Glycosidase active site mutations in human alpha-L-iduronidase.

Mucopolysaccharidosis type I (MPS I; McKusick 25280) results from a deficiency in alpha-L-iduronidase activity. Using a bioinformatics approach, we have previously predicted the putative acid/base catalyst and nucleophile residues in the active site of this human lysosomal glycosidase to be Glu182 and Glu299, respectively. To obtain experimental evidence supporting these predictions, wild-type alpha-L-iduronidase and site-directed mutants E182A and E299A were individually expressed in Chinese hamster ovary-K1 cell lines. We have compared the synthesis, processing, and catalytic properties of the two mutant proteins with wild-type human alpha-L-iduronidase. Both E182A and E299A transfected cells produced catalytically inactive human alpha-L-iduronidase protein at levels comparable to the wild-type control. The E182A protein was synthesized, processed, targeted to the lysosome, and secreted in a similar fashion to wild-type alpha-L-iduronidase. The E299A mutant protein was also synthesized and secreted similarly to the wild-type enzyme, but there were alterations in its rate of traffic and proteolytic processing. These data indicate that the enzymatic inactivity of the E182A and E299A mutants is not due to problems of synthesis/folding, but to the removal of key catalytic residues. In addition, we have identified a MPS I patient with an E182K mutant allele. The E182K mutant protein was expressed in CHO-K1 cells and also found to be enzymatically inactive. Together, these results support the predicted role of E182 and E299 in the catalytic mechanism of alpha-L-iduronidase and we propose that the mutation of either of these residues would contribute to a very severe clinical phenotype in a MPS I patient.

Use of molecular beacons to detect an antifolate resistance-associated mutation in Plasmodium falciparum.

A PCR-based technique using new fluorescent probes, called molecular beacons, was developed to detect the antifolate resistance-associated S108N point mutation in Plasmodium falciparum. One hundred African clinical isolates were tested by the new method in comparison with the PCR-restriction fragment length polymorphism method. This new molecular technique appears to be a promising tool for epidemiological studies.

[Antibiotic sensitivity of gram negative bacilli isolated from urinary tract infections at NUHC of Cotonou (Benin) from March to December 1992]. / Sensibilité aux antibiotiques de bacilles a gram négatif isolés d'infections urinaires au CHNU de Cotonou (Bénin) de mars à Décembre 1992.

Eleven antibiotics were tested against 1,194 Gram negative bacilli isolated from urinary tract infections at the National University Hospital Center at Cotonou. Among the betalactams tested, only cefotaxime remained active against most of the bacteria tested: 90% of the strains of Escherichia coli and 75% of the strains of Enterobacter cloacae were sensitive. Ampicilline, on the other hand, had lost its activity even on strains which are usually the most susceptible. Thirteen percent of the E. coli strains were sensitive. This reduction in antibiotic activity against bacterial strains in Cotonou, which concerned to various degrees the tetracyclines, chloramphenicol, cotrimoxazole, is less pronounced for the amino-sides (gentamicine and netilmicine), and the quinolones of which nalidixique acid was active against 83.9% of the strains of E. coli. The low frequency of isolation of wild type strains (sensitive to betalactams) is probably the consequence of strong selection pressure due to a massive, and uncontrolled use of antibiotics in Cotonou.