Summary Pyruvate kinase (PK) deficiency is the most common enzyme defect affecting the glycolytic pathway of the erythrocyte. Usually, it is clinically silent in heterozygotes but serious disorders are described at birth in homozygotes or compound heterozygotes. Including the mutants herein reported, more than 180 mutations of the PK‐LR gene have now been identified. This 3‐year study was carried out to detect mutations associated with disease‐affecting families. Haematological indices, erythrocyte PK and glucose‐6‐phosphate dehydrogenase activities were measured. Molecular characterisation of the PK gene mutations included restriction enzyme analysis, mutation scanning and gene sequencing. Among the 56 families studied, nine homozygous cases and 41 different mutations were found. Eight mutations involved a splice site, 31 missense mutations were located in crucial domains of the molecule (catalytic site, cleft between the A and C domains, A/A’ interface) and two cases of insertion–deletion were found. In total, 20 new mutations modifying the structure of the enzyme and seven affecting a splice site are reported. PK deficiency is an under diagnosed disease. However, deficiency could be life threatening in perinatal period and we report two lethal cases. These results support the characterisation of PK mutations, and show that prenatal diagnosis can identify affected infants and prepare safer conditions for the birth.
Enzymes of the glycolytic pathway as well as some ancillary enzymes were studied in normal red cells parasitized with Plasmodium falciparum in culture at varying parasitemias as well as in isolated parasites. The levels of all enzymes except diphosphoglycerate mutase, glucose-6- phosphate dehydrogenase, and adenylate kinase were elevated. Extreme elevations of hexokinase, aldolase, enolase, pyruvate kinase, and adenosine deaminase concentrations were noted. In most cases, electrophoretically distinct bands of enzyme activity were also seen. These findings partly explain the previously noted 50- to 100-fold increase in glucose consumption of infected red cells and suggest that further knowledge of these parasite enzymes and their genetic basis may aid both in designing new chemotherapy and in understanding the evolution of these parasites.
Using a transgenic approach, we studied the role of GATA-3 in T cells. As previously shown, enforced GATA-3 expression in transgenic mice inhibits Th1 differentiation of CD4 T cells, but unexpectedly, both type 1 (interferon ␥) and type 2 (interleukin (IL)-4 and IL-13) cytokine genes were activated in the transgenic CD8 T cells. Because IL-13 gene expression was highly enhanced in vivo by GATA-3 expression, we studied the human and the mouse IL-13 gene promoters and found an evolutionary-conserved association of a consensus GATA binding site and two GATG motifs. We showed that efficient GATA-3 binding to this regulatory sequence required these three motifs and that the affinity of the GATA zinc fingers for this association was five times higher than for the consensus GATA binding site alone. Transfections in a T cell line or transactivation by GATA-3 showed that the combination of the three sites was required for full transcriptional activity of the IL-13 gene promoter. Finally we showed that this association of binding sites causes a very high sensitivity of the IL-13 gene promoter to small variations in the level of GATA-3 protein. Altogether, these results indicate an important role of GATA-3 in CD8 cytokine gene expression and demonstrate that a critical network of GATA binding sites highly modulates GATA-3 activity.
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