Improvement of the liquid-chromatographic analysis of protein tryptic digests by the use of long-capillary monolithic columns with UV and MS detection

Optimisation of peak capacity is an important strategy in gradient liquid chromatography (LC). This can be achieved by using either long columns or columns packed with small particles. Monolithic columns allow the use of long columns at relatively low back-pressure. The gain in peak capacity using long columns was evaluated by the separation of a tryptic bovine serum albumin digest with an LC–UV–mass spectrometry (MS) system and monolithic columns of different length (150 and 750 mm). Peak capacities were determined from UV chromatograms and MS/MS data were used for Mascot database searching. Analyses with a similar gradient slope for the two columns produced ratios of the peak capacities that were close to the expected value of the square root of the column length ratio. Peak capacities of the short column were 12.6 and 25.0 with 3 and 15 min gradients, respectively, and 29.7 and 41.0 for the long column with 15 and 75 min gradients, respectively. Protein identification scores were also higher for the long column, 641 and 750 for the 3- and 15-min gradients with the short column and 1,376 and 993 for the 15- and 75-min gradients with the long column. Thus, the use of long monolithic columns provides improved peptide separation and increased reliability of protein identification

Common Variants at 10 Genomic Loci Influence Hemoglobin A(1C) Levels via Glycemic and Nonglycemic Pathways

OBJECTIVE Glycated hemoglobin (HbA1c), used to monitor and diagnose diabetes, is influenced by average glycemia over a 2- to 3-month period. Genetic factors affecting expression, turnover, and abnormal glycation of hemoglobin could also be associated with increased levels of HbA1c. We aimed to identify such genetic factors and investigate the extent to which they influence diabetes classification based on HbA1c levels. RESEARCH DESIGN AND METHODS We studied associations with HbA1c in up to 46,368 nondiabetic adults of European descent from 23 genome-wide association studies (GWAS) and 8 cohorts with de novo genotyped single nucleotide polymorphisms (SNPs). We combined studies using inverse-variance meta-analysis and tested mediation by glycemia using conditional analyses. We estimated the global effect of HbA1c loci using a multilocus risk score, and used net reclassification to estimate genetic effects on diabetes screening. RESULTS Ten loci reached genome-wide significant association with HbA1c, including six new loci near FN3K (lead SNP/P value, rs1046896/P = 1.6 × 10−26), HFE (rs1800562/P = 2.6 × 10−20), TMPRSS6 (rs855791/P = 2.7 × 10−14), ANK1 (rs4737009/P = 6.1 × 10−12), SPTA1 (rs2779116/P = 2.8 × 10−9) and ATP11A/TUBGCP3 (rs7998202/P = 5.2 × 10−9), and four known HbA1c loci: HK1 (rs16926246/P = 3.1 × 10−54), MTNR1B (rs1387153/P = 4.0 × 10−11), GCK (rs1799884/P = 1.5 × 10−20) and G6PC2/ABCB11 (rs552976/P = 8.2 × 10−18). We show that associations with HbA1c are partly a function of hyperglycemia associated with 3 of the 10 loci (GCK, G6PC2 and MTNR1B). The seven nonglycemic loci accounted for a 0.19 (% HbA1c) difference between the extreme 10% tails of the risk score, and would reclassify ∼2% of a general white population screened for diabetes with HbA1c. CONCLUSIONS GWAS identified 10 genetic loci reproducibly associated with HbA1c. Six are novel and seven map to loci where rarer variants cause hereditary anemias and iron storage disorders. Common variants at these loci likely influence HbA1c levels via erythrocyte biology, and confer a small but detectable reclassification of diabetes diagnosis by HbA1c

Association between genetic variants in the Coenzyme Q10 metabolism and Coenzyme Q10 status in humans

<p>Abstract</p> <p>Background</p> <p>Coenzyme Q₁₀(CoQ₁₀) is essential for mitochondrial energy production and serves as an antioxidants in extra mitochondrial membranes. The genetics of primary CoQ₁₀deficiency has been described in several studies, whereas the influence of common genetic variants on CoQ₁₀status is largely unknown. Here we tested for non-synonymous single-nucleotidepolymorphisms (SNP) in genes involved in the biosynthesis (CoQ3^G272S, CoQ6^M406V, CoQ7^M103T), reduction (NQO1^P187S, NQO2^L47F) and metabolism (apoE3/4) of CoQ₁₀and their association with CoQ₁₀status. For this purpose, CoQ₁₀serum levels of 54 healthy male volunteers were determined before (T₀) and after a 14 days supplementation (T₁₄) with 150 mg/d of the reduced form of CoQ₁₀.</p> <p>Findings</p> <p>At T₀, the CoQ₁₀level of heterozygous NQO1^P187Scarriers were significantly lower than homozygous S/S carriers (0.93 ± 0.25 μM versus 1.34 ± 0.42 μM, p = 0.044). For this polymorphism a structure homology-based method (PolyPhen) revealed a possibly damaging effect on NQO1 protein activity. Furthermore, CoQ₁₀plasma levels were significantly increased in apoE4/E4 genotype after supplementation in comparison to apoE2/E3 genotype (5.93 ± 0.151 μM versus 4.38 ± 0.792 μM, p = 0.034). Likewise heterozygous CoQ3^G272Scarriers had higher CoQ₁₀plasma levels at T₁₄compared to G/G carriers but this difference did not reach significance (5.30 ± 0.96 μM versus 4.42 ± 1.67 μM, p = 0.082).</p> <p>Conclusions</p> <p>In conclusion, our pilot study provides evidence that NQO1^P187Sand apoE polymorphisms influence CoQ₁₀status in humans.</p

Coexpression of vesicular glutamate transporters 1 and 2, glutamic acid decarboxylase and calretinin in rat entorhinal cortex

We studied the distribution and coexpression of vesicular glutamate transporters (VGluT1, VGluT2), glutamic acid decarboxylase (GAD) and calretinin (CR, calcium-binding protein) in rat entorhinal cortex, using immunofluorescence staining and multichannel confocal laser scanning microscopy. Images were computer processed and subjected to automated 3D object recognition, colocalization analysis and 3D reconstruction. Since the VGluTs (in contrast to CR and GAD) occurred in fibers and axon terminals only, we focused our attention on these neuronal processes. An intense, punctate VGluT1-staining occurred everywhere in the entorhinal cortex. Our computer program resolved these punctae as small 3D objects. Also VGluT2 showed a punctate immunostaining pattern, yet with half the number of 3D objects per tissue volume compared with VGluT1, and with statistically significantly larger 3D objects. Both VGluTs were distributed homogeneously across cortical layers, with in MEA VGluT1 slightly more densely distributed than in LEA. The distribution pattern and the size distribution of GAD 3D objects resembled that of VGluT2. CR-immunopositive fibers were abundant in all cortical layers. In double-stained sections we noted ample colocalization of CR and VGluT2, whereas coexpression of CR and VGluT1 was nearly absent. Also in triple-staining experiments (VGluT2, GAD and CR combined) we noted coexpression of VGluT2 and CR and, in addition, frequent coexpression of GAD and CR. Modest colocalization occurred of VGluT2 and GAD, and incidental colocalization of all three markers. We conclude that the CR-containing axon terminals in the entorhinal cortex belong to at least two subpopulations of CR-neurons: a glutamatergic excitatory and a GABAergic inhibitory

Tauopathic Changes in the Striatum of A53T α-Synuclein Mutant Mouse Model of Parkinson's Disease

Tauopathic pathways lead to degenerative changes in Alzheimer's disease and there is evidence that they are also involved in the neurodegenerative pathology of Parkinson's disease [PD]. We have examined tauopathic changes in striatum of the α-synuclein (α-Syn) A53T mutant mouse. Elevated levels of α-Syn were observed in striatum of the adult A53T α-Syn mice. This was accompanied by increases in hyperphosphorylated Tau [p-Tau], phosphorylated at Ser202, Ser262 and Ser396/404, which are the same toxic sites also seen in Alzheimer's disease. There was an increase in active p-GSK-3β, hyperphosphorylated at Tyr216, a major and primary kinase known to phosphorylate Tau at multiple sites. The sites of hyperphosphorylation of Tau in the A53T mutant mice were similar to those seen in post-mortem striata from PD patients, attesting to their pathophysiological relevance. Increases in p-Tau were not due to alterations on protein phosphatases in either A53T mice or in human PD, suggesting lack of involvement of these proteins in tauopathy. Extraction of striata with Triton X-100 showed large increases in oligomeric forms of α-Syn suggesting that α-Syn had formed aggregates the mutant mice. In addition, increased levels of p-GSK-3β and pSer396/404 were also found associated with aggregated α-Syn. Differential solubilization to measure protein binding to cytoskeletal proteins demonstrated that p-Tau in the A53T mutant mouse were unbound to cytoskeletal proteins, consistent with dissociation of p-Tau from the microtubules upon hyperphosphorylation. Interestingly, α-Syn remained tightly bound to the cytoskeleton, while p-GSK-3β was seen in the cytoskeleton-free fractions. Immunohistochemical studies showed that α-Syn, pSer396/404 Tau and p-GSK-3β co-localized with one another and was aggregated and accumulated into large inclusion bodies, leading to cell death of Substantia nigral neurons. Together, these data demonstrate an elevated state of tauopathy in striata of the A53T α-Syn mutant mice, suggesting that tauopathy is a common feature of synucleinopathies

Municipal distribution of ovarian cancer mortality in Spain

<p>Abstract</p> <p>Background</p> <p>Spain was the country that registered the greatest increases in ovarian cancer mortality in Europe. This study describes the municipal distribution of ovarian cancer mortality in Spain using spatial models for small-area analysis.</p> <p>Methods</p> <p>Smoothed relative risks of ovarian cancer mortality were obtained, using the Besag, York and Molliè autoregressive spatial model. Standardised mortality ratios, smoothed relative risks, and distribution of the posterior probability of relative risks being greater than 1 were depicted on municipal maps.</p> <p>Results</p> <p>During the study period (1989–1998), 13,869 ovarian cancer deaths were registered in 2,718 Spanish towns, accounting for 4% of all cancer-related deaths among women. The highest relative risks were mainly concentrated in three areas, i.e., the interior of Barcelona and Gerona (north-east Spain), the north of Lugo and Asturias (north-west Spain) and along the Seville-Huelva boundary (in the south-west). Eivissa (Balearic Islands) and El Hierro (Canary Islands) also registered increased risks.</p> <p>Conclusion</p> <p>Well established ovarian cancer risk factors might not contribute significantly to the municipal distribution of ovarian cancer mortality. Environmental and occupational exposures possibly linked to this pattern and prevalent in specific regions, are discussed in this paper. Small-area geographical studies are effective instruments for detecting risk areas that may otherwise remain concealed on a more reduced scale.</p

Cell Survival from Chemotherapy Depends on NF-κB Transcriptional Up-Regulation of Coenzyme Q Biosynthesis

9 pages and 6 figures.[Background] Coenzyme Q (CoQ) is a lipophilic antioxidant that is synthesized by a mitochondrial complex integrated by at least ten nuclear encoded COQ gene products. CoQ increases cell survival under different stress conditions, including mitochondrial DNA (mtDNA) depletion and treatment with cancer drugs such as camptothecin (CPT). We have previously demonstrated that CPT induces CoQ biosynthesis in mammal cells.[Methodology/Principal Findings] CPT activates NF-κB that binds specifically to two κB binding sites present in the 5′-flanking region of the COQ7 gene. This binding is functional and induces both the COQ7 expression and CoQ biosynthesis. The inhibition of NF-κB activation increases cell death and decreases both, CoQ levels and COQ7 expression induced by CPT. In addition, using a cell line expressing very low of NF-κB, we demonstrate that CPT was incapable of enhancing enhance both CoQ biosynthesis and COQ7 expression in these cells.[Conclusions/Significance] We demonstrate here, for the first time, that a transcriptional mechanism mediated by NF-κB regulates CoQ biosynthesis. This finding contributes new data for the understanding of the regulation of the CoQ biosynthesis pathway.This work was supported by spanish Ministerio de Educacion y Ciencia Grant BFU2005-03017.Peer reviewe

Olives and olive oil are sources of electrophilic fatty acid nitroalkenes

Extra virgin olive oil (EVOO) and olives, key sources of unsaturated fatty acids in the Mediterranean diet, provide health benefits to humans. Nitric oxide (•NO) and nitrite (NO2-)-dependent reactions of unsaturated fatty acids yield electrophilic nitroalkene derivatives (NO 2-FA) that manifest salutary pleiotropic cell signaling responses in mammals. Herein, the endogenous presence of NO2-FA in both EVOO and fresh olives was demonstrated by mass spectrometry. The electrophilic nature of these species was affirmed by the detection of significant levels of protein cysteine adducts of nitro-oleic acid (NO2-OA-cysteine) in fresh olives, especially in the peel. Further nitration of EVOO by NO2- under acidic gastric digestive conditions revealed that human consumption of olive lipids will produce additional nitro-conjugated linoleic acid (NO2-cLA) and nitro-oleic acid (NO2-OA). The presence of free and protein-adducted NO2-FA in both mammalian and plant lipids further affirm a role for these species as signaling mediators. Since NO2-FA instigate adaptive anti-inflammatory gene expression and metabolic responses, these redox-derived metabolites may contribute to the cardiovascular benefits associated with the Mediterranean diet. © 2014 Fazzari et al