Evolutionary conservation of redundancy between a diverged pair of forkhead transcription factor homologues.

The Caenorhabditis elegans gene pes-1 encodes a transcription factor of the forkhead family and is expressed in specific cells of the early embryo. Despite these observations suggesting pes-1 to have an important regulatory role in embryogenesis, inactivation of pes-1 caused no apparent phenotype. This lack of phenotype is a consequence of genetic redundancy. Whereas a weak, transitory effect was observed upon disruption of just T14G12.4 (renamed fkh-2) gene function, simultaneous disruption of the activity of both fkh-2 and pes-1 resulted in a penetrant lethal phenotype. Sequence comparison suggests these two forkhead genes are not closely related and the functional association of fkh-2 and pes-1 was only explored because of the similarity of their expression patterns. Conservation of the fkh-2/pes-1 genetic redundancy between C. elegans and the related species C. briggsae was demonstrated. Interestingly the redundancy in C. briggsae is not as complete as in C. elegans and this could be explained by alterations of pes-1 specific to the C. briggsae ancestry. With overlapping function retained on an evolutionary time-scale, genetic redundancy may be extensive and expression pattern data could, as here, have a crucial role in characterization of developmental processes.

Direct determination of some phenothiazine sedatives in greyhound urine by fluoroimmunoassay.

Antibodies have been raised in rabbits to a chlorpromazine sulfoxide-bovine serum albumin immunogen and the resulting antiserum used to develop a magnetizable solid-phase antibody fluoroimmunoassay for the detection of sulfoxide metabolites of commonly used phenothiazine and thioxanthine neuroleptics. These assays were used to screen metabolite levels in the urine of a greyhound following oral medication with chlorpromazine in order to assess the potential of these assays as simple screens for detecting exposure of racing greyhounds to such sedatives. The urine samples were also screened for neuroleptic content using an established radioreceptor assay and by TLC. The immunoassay described represents a relatively simple, sensitive and group-specific alternative method for screening for medication with phenothiazine and structurally similar sedatives in urine samples.

Kinetic modelling of isometamidium chloride (Samorin) uptake by Trypanosoma congolense.

Clones of Trypanosoma congolense which express resistance to the widely used trypanocide isometamidium chloride accumulate less of the drug than clones which are sensitive to drug treatment. A mathematical model has been developed which was able to predict theoretical lines representing the uptake kinetics in trypanosomes which were sensitive to isometamidium, as well as for resistant trypanosomes in which reduced accumulation was a result of either reduced uptake or enhanced efflux of the drug. Data from drug uptake experiments were then fitted to these theoretical lines. While the value for drug efflux could not be separated from the dissociation constant of the trypanosomes for isometamidium, it was demonstrated that reduced accumulation is not a result of reduced uptake of isometamidium by drug-resistant trypanosomes.

Transport of isometamidium (Samorin) by drug-resistant and drug-sensitive Trypanosoma congolense.

The uptake kinetics of a 14C-labelled trypanocidal compound isometamidium chloride (Samorin, RMB Animal Health Ltd, UK) was measured in drug-resistant and drug-sensitive Trypanosoma congolense. It was established that drug uptake was significantly more rapid and quantitatively greater in drug-sensitive parasites. There was clear evidence that drug uptake in both the resistant and sensitive trypanosomes was by a specific, receptor-mediated process. This specific drug transport was energy-dependent, being sensitive to metabolic inhibition with SHAM/glycerol. Significant differences in drug transport were observed which could be correlated with resistance to isometamidium. The optimal pH for drug accumulation was lowered in the resistant trypanosomes; this finding, along with an observed change in specificity for the related compound homidium bromide, suggested that the specific receptor for isometamidium is altered in the resistant trypanosomes, possibly resulting in a reduction in drug uptake. In addition to these alterations in drug uptake, efflux of isometamidium also appears to occur in the resistant trypanosomes. Both a reduction in incubation temperature and metabolic inhibition increased the level of trypanosome-associated isometamidium in the resistant parasites. This was in contrast to observations using drug-sensitive parasites. Furthermore, the addition of calcium flux-modulating agents to the incubation medium also resulted in an increase in accumulation by the resistant parasites.

Effect of isometamidium on Trypanosoma congolense infectivity.

Isometamidium chloride (Samorin, RMB, England) is a widely used and highly effective trypanocide for the treatment of bovine trypanosomiases. However, the appearance of isometamidium-resistant populations of T. congolense in Africa makes it necessary to develop methods for the rapid and reliable detection of drug resistance in the laboratory. Currently available tests are time-consuming and/or expensive. In the present study, the short-term in vitro incubation of trypanosomes in a range of isometamidium concentrations and the infectivity of the parasites in mice has been assessed. A series of T. congolense isolates were used which were known to differ in their in vivo sensitivity to the drug. The results showed a close correlation between the known level of resistance and the capability of trypanosomes to remain infective after incubation in isometamidium. Thus isolates displaying a high level of resistance in vivo remained infective following incubation in higher concentrations of drug. This assay may provide a simple and reliable method for detecting drug resistance in T. congolense.