Annick Thompson

cDNA cloning of a novel secreted isoform of the human receptor for advanced glycation end products and characterization of cells co-expressing cell-surface scavenger receptors and Swedish mutant amyloid precursor protein.

The receptor for advanced glycation end products (RAGE) has been proposed as a cell surface receptor that binds amyloid-beta protein (Abeta), thereby triggering its cytotoxic effects [S.D. Yan, X. Chen, J. Fu, M. Chen, H. Zhu, A. Roher, T. Slattery, L. Zhao, M. Nagashima, J. Morser, A. Migheli, P. Nawroth, D. Stern, A.M. Schmidt, RAGE and amyloid-beta peptide neurotoxicity in Alzheimers disease, Nature 382 (1996) 685-691.]. A cDNA library of human lung was screened for RAGE with an appropriate hybridization probe. In addition to cell surface RAGE, one clone was found which encodes a new version of RAGE, termed hRAGEsec, which lacks the 19 amino acids of the membrane-spanning region and is therefore secreted. Comparison with the genomic sequence revealed that the synthesis of the secreted isoform requires alternative splicing. The deduced protein sequence of the mature hRAGEsec consists of 321 amino acids with a predicted molecular mass of 35.66 kDa. The pattern of expression of hRAGEsec in human brain was analyzed by in situ hybridization histochemistry. The most intense expression of the gene in contrast to cell surface RAGE was detected in hippocampal CA3 pyramidal cells, dentate gyrus granule cells, cortical neurons as well as glial cells in white matter. To investigate the interaction between Abeta and RAGE and another scavenger receptor, SRA, under physiological conditions, they were co-expressed with human betaAPP(695)-SFAD in a human cell and the level of Abeta in the condition medium was assessed by immunoprecipitation and enzyme-linked immunosorbent assay (ELISA) analysis. A nearly 100% reduction of Abeta from the conditioned medium of hRAGE cells and approximately 40% reduction from the SRA-cells implied that hRAGE could be a prominent cell surface receptor interacting with Abeta.

Pharmacogenetic analysis of adverse drug effect reveals genetic variant for susceptibility to liver toxicity.

A retrospective pharmacogenetic study was conducted to identify possible genetic susceptibility factors in patients in whom the administration of the anti-Parkinson drug, tolcapone (TASMAR®), was associated with hepatic toxicity. We studied 135 cases of patients with elevated liver transaminase levels (ELT) of ≥1.5 times above the upper limit of normal, in comparison with matched controls that had also received the drug but had not experienced ELT. DNA samples were genotyped for 30 previously described or newly characterized bi-allelic single nucleotide polymorphisms (SNPs), representing 12 candidate genes selected based on the known metabolic pathways involved in the tolcapone elimination. SNPs located within the UDP-glucuronosyl transferase 1A gene complex, which codes for the enzymes involved in the main elimination pathway of the drug, were found to be significantly associated with the occurrence of tolcapone-associated ELTs.

X-linked immunodeficiency with hyperimmunoglobulinemia M appears to be linked to the DXS42 restriction fragment length polymorphism locus

SummaryThe gene involved in X-linked immunodeficiency with hyperimmunoglobulinemia M (XHM) was localized by the use of nine restriction fragment length polymorphic (RFLP) markers covering the entire X chromosome. Multipoint linkage analysis of RFLP data obtained in a three generation XHM pedigree indicates the Xq24-q27 area around the DXS42 RFLP locus as the most likely localization of the XHM locus.

Genetic Polymorphism of CYP2C19 Gene in the Stanislas Cohort. A link with Inflammation

CYP2C19, a member of the cytochrome P450 family, metabolises arachidonic acid to produce epoxyeicosanoid acids, which are involved in vascular tone and inflammation. Thus, this study describes the possible relationship between a CYP2C19 polymorphism (681G>A) and three inflammatory markers: interleukin (IL)‐6, tumor necrosis factor‐α (TNF‐α) and high sensitivity C‐reactive protein (hs‐CRP) in healthy individuals.

cDNA cloning and molecular characterization of human brain metalloprotease MP100: a beta-secretase candidate?

Abstract : Metalloprotease MP100 was originally isolated as a β‐secretase candidate from human brain using a β‐amyloid precursor protein (β‐APP)‐derived p‐nitroanilide (pNA) peptide substrate. Peptide sequences from purified MP100 were now found to resemble sequences reported for a puromycin‐sensitive aminopeptidase (PSA) highly enriched in brain, and cDNA cloning revealed nearly complete homology of MP100 to PSA, with only a single bp difference resulting in an amino acid change at position 184. Another MP100 cDNA encoded a protein with a 36‐amino acid deletion (positions 180‐217) and a two‐amino acid insertion after Val533. Purified recombinant human MP100 cleaved the original pNA substrate as well as a free β‐site‐spanning amyloid β (Aβ) peptide (Aβ‐10/+10), generating Aβ1‐10. The latter substrate, however, remained uncleaved, if N‐ and C‐terminally blocked, and also purified β‐APP was not cleaved. Double immunoimaging revealed partial, patchy, colocalization of β‐APP and MP100 in doubly transfected human embryonic kidney cells (HEK cells) and in normal neuroblastoma cells, and both proteins could be coimmunoprecipitated from rat brain extracts, suggesting their close vicinity in vivo. Coexpression of MP100 and β‐APP695, however, did not boost Aβ levels in HEK cells, although active enzyme was produced. Thus, MP100 does not exert true β‐secretase‐like function in cells, although it may well act as a secondary exoprotease in a complex β‐APP/Aβ metabolism.

Interaction between CYP1A1 T3801C and AHR G1661A polymorphisms according to smoking status on blood pressure in the Stanislas cohort.

Background CYP1A1, one of the key enzymes in detoxifying toxic components produced during cigarette smoking, is regulated by aromatic hydrocarbon receptor (AHR). A CYP1A1 T3801C polymorphism, associated with a higher CYP1A1 inducibility and enhanced catalytic activity, has been linked to stroke, triple vessel disease and may, therefore, be associated with blood pressure (BP). The relation of the widely studied G1661A polymorphism of the human AHR gene with BP is unknown. Objectives To investigate the genetic influence of CYP1A1 T3801C and AHR G1661A polymorphisms on BP in relation to tobacco consumption. Design and participants Study participants were selected from a French longitudinal cohort of volunteers for a free health check-up. These individuals (302 men and 311 women) were not taking medication that can affect blood pressure. Information about active smoking status was obtained by a self-administered questionnaire. Results After multiple regression analysis, systolic blood pressure (SBP) and diastolic blood pressure (DBP) did not differ significantly according to their tobacco status excepted for DBP in men. In addition, neither CYP1A1 T3801C nor AHR G1661A polymorphism was linked to blood pressure. However, systolic and diastolic blood pressures differed significantly according to CYP1A1 T3801C genotype between ex-smokers and smokers. Finally, the interaction between CYP1A1 T3801C and AHR G1661A polymorphisms explained a significant difference of SBP and DBP between carriers of both CYP1A1-C3801 and AHR-A1661 alleles. Conclusion This study is the first to show an interaction between the CYP1A1 T3801C and AHR G1661A polymorphisms. This interaction could explain the difference in blood pressure level between smokers and non-smokers/ex-smokers but needs to be confirmed in a large sample.

Heterogeneity in the map distance between X-linked agammaglobulinemia and a map of nine RFLP loci

SummaryIn nine family pedigrees in which X-linked agammaglobulinemia (XLA) is segregating, a multi-point linkage analysis has been carried out. In each family, the map distance, d, between XLA and a fixed point in a known map of nine RFLP loci on the X chromosome was estimated by calculating the log likelihoods, L(d). Using a new method, the 10-point likelihood was approximated by appropriately combining three 4-point likelihoods. Homogeneity tests (admixture tests) were performed showing clear evidence for heterogeneity of XLA.

Expression and characterization of human β-secretase candidates metalloendopeptidase MP78 and cathepsin D in βAPP-overexpressing cells

Abstract Human β-secretase candidates, MP78 (h-MP78, EC 3.4.24.15) and cathepsin D (Cat D, EC 3.4.23.5), were evaluated for their ability to enhance amyloid-β-protein (Aβ) secretion when overexpressed in βAPP-containing cells. HEK-293 cells stably co-expressing h-MP78 or Cat D and h-βAPP695 were metabolically labeled with [35S]methionine and Aβ secretion was quantified in the conditioned media by immunoprecipitation and ELISA without showing any significant increase in Aβ production. Because Cat D is known to have a higher affinity for APP-substrate containing the Swedish familial Alzheimers disease double mutation (SFAD, K595N and M596L substitutions in βAPP695) than for the wild type substrate [Dreyer et al., Eur. J. Biochem., 224 (1994) 265–271], the effect of Cat D overexpression was tested in a HEK293/βAPPSFAD stable cell line. ELISA analysis of the conditioned media from these cells did also not reveal any increase in Aβ generation. In addition, recombinant h-MP78 purified from E. coli cleaved an APP-derived substrate spanning the β-secretase site (ISEVKMD1AEFRHDS) at multiple sites, but the β-site cleavage was only a minor one; cleavage occurred predominantly at K–M and E–F bonds. Human liver Cat D also cleaved the same substrate at multiple sites, yet the major cleavage at pH 4.0 occurred at the amyloidogenic D1 site. These findings indicate that h-MP78 does not have the cleavage specificity required for a β-secretase protease and although Cat D fulfilled the amyloidogenic cleavage specificity, the results of the co-expression experiments make both enzymes less likely candidates as relevant β-secretases.

Evidence for male X chromosomal mosaicism in X-linked agammaglobulinemia

SummaryX-Linked agammaglobulinemia (XLA) is a severe antibody deficiency disease in man, resulting from an arrest in differentiation of pre-B cells. XLA is recessive: female carriers do not exhibit antibody deficiency, but manifest an exclusive inactivation of the XLA-carrying X chromosome in all peripheral blood B lymphocytes. An exclusive inactivation of the paternal X chromosome in the B lymphocytes of all daugthers thers of a male who had no agammalobulineamia demonstrated that the XLA defect can originate from healthy males. These males are X chromosomal mosaics. X-Chromosomal RFLP segregation analyses in other XLA pedigrees suggest a frequent introduction of XLA by healthy males. This implies that XLA often originates from mitotic errors, either at postmeiotic or early postzygotic stages.