Association analyses of DNA polymorphisms in bovine SREBP-1, LXRα,FADS1 genes with fatty acid composition in Canadian commercial crossbred beef steers

Two previously reported DNA polymorphisms of sterol regulatory element binding transcription factor 1 (SREBP1) and liver X receptor alpha (LXRα) and two DNA polymorphisms of fatty acid desaturase 1 (FADS1) were evaluated for associations with fatty acids in brisket adipose tissue of Canadian cross-bred beef steers. The polymorphism of 84 bp insert/deletion in intron 5 of SREBP1 was significantly associated with the concentration of 9c C17:1 (P=0.013). The G>A single nucleotide polymorphism (SNP) in the exon 4 of LXRα gene was associated with the concentration of 9c, 11t C18:2 (P=0.04), sum of conjugated linoleic acids (CLA) (P=0.025) and 11c C20:1(P=0.042). Two DNA polymorphisms in the promoter region of FADS1, deletion/insertion of ->GTG in rs133053720 and SNP A>G in rs42187276, were significantly associated with concentrations of C17:0 iso, C17:0 ai, total branched chain fatty acids (BFA), 12t C18:1, 13t/14t C18:1, 15t C18:1, and 13c C18:1 (P<0.05). Further studies are needed to validate the associations and to delineate the roles of the gene polymorphisms in determining the fatty acid composition in beef tissues.

The frequency of muscle protein polymorphism in Menidia menidia (Atherinidae) along the Atlantic coast

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Candida albicans Increases Tumor Cell Adhesion to Endothelial Cells In Vitro: Intraspecific Differences and Importance of the Mannose Receptor

The dimorphic fungus Candida albicans is able to trigger a cytokine-mediated pro-inflammatory response that increases tumor cell adhesion to hepatic endothelium and metastasis. To check the intraspecific differences in this effect, we used an in vitro murine model of hepatic response against C. albicans, which made clear that tumor cells adhered more to endothelium incubated with blastoconidia, both live and killed, than germ tubes. This finding was related to the higher carbohydrate/protein ratio found in blastoconidia. In fact, destruction of mannose ligand residues on the cell surface by metaperiodate treatment significantly reduced tumor cell adhesion induced. Moreover, we also noticed that the effect of clinical strains was greater than that of the reference one. This finding could not be explained by the carbohydrate/protein data, but to explain these differences between strains, we analyzed the expression level of ten genes (ADH1, APE3, IDH2, ENO1, FBA1, ILV5, PDI1, PGK1, QCR2 and TUF1) that code for the proteins identified previously in a mannoprotein-enriched pro-metastatic fraction of C. albicans. The results corroborated that their expression was higher in clinical strains than the reference one. To confirm the importance of the mannoprotein fraction, we also demonstrate that blocking the mannose receptor decreases the effect of C. albicans and its mannoproteins, inhibiting IL-18 synthesis and tumor cell adhesion increase by around 60%. These findings could be the first step towards a new treatment for solid organ cancers based on the role of the mannose receptor in C. albicans-induced tumor progression and metastasis.

Fine mapping and functional analysis of the multiple sclerosis risk gene CD6

CD6 has recently been identified and validated as risk gene for multiple sclerosis (MS), based on the association of a single nucleotide polymorphism (SNP), rs17824933, located in intron 1. CD6 is a cell surface scavenger receptor involved in T-cell activation and proliferation, as well as in thymocyte differentiation. In this study, we performed a haptag SNP screen of the CD6 gene locus using a total of thirteen tagging SNPs, of which three were non-synonymous SNPs, and replicated the recently reported GWAS SNP rs650258 in a Spanish-Basque collection of 814 controls and 823 cases. Validation of the six most strongly associated SNPs was performed in an independent collection of 2265 MS patients and 2600 healthy controls. We identified association of haplotypes composed of two non-synonymous SNPs [rs11230563 (R225W) and rs2074225 (A257V)] in the 2nd SRCR domain with susceptibility to MS (Pmax(T) permutation=161024). The effect of these haplotypes on CD6 surface expression and cytokine secretion was also tested. The analysis showed significantly different CD6 expression patterns in the distinct cell subsets, i.e. – CD4+ naı¨ve cells, P = 0.0001; CD8+ naı¨ve cells, P,0.0001; CD4+ and CD8+ central memory cells, P = 0.01 and 0.05, respectively; and natural killer T (NKT) cells, P = 0.02; with the protective haplotype (RA) showing higher expression of CD6. However, no significant changes were observed in natural killer (NK) cells, effector memory and terminally differentiated effector memory T cells. Our findings reveal that this new MS-associated CD6 risk haplotype significantly modifies expression of CD6 on CD4+ and CD8+ T cells.

Papel de las GTPasas de la familia rho en la señalización mediada por el receptor P2X7

El objetivo de este proyecto es estudiar la señalización del receptor P2X7 en respuesta a ATP en macrófagos J774A.1 y en células CHO K1. Para ello, se subclonó el gen que codifica para la proteína P2X7 en el vector PMT2 HA AA. Este plásmido fue transfectado a células CHO K1, J774.A1 y HEK 293T para distintas pruebas como la movilización de calcio intracelular para comprobar si ambos tipos celulares muestran la misma señal, y además se miró la expresión de este receptor y si su activación mediada por ATP se traduce en la activación de proteínas de la familia Rho. Se ha visto que las J774A.1 expresan funcionalmente el receptor P2X7, mientras que las CHO K1 muestran una respuesta funcional diferente que no se corresponde con la clásica señalización del P2X7 asociado a la apertura del canal y posterior poro. Además, al expresar el receptor en celúlas HEK 293T, se ha visto de una manera indirecta, midiendo la fosforilación de PAK, que la ruta de rac se regula de forma positiva cuando se activa el receptor P2X7 en macrófagos.

Evaluation of Alpha 1-Antitrypsin and the Levels of mRNA Expression of Matrix Metalloproteinase 7, Urokinase Type Plasminogen Activator Receptor and COX-2 for the Diagnosis of Colorectal Cancer

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The Influence of the Val158Met Catechol-O-Methyltransferase Polymorphism on the Personality Traits of Bipolar Patients

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Association between CASP8-652 6N Del Polymorphism (rs3834129) and Colorectal Cancer Risk: Results from a Multi-Centric Study

The common 2652 6N del variant in the CASP8 promoter (rs3834129) has been described as a putative low-penetrance risk factor for different cancer types. In particular, some studies suggested that the deleted allele (del) was inversely associated with CRC risk while other analyses failed to confirm this. Hence, to better understand the role of this variant in the risk of developing CRC, we performed a multi-centric case-control study. In the study, the variant 2652 6N del was genotyped in a total of 6,733 CRC cases and 7,576 controls recruited by six different centers located in Spain, Italy, USA, England, Czech Republic and the Netherlands collaborating to the international consortium COGENT (COlorectal cancer GENeTics). Our analysis indicated that rs3834129 was not associated with CRC risk in the full data set. However, the del allele was under-represented in one set of cases with a family history of CRC (per allele model OR = 0.79, 95% CI = 0.69-0.90) suggesting this allele might be a protective factor versus familial CRC. Since this multi-centric case-control study was performed on a very large sample size, it provided robust clarification of the effect of rs3834129 on the risk of developing CRC in Caucasians.

Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes

Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca2+ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca2+ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP(3)Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP(3)Rs. Blockade of Ca2+-induced Ca2+ release by TMB-8 following alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca2+ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca2+ overload whereas IP3R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by alpha subunit of the eukaryotic initiation factor 2 alpha phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca2+ release through RyRs contributes to cytosolic Ca2+ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes. Cell Death and Disease (2010) 1, e54; doi:10.1038/cddis.2010.31; published online 15 July 2010

The COMT Val158 Met polymorphism as an associated risk factor for Alzheimer disease and mild cognitive impairment in APOE 4 carriers

Background: The aim of this study is to examine the influence of the catechol-O-methyltranferase (COMT) gene (polymorphism Val158 Met) as a risk factor for Alzheimer's disease (AD) and mild cognitive impairment of amnesic type (MCI), and its synergistic effect with the apolipoprotein E gene (APOE). A total of 223 MCI patients, 345 AD and 253 healthy controls were analyzed. Clinical criteria and neuropsychological tests were used to establish diagnostic groups. The DNA Bank of the University of the Basque Country (UPV-EHU) (Spain) determined COMT Val158 Met and APOE genotypes using real time polymerase chain reaction (rtPCR) and polymerase chain reaction (PCR), and restriction fragment length polymorphism (RFLPs), respectively. Multinomial logistic regression models were used to determine the risk of AD and MCI. Results: Neither COMT alleles nor genotypes were independent risk factors for AD or MCI. The high activity genotypes (GG and AG) showed a synergistic effect with APOE epsilon 4 allele, increasing the risk of AD (OR = 5.96, 95% CI 2.74-12.94, p < 0.001 and OR = 6.71, 95% CI 3.36-13.41, p < 0.001 respectivily). In AD patients this effect was greater in women. In MCI patients such as synergistic effect was only found between AG and APOE epsilon 4 allele (OR = 3.21 95% CI 1.56-6.63, p = 0.02) and was greater in men (OR = 5.88 95% CI 1.69-20.42, p < 0.01). Conclusion: COMT (Val158 Met) polymorphism is not an independent risk factor for AD or MCI, but shows a synergistic effect with APOE epsilon 4 allele that proves greater in women with AD.

Soluble Angiopoietin Receptor Tie-2 In Patients With Acute Myocardial Infarction And Its Detection By Optical Protein-Chip

The Tie-2 receptor has been shown to play a role in angiogenesis in atherosclerosis. The conventional method assaying the level of soluble Tie-2 (sTie-2) was ELISA. However, this method has some disadvantages. The aims of this research are to establish a more simple detection method, the optical protein-chip based on imaging ellipsomtry (OPC-IE) applying to Tie-2 assay. The sTie-2 biosensor surface on silicon wafer was prepared first, and then serum levels of sTie-2 in 38 patients with AMI were measured on admission (day 1), day 2, day 3 and day 7 after onset of chest pain and 41 healthy controls by ELISA and OPC-IE in parallel. Median level of sTie-2 increased significantly in the AMI patients when compared with the controls. Statistics showed there was a significant correlation in sTie-2 results between the two methods (r=0.923, P0.01). The result of this study showed that the level of sTie-2 increased in AMI, and OPC-IE assay was a fast, reliable, and convenient technique to measure sTie-2 in serum.

Parametric Analysis for Monitoring 2D Kinetics of Receptor-Ligand binding

Thermal fluctuation approach is widely used to monitor association kinetics of surface-bound receptor-ligand interactions. Various protocols such as sliding standard deviation (SD) analysis (SSA) and Page's test analysis (PTA) have been used to estimate two-dimensional (2D) kinetic rates from the time course of displacement of molecular carrier. In the current work, we compared the estimations from both SSA and modified PTA using measured data from an optical trap assay and simulated data from a random number generator. Our results indicated that both SSA and PTA were reliable in estimating 2D kinetic rates. Parametric analysis also demonstrated that such the estimations were sensitive to parameters such as sampling rate, sliding window size, and threshold. These results furthered the understandings in quantifying the biophysics of receptor-ligand interactions.

Structural and mechanistic motifs in membrane proteins: the three-dimensional modelling of rhodopsin, band 3, and the nicotinic acetylcholine receptor

Because so little is known about the structure of membrane proteins, an attempt has been made in this work to develop techniques by which to model them in three dimensions. The procedures devised rely heavily upon the availability of several sequences of a given protein. The modelling procedure is composed of two parts. The first identifies transmembrane regions within the protein sequence on the basis of hydrophobicity, β-turn potential, and the presence of certain amino acid types, specifically, proline and basic residues. The second part of the procedure arranges these transmembrane helices within the bilayer based upon the evolutionary conservation of their residues. Conserved residues are oriented toward other helices and variable residues are positioned to face the surrounding lipids. Available structural information concerning the protein's helical arrangement, including the lengths of interhelical loops, is also taken into account. Rhodopsin, band 3, and the nicotinic acetylcholine receptor have all been modelled using this methodology, and mechanisms of action could be proposed based upon the resulting structures.

Specific residues in the rhodopsin and iodopsin sequences were identified, which may regulate the proteins' wavelength selectivities. A hinge-like motion of helices M3, M4, and M5 with respect to the rest of the protein was proposed to result in the activation of transducin, the G-protein associated with rhodopsin. A similar mechanism is also proposed for signal transduction by the muscarinic acetylcholine and β-adrenergic receptors.

The nicotinic acetylcholine receptor was modelled with four trans-membrane helices per subunit and with the five homologous M2 helices forming the cation channel. Putative channel-lining residues were identified and a mechanism of channel-opening based upon the concerted, tangential rotation of the M2 helices was proposed.

Band 3, the anion exchange protein found in the erythrocyte membrane, was modelled with 14 transmembrane helices. In general the pathway of anion transport can be viewed as a channel composed of six helices that contains a single hydrophobic restriction. This hydrophobic region will not allow the passage of charged species, unless they are part of an ion-pair. An arginine residue located near this restriction is proposed to be responsible for anion transport. When ion-paired with a transportable anion it rotates across the barrier and releases the anion on the other side of the membrane. A similar process returns it to its original position. This proposed mechanism, based on the three-dimensional model, can account for the passive, electroneutral, anion exchange observed for band 3. Dianions can be transported through a similar mechanism with the additional participation of a histidine residue. Both residues are located on M10.

Protein-protein recognition: The neonatal Fe receptor and immunoglobulin G

The neonatal Fe receptor (FeRn) binds the Fe portion of immunoglobulin G (IgG) at the acidic pH of endosomes or the gut and releases IgG at the alkaline pH of blood. FeRn is responsible for the maternofetal transfer of IgG and for rescuing endocytosed IgG from a default degradative pathway. We investigated how FeRn interacts with IgG by constructing a heterodimeric form of the Fe (hdFc) that contains one FeRn binding site. This molecule was used to characterize the interaction between one FeRn molecule and one Fe and to determine under what conditions FeRn forms a dimer. The hdFc binds one FeRn molecule at pH 6.0 with a K_d of 80 nM. In solution and with FeRn anchored to solid supports, the heterodimeric Fe does not induce a dimer of FeRn molecules. FcRnhdFc complex crystals were obtained and the complex structure was solved to 2.8 Å resolution. Analysis of this structure refined the understanding of the mechanism of the pH-dependent binding, shed light on the role played by carbohydrates in the Fe binding, and provided insights on how to design therapeutic IgG antibodies with longer serum half-lives. The FcRn-hdFc complex in the crystal did not contain the FeRn dimer. To characterize the tendency of FeRn to form a dimer in a membrane we analyzed the tendency of the hdFc to induce cross-phosphorylation of FeRn-tyrosine kinase chimeras. We also constructed FeRn-cyan and FeRn-yellow fluorescent proteins and have analyzed the tendency of these molecules to exhibit fluorescence resonance energy transfer. As of now, neither of these analyses have lead to conclusive results. In the process of acquiring the context to appreciate the structure of the FcRn-hdFc interface, we developed a study of 171 other nonobligate protein-protein interfaces that includes an original principal component analysis of the quantifiable aspects of these interfaces.