Tania Aguirre

D90A heterozygosity in the SOD1 gene is associated with familial and apparently sporadic amyotrophic lateral sclerosis

All mutations in the SOD1 gene associated with familial ALS behave as dominant traits.One mutation, however, giving rise to an aspartic acid to alanine substitution in codon 90 (D90A), was reported only to induce motor neuron disease in homozygous individuals in the Scandinavian population. We describe two families with ALS and one apparently sporadic ALS patient who are heterozygous for the D90A mutation. One patient had the unusual phenotype of focal nonprogressing motor neuron disease. NEUROLOGY 1996;47: 1336-1339

Cloning and sequencing of a 24-kDa Trypanosoma cruzi specific antigen released in association with membrane vesicles and defined by a monoclonal antibody

Summary— In the present study we have used the Tcr7 monoclonal antibody (mAb) previously characterized as directed against Trypanosoma cruzi 24–25‐kDa specific antigens, both are immunogenic in man and during experimental T cruzi infections. We have demonstrated that mAb Tcr7 was able to recognize two in vitro translation products of molecular weights of 24 and 25 kDa. This suggested the holoproteic nature of these two related antigens bearing at least a common epitope and allowed us to use Tcr7 for an immunoscreening of a lambda ZAPII T cruzi cDNA library. Indeed, we have obtained several positive clones and completly sequenced the largest one which encoded theoretically for a protein of 23.7 kDa. The sequence analysis revealed a nearly perfect homology between this clone and one already described by other investigators and was shown to express a major flagellar protein of T cruzi able to bind calcium. Using different overlapping peptides derived from the sequence of the cDNA clone, we have localized the immunoreactivity of mAb Tcr7 mainly on several primary sequences present in the N‐terminal part of the sequence, suggesting that the mAb could recognize a discontinuous epitope. Moreover, the immunoelectron microscopy allowed us to show that the antigen(s) carrying the epitope reacting with mAb Tcr7 is (are) released in association with membrane vesicles which protruded from the parasite surface and the flagellar pocket. This new mechanism of antigen shedding is likely to be independent of phospholipase C‐mediated release of GPI‐anchored molecules.

Antiproliferative effects and mechanism of action of ICI 195,739, a novel bis-triazole derivative, on epimastigotes and amastigotes of Trypanosoma (Schizotrypanum) cruzi.

The in vitro antiproliferative effects of ICI 195,739, a recently developed bis-triazole derivative (T. Boyle, D. J. Gilman, M. B. Gravestock, and J. M. Wardleworth, Ann. N.Y. Acad. Sci. 544:86-100, 1988; J. F. Ryley, S. McGregor, and R. G. Wilson, Ann. N.Y. Acad. Sci. 544:310-328, 1988), on epimastigotes and amastigotes of Trypanosoma (Schizotrypanum) cruzi and some aspects of its mechanism of action are described. Despite previous claims that triazole compounds act on susceptible organisms by essentially the same mechanism demonstrated for the imidazole compounds, i.e., by interfering with the synthesis of ergosterol at the level of the cytochrome P-450-dependent C-14 demethylation of lanosterol, our results indicate that ICI 195,739 acts on T. cruzi epimastigotes by a dual mechanism which involves blockade of ergosterol byosynthesis and a second, still-unidentified target whose alteration leads to immediate growth arrest. Although ICI 195,739 blocks ergosterol biosynthesis at the level of C-14 lanosterol demethylation, as shown by gas-liquid and thin-layer chromatography, growth arrest in ICI 195,739-treated cells is not related to the depletion of the endogenous ergosterol pool, contrary to what was previously found for ketoconazole, the reference compound among antifungal and antiprotozoal azole derivatives. Consistent with this observation is the fact that the concentration of ICI 195,739 required to inhibit de novo synthesis of ergosterol in epimastigotes by 50% is 60 nM, which is essentially identical to that previously found for ketoconazole under identical conditions, while the minimum concentration required to produce complete growth inhibition is 0.1 microM, which is 300 times lower than that of ketoconazole. With respect to the intracellular amastigote form proliferating inside vertebrate (Vero) cells, 10 nM is sufficient to eradicate the parasite completely in 96 h, with no effects on the host cells; this concentration is identical to that previously found for ketoconazole. Growth inhibition and morphological alterations induced by ketoconazole can be reserved by exogenously added ergosterol but not by cholesterol; for ICI 195, 739, neither sterol is capable of reserving the drug effects. Contrary to what was observed for ketoconazole, the in vitro antiproliferative effects of ICI 195, 739 on both forms of the parasite are not potentiated by the simultaneous presence of terbinafine, an allylamine which blocks ergosterol production by the parasite at a different level of the sterol biosynthetic pathway. These results, together with those of an accompanying study of the ultrastructural alterations induced by the drug, strongly support the notion that ICI 195, 739 acts on T. cruzi by a novel combination of biochemical and cellular effects, which could explain its extraordinary potency in vivo against the parasite. Images

Mutational analysis of the Cu/Zn superoxide dismutase gene in 23 familial and 69 sporadic cases of amyotrophic lateral sclerosis in Belgium.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder caused by degeneration of motor neurons of the spinal cord and brain. The majority of ALS cases are sporadic (SALS). However, in 10–15% of ALS cases the disease is inherited as an autosomal dominant trait (familial ALS or FALS). We used a non-radioactive SSCP method, in combination with solid phase sequencing, to screen the entire SOD1 (Cu/Zn superoxide dismutase) coding region and flanking intronic sequences for mutations. Twenty-three patients from 11 ALS families and 69 SALS patients of Belgian origin were studied. Three different mutations were identified (L38V, D90A and G93C) in seven families. Importantly, the D90A was only found in the heterozygous state. In addition two single base pair variants (IVS1 +19G>A and AAC139 AAT) were identified in two SALS patients. These results suggest that the SOD1 analysis is useful in FALS but less so in SALS cases. The SSCP analysis has proved fast and reliable for this purpose.

Fluorescent chemical cleavage of mismatches for efficient screening of the factor VIII gene

The detection of mutations in large and complex genes represents a practical challenge in research and diagnostic laboratories. Available methods are either time‐consuming or lack sensitivity. Mutation detection in the factor VIII gene, responsible for haemophilia A, is hampered by its large size, its many exons, and the high frequency of de novo mutations that result in different mutations in unrelated patients. For an exhaustive analysis of mutations in the factor VIII gene, we established a nonradioactive screening method based on chemical cleavage of mismatches (CCM). PCR‐fragments of ˜ 1 kb were generated from genomic DNA (exon 14) or after reverse transcription from mRNA isolated from blood cells. Some modifications have been made to improve the CCM strategy. First, using a fluorescent tag, the method gains safety and flexibility. Second, fluorescent detection allows an accurate sizing of digested fragments when measured on an automated DNA sequencer. Third, by labelling both 5′ ends of the PCR‐fragment, the detection rate is virtually 100%. Finally, in the case of an X‐linked disease, samples from two patients can be mixed, which reduces the workload without losing information. In a pilot experiment, mutations were detected in 20 of 20 patients. In this series, three small insertions, two small deletions, one nonsense mutation, 13 missense mutations, and one splice mutation were found. Fifteen of these mutations are new. Thus virtually all kind of mutations are detectable by this method. Moreover, the analysis of the gene can be completed in 2 days. Hum Mutat 11:470–479, 1998.