Frequency of the AGT Pro11Leu Polymorphism in Humans: does Diet Matter?

The Pro11Leu substitution in the AGXT gene, which causes primary hyperoxaluria type 1, is found with high frequency in some human populations (e.g., 5–20% in Caucasians). It has been suggested that this detrimental mutation could have been positively selected in populations with a meat-rich diet. In order to test this hypothesis, we investigated the occurrence of Pro11Leu in both herder and agriculturalist populations from Central Asia. We found a lower frequency of this detrimental mutation in herders, whose diet is more meat-rich, as compared to agriculturalists, which therefore challenges the universality of the previous claim. Furthermore, when combining our original data with previously published results, we could show that the worldwide genetic differentiation measured at the Pro11Leu polymorphism does not depart from neutrality. Hence, the distribution of the variation observed in the AGXT gene could be due to demographic history, rather than local adaptation to diet.     

Manipulation of the adhesive behaviour of skeletal muscle cells on soft and stiff polyelectrolyte multilayers

Polyelectrolyte multilayer coatings have emerged as substrates to control a variety of cell behaviour, including adhesion, proliferation and differentiation. In particular, it is possible to modulate film stiffness by physical or chemical cross-linking. In this study, we evaluate the adhesive behaviour of skeletal muscle cells (C2C12 myoblasts) during the initial steps of spreading on layer-by-layer films of controlled stiffness made of poly(l-lysine) and hyaluronan as model biomaterial surfaces for muscle tissue engineering. We show that integrin clustering, integrin actin cytoskeleton connection and focal adhesion formation for cell spreading can be decoupled by controlling film stiffness. This made it possible to switch the cells morphologically between round and spreading shapes depending on the stiffness of the microenvironment. Although hyaluronan is one of the main components of cross-linked multilayer films, the HA receptor CD44 did not appear to mediate early adhesion as suggested by the use of blocking antibodies. In contrast, integrins were found to play a pivotal role in early adhesion: their activation significantly enhanced C2C12 myoblast spreading on soft films, where they were otherwise round. Integrin clustering was also induced by the softer films and enhanced on the stiffest films. Conversely, the use of soluble inhibitors or blocking antibodies directed against integrins induced a round phenotype on stiff films, where cells were well spread out in control conditions. We show that specific integrins were involved in the adhesion process as blocking β₃, but not β₁, integrins inhibited cell adhesion. These soft, stiff films can thus be used to tune the adhesion of C2C12 myoblasts, an early key event in myogenesis, via integrin clustering and subsequent signalling. They may be further used to decorticate the signalling pathways associated with β₃ integrins.     

Uranium induces apoptosis and is genotoxic to normal rat kidney (NRK-52E) proximal cells.

Uranium (U) is a heavy metal used in the nuclear industry and for military applications. U compounds are toxic. Their toxicity is mediated either by their radioactivity or their chemical properties. Mammalian kidneys and bones are the main organs affected by U toxicity. Although the most characteristic response to U exposure is renal dysfunction, little information is available on the mechanisms of its toxicity at the molecular level. This report studied the genotoxicity of U. Apoptosis induction in normal rat kidney (NRK-52(E)) proximal cells was investigated as a function of exposure time or concentrations (0-800microM). In parallel, DNA damage was evaluated by several methods. In order to distinguish between the intrinsic and the extrinsic pathways of apoptosis, caspases-8, -9, -10 assays were conducted and the mitochondrial membrane potential was measured. Three methods were selected for their complementarities in the detection of genetic lesions. The comet assay was used for the detection of primary lesions of DNA. gamma-H2AX immunostaining was achieved to detect DNA double-strand breaks. The micronucleus assay was used to detect chromosomic breaks or losses. DNA damage and apoptosis were observed in a concentration-dependent manner. This study demonstrated that U is genotoxic from 300microM and induces caspase-dependent apoptosis cell death from 200microM mainly through the intrinsic pathway in NRK-52(E) cells. These results suggest that the DNA damage caused by U is reversible at low concentration (200-400microM) but becomes irreversible and leads to cell death for higher concentrations (500-800microM).     

Phosphodiesterase 11A (PDE11A) and genetic predisposition to adrenocortical tumors.

PURPOSE: We have reported previously nonsense inactivating mutations of the phosphodiesterase 11A (PDE11A) gene in patients with micronodular adrenocortical hyperplasia and Cushing syndrome. The aim of this study is to investigate the presence of somatic or germ-line PDE11A mutations in various types of adrenocortical tumors: ACTH-independent macronodular adrenocortical hyperplasia (AIMAH), adrenocortical adenoma (ACA), and adrenocortical cancer (ACC). EXPERIMENTAL DESIGN: PDE11A was sequenced in 117 adrenocortical tumors and 192 controls subjects; immunohistochemistry for PDE11A and tumor cyclic AMP levels were studied in a subgroup of adrenocortical tumors. RESULTS: One PDE11A inactivating mutation (R307X) was found in one ACA, 22 germ-line missense variants (18.8%) were found in adrenocortical tumors, and only 11 missense variants (5.7%) were found in controls. By comparing the common mutations, a higher frequency of mutations in adrenocortical tumors than in age/sex-matched controls were observed [16% versus 10% in ACC, 19% versus 10% in ACA, and 24% versus 9% in AIMAH; odds ratio (OR), 3.53; P = 0.05]. Somatic DNA from adrenocortical tumors with missense variants showed a wild-type allelic loss. A significant difference between ACC and controls was observed for a polymorphism in exon 6 (E421E; OR, 2.1; P = 0.03) and three associated polymorphisms located in intron 10-exon 11-intron 11 (OR, 0.5; P = 0.01). In AIMAH/ACA, cyclic AMP levels were higher than in normal adrenals and decreased PDE11A immunostaining was present in adrenocortical tumors with PDE11A variants. CONCLUSIONS: The present investigation of a large cohort of adrenocortical tumors suggests that PDE11A sequence defects predispose to a variety of lesions (beyond micronodular adrenocortical hyperplasia) and may contribute to the development of these tumors in the general population.     

Role of oxygen derivatives in the cytotoxicity and DNA damage produced by asbestos on rat pleural mesothelial cells in vitro

The role of reactive oxygen metabolites in the toxic effectsof asbestos on pleural mesothelial cells is not well defined.We exposed rat pleural mesothelial cells (RPMC) to chrysotileand crocidolite fibers (0–40 µg/cm²) in the presenceor absence of catalase and superoxide dismutase (SOD). Cellinjury was measured using the colorimetric 3–4 (5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay and DNA damage was evaluated in terms of unscheduledDNA synthesis (UDS). Catalase (100 U/ml) and SOD (250 U/ml)protected RPMC against asbestos-induced cytotoxicity and DNAdamage. However, the inactivated enzymes and bovine serum albuminalso showed some protection, suggesting that the effect of antioxidantenzymes may be partly related to their protein nature. Theseresults suggest that oxygen derivatives are partly involvedin the toxic effects of asbestos on cultures of RPMC. The presenceof extracellular proteins may also decrease asbestos-producedtoxicity by reducing the degree of RPMC–fiber interaction.