Head-up tilt induced syncope and adenosine A2A receptor gene polymorphism.

AIMS: High adenosine plasma levels and high expression of adenosine A(2A) receptors are observed in patients with unexplained syncope and a positive head-up tilt test (HUT). This study aimed to evaluate the single nucleotide polymorphism (SNP) (c.1364 T>C) which is the most commonly found polymorphism in the A(2A) receptor gene, in patients with unexplained syncope undergoing HUT. METHODS AND RESULTS: One hundred and five patients with unexplained syncope who underwent HUT were included. Fifty-two had a positive test. Receptor genotype determinations were performed in patients and in 121 healthy subjects. Genotype (TT, CC, TC) was determined from DNA leucocytes. The distribution of the polymorphism showed significant (P < 0.0001) difference when the results of HUT were analysed. Fifty-two per cent of patients with a positive HUT had a CC genotype and 34.6% a TC genotype, whereas 13.2% of the patients with a negative HUT had a CC genotype and 71.7% a TC genotype. Patients with a CC genotype had a higher incidence of spontaneous syncopal episodes. CONCLUSION: In patients with unexplained syncope, a significant association between high incidence of syncopal episodes, positive HUT, and the presence of the CC variant in the adenosine A(2A) receptor gene was elicited.

DNA-repair and carcinogen-metabolising enzymes genetic polymorphisms as an independent risk factor for hepatocellular carcinoma in Caucasian liver-transplanted patients.

We studied polymorphisms of three genes, UDP-glucuronosyltransferase1A7 (UGT1A7), Glutathione-S-transferaseM1 (GSTM1) and X-Ray Cross Complementing group 1 (XRCC1), involved in detoxification of xenobiotics or DNA-repair in a population of 133 liver-transplanted patients, including 56 patients with hepatocellular carcinoma (HCC) and 77 without HCC, and in 89 healthy controls originating from the south of France. Multiple logistic regression analysis showed that, among liver-transplanted patients, interactions between XRCC1-G/G or -G/A and GSTM1-nul polymorphisms were independently associated with hepatocellular carcinoma (p interaction=0.027) concurrently with increasing age (p<0.001), male sex (p=0.037) and chronic hepatitis B or C virus infection (p=0.018 and p=0.001 respectively). On the contrary, no relationship was observed between UGT1A7 polymorphisms considered alone or in interaction with GSTM1 or XRCC1 polymorphisms and HCC. This suggests that concomitant impaired metabolism of carcinogenic compounds and impaired DNA-repair function play an important role in liver carcinogenesis in high-risk cirrhotic patients originating from the south of France.

Molecular characterization of myophosphorylase deficiency (McArdle disease) in 34 patients from Southern France: identification of 10 new mutations. Absence of genotype-phenotype correlation.

We report on 31 patients and 3 affected siblings (17 males and 17 females) from Southern France with McArdle disease (two from Spanish and three from Portuguese background). Molecular analysis revealed the presence of five previously described mutations: the common p.R50X nonsense mutation, the p.R94W and p.V456M missense mutations, the p.K609K conservative mutation which generates an aberrant splicing, and the p.K754fs frameshift mutation; and 10 new molecular defects: eight missense mutations at homozygous (p.G136D) or heterozygous state (p.T379M, p.G449R, p.T488I, p.R490Q, p.R570Q, p.R590H, and p.R715W), one nonsense mutation p.R650X and one deletion (p.delK170). Our results confirm that the p.R50X nonsense mutation is also the most common associated with myophosphorylase deficiency in the Southern French population: 21 of 25 French unrelated patients (15 homozygous and six heterozygous, i.e., 72% of the mutated alleles). Two patients, one from Algeria and one from Tunisia, were homozygous for a previously identified missense mutation p.V456M in a Moroccan subject. Our findings further demonstrate molecular heterogeneity of myophosphorylase deficiency, absence of genotype-phenotype correlation and expand the already crowded map of mutations within the myophosphorylase gene. Our study also provides evidence for increased medical interest of malignant hyperthermia susceptibility (MHS) because of 34 McArdle disease patients, three and two affected siblings were contracture-tested and found to be positive.

Increased expression of adenosine A2A receptors in patients with spontaneous and head-up-tilt-induced syncope.

BACKGROUND: Adenosine may play a role in the triggering of neurocardiogenic syncope, but no information on adenosine receptors is available at the present time. OBJECTIVE: The purpose of this study was to investigate whether adenosine A2A receptors expression is altered in patients with neurocardiogenic syncope. METHODS: Adenosine plasma levels (APLs), the expression of A2A receptors, were measured (mean +/- standard error of the mean) during tilt testing. Expression of receptors was assessed on mononuclear cells using a selective receptor ligand. RESULTS: At baseline, the APLs of 16 patients with a positive test were higher than those of 17 patients with a negative test and of those of a control group (2.10 +/- 0.30 vs. 0.40 +/- 0.05 and 0.41 +/- 0.06 muM, respectively; P <.0001). The number of receptors was higher in patients tested positive than in patients tested negative or in the control group (122 +/- 10 vs. 38 +/- 4 and 44 +/- 4 fmol/g of proteins, respectively; P <.0001). No difference was found in the affinity or synthesis among the three groups. CONCLUSION: This study showed an increased number and an up-regulation of adenosine A2A receptors in patients with spontaneous syncope and a positive head-up tilt, which in the context of high APLs may play a role in the recurrence of syncopal episodes.

PAP IB, a new member of the Reg gene family: cloning, expression, structural properties, and evolution by gene duplication.

Reg proteins are expressed in various organs and are involved in cancers and neurodegenerative diseases. They display a typical C-type lectin-like domain but possess additional highly conserved amino acids. By studying human databases and Expressed Sequence Tags library, we identified a new member called PAP IB. Using probabilistic approaches, we established a phylogenetic tree of eighteen Reg proteins. The dendogram showed that they constitute a superfamily composed of three distinct families (FI to FIII) of paralogues that resulted from duplication. We therefore focused on two proteins, REG Ialpha and PAP IB, belonging to the more closely related FI and FII families, respectively. REG Ialpha and PAP IB share 50% sequence identity. After cloning PAP IB, however, we found that it was expressed almost only in pancreas, unlike REG Ialpha, whose expression is ubiquitous. In addition, by building a model of the structure of PAP IB based on the X-ray structure of REG Ialpha, we observed that the two proteins displayed distinctive surface charge distribution, which may lead to different ligands binding. In spite of their common fold that should result in closely related functions, REG Ialpha and PAP IB are a good example of duplication and divergence, probably with the acquisition of new functions, thus participating in the evolution of the protein repertoire.

Assessment of occupational exposure to welding fumes by inductively coupled plasma-mass spectroscopy and by the alkaline Comet assay.

Welding fumes are classified as possibly carcinogenic to humans (Group 2B) by the International Agency for Research on Cancer. In the current study, blood and urine concentrations of aluminum (Al), cadmium (Cd), cobalt (Co), chromium (Cr), manganese (Mn), nickel (Ni), lead (Pb), and zinc (Zn) were monitored by inductively coupled plasma-mass spectrometry (ICP-MS) in 30 welders and in 22 controls. In addition, DNA damage was examined in the lymphocytes of these subjects by the alkaline Comet assay. Two biological samples were taken from the welders at the beginning (BW) and at the end (EW) of a work week. In controls, collection of samples was limited to BW. Blood concentrations of Cd, Co, Cr, Ni, and Pb were higher in the welders than in the control group while higher concentrations of Al, Cd, Co, Cr, Ni, and Pb were detected in welder urines. There was no significant difference in the metal concentrations for the BW and EW welder samples. Increased levels of DNA damage were found in lymphocytes from welders as compared to the controls, and 20/30 welders had higher levels of DNA lesions in the EW than in the BW samples. Age had a significant effect on DNA damage in the control group. Spearman's rank correlation analysis indicated that there were positive correlations between blood concentrations of Al, Co, Ni, and Pb and the levels of DNA damage. A negative correlation was found between DNA damage and Mn in blood, while there was a positive correlation between urinary Mn concentration and DNA damage. These data indicate that occupational exposure to welding fumes increases DNA damage in lymphocytes.

Effect of hyperoxic and hyperbaric conditions on the adenosinergic pathway and CD26 expression in rat.

The nucleoside adenosine acts on the nervous and cardiovascular systems via the A2A receptor (A2AR). In response to oxygen level in tissues, adenosine plasma concentration is regulated in particular via its synthesis by CD73 and via its degradation by adenosine deaminase (ADA). The cell-surface endopeptidase CD26 controls the concentration of vasoactive and antioxidant peptides and hence regulates the oxygen supply to tissues and oxidative stress response. Although overexpression of adenosine, CD73, ADA, A2AR, and CD26 in response to hypoxia is well documented, the effects of hyperoxic and hyperbaric conditions on these elements deserve further consideration. Rats and a murine Chem-3 cell line that expresses A2AR were exposed to 0.21 bar O2, 0.79 bar N2 (terrestrial conditions; normoxia); 1 bar O2 (hyperoxia); 2 bar O2 (hyperbaric hyperoxia); 0.21 bar O2, 1.79 bar N2 (hyperbaria). Adenosine plasma concentration, CD73, ADA, A2AR expression, and CD26 activity were addressed in vivo, and cAMP production was addressed in cellulo. For in vivo conditions, 1) hyperoxia decreased adenosine plasma level and T cell surface CD26 activity, whereas it increased CD73 expression and ADA level; 2) hyperbaric hyperoxia tended to amplify the trend; and 3) hyperbaria alone lacked significant influence on these parameters. In the brain and in cellulo, 1) hyperoxia decreased A2AR expression; 2) hyperbaric hyperoxia amplified the trend; and 3) hyperbaria alone exhibited the strongest effect. We found a similar pattern regarding both A2AR mRNA synthesis in the brain and cAMP production in Chem-3 cells. Thus a high oxygen level tended to downregulate the adenosinergic pathway and CD26 activity. Hyperbaria alone affected only A2AR expression and cAMP production. We discuss how such mechanisms triggered by hyperoxygenation can limit, through vasoconstriction, the oxygen supply to tissues and the production of reactive oxygen species.

Patterns of gene expressions induced by arsenic trioxide in cultured human fibroblasts.

Arsenic exposure is associated with several human diseases and particularly, with neoplasia. Although the mechanism of arsenic toxicity is not fully understood, several recent works pointed out the involvement of oxidative stress in arsenic-induced DNA damage that, in living cells, correlates with changes in gene expressions. In cultured human fibroblasts exposed for 24 h to micromolar arsenic concentrations, we studied, using real-time RT-PCR, the expression profile of a limited number of genes: genes coding for a stress protein (HSP70), transcription factors (cJUN, cFOS, ETR103, ETR101 and TTP) and cell cycle or DNA repair proteins (P21, GADD153). We observed that the expression profile of genes followed individual different patterns that can be summed up in early-transient gene expression by contrast to delayed gene expression.

SKCa Channels Blockage Increases the Expression of Adenosine A2A Receptor in Jurkat Human T Cells.

Adenosine is a nucleoside displaying various biological effects via stimulation of four G-protein-coupled receptors, A1, A2A, A2B, and A3. Adenosine also modulates voltage-gated (Kv) and small conductance calcium-activated (SKCa) potassium channels. The effect of these potassium channels on the expression of adenosine receptors is poorly understood. We evaluated the action of BgK (a natural Kv channel blocker) and Lei-Dab7 (a synthetic SKCa channel blocker) on the expression of adenosine A2A receptors (A2AR) in Jurkat human T cells. We found that Lei-Dab7, but not BgK, increased the maximal binding value of the tritiated ligand ZM241385 to A2AR in a dose-dependent manner (+45% at 5 nM; +70% at 50 nM as compared to control). These results were further confirmed by Western blotting using a specific monoclonal antibody to human A2AR. The ligand affinity-related dissociation constant and A2AR mRNA amount were not significantly modified by either drug. We suggest that modulation of SKCa channels can influence membrane expression of A2AR and thus has a therapeutic potential.

Involvement of endogenous opioid system in scorpion toxin-induced antinociception in mice.

The present study analyzes the involvement of the endogenous opioid system in the antinociceptive effects produced in mammals after alpha- or beta- scorpion toxin injections. The analgesic effects on mice of the alpha-anatoxin Amm VIII, a weak modulator of Na(v)1.2 channel, and the depressant insect-selective beta-toxin LqqIT2 were evaluated by intraperitoneal route. The two toxins increased hot plate and tail flick latencies in a dose-dependent manner. We also compared the effects of the toxins with those obtained after acetic acid administration or cold-water tail immersion, which both induce pain relief through the activation of diffuse noxious inhibitory controls (DNIC) and the release of endogenous opioids. The increased latencies obtained with the toxins, acetic acid, or cold-water tail immersion were partly reversed by the co-administration of the opioid receptor antagonist naloxone. Finally, AmmVIII, LqqIT2, or acetic acid, induced increased c-fos mRNA expression in spinal cord. This increase disappeared when the toxins were co-injected with acetic acid. In conclusion, we show for the first time that an alpha-anatoxin exhibits a potent analgesic activity and confirm that depressant beta-toxins are able to reduce nociception. We hypothesize that pain relief induced by these scorpion toxins may implicate the activation of an endogenous opioid system and may be partly the result of a counter irritation phenomenon, which could be due to the activation of DNIC.

hOGG1(326), XRCC1(399) and XRCC3(241) polymorphisms influence micronucleus frequencies in human lymphocytes in vivo.

A pooled analysis of five biomonitoring studies was performed to assess the influence of hOGG1(326), XRCC1(399) and XRCC3(241) gene polymorphisms on micronuclei (MN) frequency in human peripheral blood lymphocytes, as measured by the ex vivo/in vitro cytokinesis-block micronucleus (CBMN) assay. Each study addressed a type of occupational exposure potentially able to induce DNA strand breakage (styrene, ionising radiation, cobalt/hard metal, welding fumes and inorganic arsenite compounds), and therefore MN, as a result of base excision repair and double-strand break repair deficiencies. The effect of genotype, age, exposure to genotoxic agents and smoking habit on MN induction was determined using Poisson regression analysis in 171 occupationally exposed male workers and in 132 non-exposed male referents. The analysis of genotype-genotype, genotype-smoking and genotype-exposure interactions by linear combinations of parameters showed significantly higher MN frequencies in the following subsets: (i) occupationally exposed workers carrying either the Thr/Thr or the Thr/Met XRCC3(241) genotypes compared to their referent counterparts (P < 0.001) and (ii) carriers of the Met/Met XRCC3(241) genotype compared to Thr/Thr XRCC3(241) carriers, as far as they are non-exposed and bear the variant (Ser/Cys or Cys/Cys) hOGG1(326) genotype (P < 0.01). Significantly lower MN frequencies were observed in carriers of the variant hOGG1(326) genotype compared to Ser/Ser hOGG1(326) carriers in the subgroup of non-smokers with Thr/Thr XRCC3(241) genotype (P < 0.01). Stratified analysis by occupational exposure showed a significant MN increase with smoking in occupationally exposed carriers of the Arg/Gln XRCC1(399)genotype (P < 0.001). In contrast, a significant MN decrease with smoking was observed in referents carrying the Ser/Ser hOGG1(326) genotype (P < 0.01). These findings provide evidence that the combination of different DNA repair genes and their interaction with environmental genotoxic agents may modulate MN induction. Understanding the complexity of the relationships between exposure, DNA repair and MN frequencies require larger scale studies and complementary biomarkers.

Oculocutaneous albinism type 2 (OCA2) with homozygous 2.7-kb deletion of the P gene and sickle cell disease in a Cameroonian family. Identification of a common TAG haplotype in the mutated P gene.

In this study, we report on a Cameroonian family from the Ewondo ethnic group, presenting with three oculocutaneous albinism type 2 (OCA2) patients homozygous for the 2.7-kb deletion of the P gene. In one of these patients OCA2 was associated with sickle cell anaemia and in two with the sickle cell trait. We took this opportunity to determine single nucleotide polymorphism (SNP) haplotypes within the P gene in this family in comparison with a group of 53 OCA2 patients homozygous for the same mutation and with a matched unrelated full-coloured control group of 49 subjects, originating from seven different ethnic groups of Southern Cameroon including Ewondo. A combination of five exonic and intronic SNPs in the OCA2 gene was genotyped by sequencing PCR products. We found 3 different haplotypes (TAGCT, TAGTT and TAGCC with frequencies of 0.66, 0.28 and 0.06, respectively) associated with the mutation in the 53 OCA2 patients, while 11 different haplotypes were observed in the control group. These observations suggest that the mutation appeared on the relatively frequent haplotype TAGCT, and that the two other haplotypes are derived from two independent recombination events. These haplotypic data, associated with a value of 1/15,000 for the prevalence of the 2.7-kb mutation, a present effective population size of 10,000,000 for Cameroon and a recombination rate of 0.0031, allowed us to estimate that this mutation originated 4,100-5,645 years ago.

Influence of haemodialysis and left ventricular failure on peripheral A(2A) adenosine receptor expression.

BACKGROUND: Haemodialysis (HD) sometimes accelerates left ventricular failure (LVF). As adenosine (ADO) is strongly implicated in cardiovascular functions, particularly via A(2A) receptor activation and as changes of peripheral A(2A) receptors mirror changes occurring in the cardiovascular system, we examined the influence of HD and LVF on both ADO plasma concentration and the expression of A(2A) receptors (i.e. Bmax, K(D) and mRNA amount) of peripheral blood mononuclear cells. METHODS: This cross-sectional study included 61 chronic renal failure (CRF) patients: 41 without LVF (24 haemodialysed and 17 undialysed) and 20 with LVF (9 haemodialysed and 11 undialysed). Ten LVF patients without CRF and 10 healthy subjects were also examined. RESULTS: (i) Bmax values of CRF patients without LVF were significantly decreased in undialysed patients compared with haemodialysed patients, and compared with controls (69 +/- 25 vs 98 +/- 33 vs 180 +/- 60 fmol/mg of protein, P < 0.05). Bmax values of CRF patients with LVF were lower in undialysed patients than in haemodialysed patients (60 +/- 27 vs 101 +/- 27 fmol/mg of protein, P < 0.05). Bmax values of LVF patients without CRF were lower than in controls (51 +/- 19 vs 180 +/- 60 fmol/mg of protein). (ii) A(2A) mRNA expression was increased in haemodialysed patients compared with controls (20.2 +/- 0.75 vs 17.6 +/- 1.3, P < 0.05). (iii) ADO plasma levels were high in haemodialysed patients and further increased during the HD sessions. CONCLUSION: The number of A(2A) receptors was decreased by CRF with or without LVF. However, this decrease was less important in haemodialysed patients. The changes in peripheral A(2A) receptor expression suggest a significant inflammatory response to HD and heart or kidney failure. Whether these changes do reflect alterations in cardiomyocytes needs further investigation.

In vitro interactions between DMSA-coated maghemite nanoparticles and human fibroblasts: A physicochemical and cyto-genotoxical study.

Although the current production of oxide nanoparticles may be modest, the wide range of proposed applications and forecasted growth in production has raised questions about the potential impact of these nanoparticles on the environment and human health. Iron oxide nanoparticles have been proposed for an increasing number of biomedical applications although in vitro toxicity depending on the particles coating has been evidenced. The aim of this study was to examine the potential in vitro cyto- and genotoxicity on human dermal fibroblasts of DMSA-coated maghemite nanoparticles (NmDMSA) as a function of well-defined physicochemical states. Well-stabilized NmDMSA produced weak cytotoxic and no genotoxic effects. This is attributed in part to the DMSA coating, which serves as a barrier for a direct contact between nano-oxide and fibroblasts, inhibiting a potential toxic effect.