Evaluating HapMap SNP data transferability in a large-scale genotyping project involving 175 cancer-associated genes

One of the many potential uses of the HapMap project is its application to the investigation of complex disease aetiology among a wide range of populations. This study aims to assess the transferability of HapMap SNP data to the Spanish population in the context of cancer research. We have carried out a genotyping study in Spanish subjects involving 175 candidate cancer genes using an indirect gene-based approach and compared results with those for HapMap CEU subjects. Allele frequencies were very consistent between the two samples, with a high positive correlation (R) of 0.91 (P<<1×10⁻⁶). Linkage disequilibrium patterns and block structures across each gene were also very similar, with disequilibrium coefficient (r ²) highly correlated (R=0.95, P<<1×10⁻⁶). We found that of the 21 genes that contained at least one block larger than 60 kb, nine (ATM, ATR, BRCA1, ERCC6, FANCC, RAD17, RAD50, RAD54B and XRCC4) belonged to the GO category “DNA repair”. Haplotype frequencies per gene were also highly correlated (mean R=0.93), as was haplotype diversity (R=0.91, P<<1×10⁻⁶). “Yin yang” haplotypes were observed for 43% of the genes analysed and 18% of those were identical to the ancestral haplotype (identified in Chimpazee). Finally, the portability of tagSNPs identified in the HapMap CEU data using pairwise r ² thresholds of 0.8 and 0.5 was assessed by applying these to the Spanish and current HapMap data for 66 genes. In general, the HapMap tagSNPs performed very well. Our results show generally high concordance with HapMap data in allele frequencies and haplotype distributions and confirm the applicability of HapMap SNP data to the study of complex diseases among the Spanish population. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Pig liver phosphomevalonate kinase: kinetic mechanism

Phosphomevalonate kinase catalyzes the phosphorylation of phosphomevalonate to diphosphomevalonate by ATP, one of the initial steps in the biosynthesis of steroids and isoprenoids. In previous studies, the enzyme from pig liver was purified and characterized, and preliminary work showed that the enzyme follows hyperbolic kinetics and a sequential mechanism. The present work is a more detailed analysis of its kinetic mechanism, using initial velocity and isotope exchange at equilibrium measurements. The results are compatible with a Bi Bi sequential ordered mechanism with phosphomevalonate as the first substrate and ADP the last product. The Km values estimated are 43+/-7 microM for Mg-ATP and 12+/-3 microM for phosphomevalonate, with a Vmax of 51+/-2 micromol min-1 mg of protein-1.     

Antioxidant inhibition of oxygen radicals for measurement of total antioxidant capacity in biological samples

Few methods for assessing total antioxidant capacity (TAC) include both the percentage of inhibition and the length of inhibition in the measurement. Available methods require above ambient constant temperature incubation, reaction preheating, and/or separate assays for testing hydrophilic and hydrophobic samples. We describe a high-throughput method, antioxidant inhibition of oxygen radicals (AIOR), that overcomes these difficulties. AIOR uses peroxyl radicals to trigger a decrease in fluorescence of the indicator molecule, uroporphyrin I, which is delayed by the presence of antioxidants. The area under the curve is measured by a fluorescence spectrophotometer in a 96-well microplate format, and TAC results are expressed as millimole/liter Trolox equivalents. AIOR is performed at ambient temperature and is applicable to samples in either aqueous or common organic solvents. The reaction between uroporphyrin I and the peroxyl radicals generated from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) was found to be of first-order kinetics with a mean rate constant (k) of 0.0254. Applications to measure antioxidant capacity are demonstrated on individual chemicals and biological samples. The method has good linearity, within- and between-assay precision, and recovery.     

Synthesis, characterisation and natural abundance Os NMR spectroscopy of hydride bridged triosmium clusters with chiral diphosphine ligands

Treatment of [Os₃(μ-H)₂(CO)₁₀] with the chiral diphosphines BINAP, tolBINAP [(R)-2,2′-bis(di-4-tolylphosphino)-1,1′-binaphthyl], DIOP [(4R,5R)-(−)-O-isopropenylidene-2,3-dihydroxy-1,4-bis(diphenylphosphino)butane] affords [Os₃(μ-H)₂(CO)₈(μ-L)] (L = BINAP (1), tolBINAP (2), DIOP (4)) in high yield. The X-ray structures for 1, 2 and 4 are reported, and structural and spectroscopic comparisons are made between these clusters and [Os₃(μ-H)₂(CO)₈(μ-L)] (L = dppm (5), dppe (6), dppp (7)) which were synthesised similarly. Compounds 5 to 7 were previously synthesised by hydrogenation of 1,2-[Os₃(CO)₁₀(μ-L)] but the route from [Os₃(μ-H)₂(CO)₁₀] is preferable. The H-bridged Os?Os distances are similar in 1, 2 and 4 indicating that these species are formally unsaturated 46-electron clusters. The P?P distances vary from 4.24 to 4.30 Å in 1 and 2, respectively, to 4.53 Å in 4 and there are related changes in the angles associated with the ligand set around the H-bridged osmium atoms. Introduction of the diphosphine ligands completely suppresses the ability to add CO, to insert acetylene to form a μ-η¹,η²-vinyl compound, and to exchange hydride ligands with styrene-d₈, which are reactions characteristic of [Os₃(μ-H)₂(CO)₁₀]. Clusters 2 and 5-7 were also used to examine the potential of natural abundance ¹⁸⁷Os NMR spectroscopy through techniques based on inverse detection by HMQC, HSQC and HMBC spectroscopy.     

Functional evaluation of serine 252 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase mutant Ser252Ala, affecting the conserved Walker A serine residue, was characterized to elucidate the role of this serine residue. The substitution did not result in changes in the protein structure, as indicated by circular dichroism, intrinsic fluorescence spectroscopy, and gel-exclusion chromatography. Kinetic analysis of the mutated enzyme in both directions of the main reaction and in the two secondary reactions showed an approximately 50-fold increase in apparent K(m) for oxaloacetate with minor alterations in the other kinetic parameters. These results show that the hydroxyl group of serine 252 is required for proper oxaloacetate interaction.     

Inhaled IL-2 induces systemic immunomodulation in patients with renal cell carcinoma and lung metastasis

The peripheral blood lymphocytes of eight patients with metastatic renal cell carcinoma, and of eight healthy volunteers were analyzed by four-color flow cytometry to characterize the immunophenotypic alterations manifested, determine the prevalence of lymphocyte apoptosis, and detect evidence of the systemic effect of inhaled IL-2. The T, B and NK lymphocytes of untreated patients were found to have undergone profound changes characterized by an increase in susceptibility to both spontaneous and mitogen-induced ex vivo apoptosis, a modified distribution of the main lymphocyte populations in the peripheral blood, and alterations in activation status. An increase in the proportion of regulatory T cells was also seen in these patients. Treatment with inhaled IL-2, however, normalized the rate of apoptosis in all the lymphocyte subpopulations studied, as well as their distribution and activation status. These findings demonstrate that inhaled IL-2 has systemic immunomodulatory effects.     

Validation of trans-rectal ultrasonography for counting preovulatory follicles in weaned sows

The present study investigated the accuracy of trans-rectal ultrasonography (TRU) for assessing the exact number of preovulatory follicles (POFs, with a diameter from 6 to 10mm) present in the ovaries of weaned sows. The ovaries of 63 hormonally treated (1500 IU of eCG) weaned sows were checked with TRU (7.5-MHz multiple scan angle transducer) in two successive scanning sessions performed at 26-27 and 30-31h after the beginning of oestrus signs, and the maximum number of POFs were counted. Sows were subjected to laparoscopy (LAP) immediately after the last TRU scan to confirm the number of POFs. The differences (mean+/-S.D.) in the number of POFs counted with TRU and LAP on each ovary were analyzed as a whole and after sorting the ovaries into three classes according to the number of POFs visualized by LAP: (1) less than 7; (2) from 7 to 13; and (3) more than 13. A significant correlation (P<0.01) was found between TRU and LAP for both the whole data set (126 ovaries) and in each of the three ovarian classes. Despite the significant correlation, TRU underestimated the number of POFs by 1.40+/-1.67 compared with LAP (P<0.001). However, the underestimation varied among the ovarian classes. This difference was not significant (P>0.05) in class 1 and was significant (P<0.001) in classes 2 (1.11+/-1.30 less POFs than counted by TRU) and 3 (3.19+/-1.54 less POFs than counted by TRU). In conclusion, TRU is a valuable tool to count the number of POFs present in the ovaries of weaned sows, but a certain degree of underestimation should be expected when the number of POFs is large.