater snails, mussels, lugworms, and oysters. It was shown that the measured RQ/Q ratio correlated with the importance of fumarate reduction in vivo. This is the first demonstration of the role of RQ in eukaryotes, other than parasitic helminths. Furthermore, throughout the development of the liver fluke Fasciola hepatica, a strong correlation was found between the quinone composition and the type of metabolism: the amount of Q was correlated with the use of the aerobic respiratory chain, and the amount of RQ with the use of fumarate reduction. It can be concluded that RQ is an essential component for fumarate reduction in eukaryotes, in contrast to prokaryotes, which use menaquinone in this process. Analyses of enzyme kinetics, as well as the known differences in primary structures of prokaryotic and eukaryotic complexes that reduce fumarate, support the idea that fumarate-reducing eukaryotes possess an enzyme complex for the reduction of fumarate, structurally related to the succinate dehydrogenase-type complex II, but with the functional characteristics of the prokaryotic fumarate reductases.
Asunto(s)
Complejos Multienzimáticos/metabolismo , Oxidorreductasas/metabolismo , Succinato Deshidrogenasa/metabolismo , Ubiquinona/análogos & derivados , Animales , Transporte de Electrón , Complejo II de Transporte de Electrones , Helmintos/enzimología , Helmintos/metabolismo , Cinética , Oxidación-Reducción , Ubiquinona/metabolismoRESUMEN
Resistance to antimitotic chemotherapeutics in pathogenic nematodes, fungi and mammalian cells is closely associated with structural changes in cytoskeletal beta-tubulin. We investigated the possibility of using the well-characterised free-living nematode Caenorhabditis elegans as a model for studying the mechanism of resistance against benzimidazole (BZ) drugs in the parasitic nematode Haemonchus contortus. Functional analysis of a conserved beta-tubulin isotype (tub-1) mutation near GTP-binding domain II, which is linked to BZ resistance, was carried out in C. elegans by heterologous expression of: (1) parasite BZ-sensitive alleles; (2) BZ-resistant alleles; and (3) in vitro mutagenised beta-tubulin gene constructs. The injected heterologous gene constructs were not only stably maintained, but also expressed as shown by reverse transcriptase-polymerase chain reaction analysis. The degree of BZ drug susceptibility of the transformants was assayed and quantified by incubation with both benomyl and thiabendazol. All H. contortus tub-1 constructs, which encoded Phe at position 200, conferred susceptibility to thiabendazole in BZ-resistant C. elegans ben-1 mutants. In contrast, constructs carrying Tyr200 did not alter the BZ drug phenotype. From these experiments we conclude that: (1) C. elegans can be used as an expression host, since injected parasite genes were biologically active; and (2) the single Phe to Tyr mutation at position 200 in beta-tubulin isotype 1 is the cause of BZ resistance in H. contortus.
Asunto(s)
Antinematodos/farmacología , Caenorhabditis elegans/genética , Haemonchus/efectos de los fármacos , Haemonchus/genética , Proteínas del Helminto/genética , Tubulina (Proteína)/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/efectos de los fármacos , Resistencia a Medicamentos/genética , Datos de Secuencia Molecular , Mutación , Relación Estructura-Actividad , Transformación GenéticaRESUMEN
The carbohydrate metabolism of free-living and parasitic stages of the sheep nematode Haemonchus contortus was studied, and it was demonstrated that during development a switch occurred from Krebs-cycle activity towards a more fermentative metabolism. During this switch a transition might take place in complex II of the respiratory chain. In the free-living (L3) and early parasitic (XL3) stages, complex II catalyses the oxidation of succinate to fumarate via the Krebs cycle, whereas in adults complex II functions in the reverse reaction, the reduction of fumarate to succinate. L3 and XL3 were shown to already possess a large anaerobic capacity. They survived well in the absence of oxygen or in the presence of cyanide, which completely blocked respiration. Krebs-cycle activity, however, was only partially inhibited by cyanide; the XL3s in particular produced in the presence of cyanide large amounts of propanol, the production of which probably functions as an alternative electron sink. For further investigation of the observed metabolic switch, complex II of the respiratory chain, a key enzyme involved in this switch, was studied. The B subunit of complex II was cloned and sequenced. These clones all showed sequences similar to the B subunit of succinate dehydrogenase from other species, and included the amino-terminal signal sequence for importation into mitochondria. Two genes were identified, types 1 and 2, based on the DNA and amino acid sequences and on the lack of cross-reaction to each other when used as probes on Southern blots. On Northern blots, the two genes showed a different expression pattern during the development of the parasite.(ABSTRACT TRUNCATED AT 250 WORDS)