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Binding Analyses (binding + analysis)
Selected AbstractsHaplotype Analysis Confirms the Association Between the HCRTR2 Gene and Cluster HeadacheHEADACHE, Issue 7 2008Innocenzo Rainero MD Background., Several studies suggested that genetic factors play a role in cluster headache (CH) susceptibility. We found a significant association between the 1246 G>A polymorphism of the hypocretin receptor-2 (HCRTR2) gene and the disease. This association was confirmed in a large study from Germany but was not replicated in a dataset of CH patients from Northern Europe. Objective., The purpose of this study was to further evaluate the association between CH and the HCRTR2 gene using new polymorphisms, estimating the frequency of different gene haplotypes, searching for gene mutations, and evaluating the effects of the examined polymorphisms on hypocretin binding sites. Methods., We genotyped 109 CH patients and 211 healthy controls for 5 new polymorphisms of the HCRTR2 gene and we inferred different gene haplotypes. Complete HCRTR2 sequencing was undertaken for 11 independent CH patients, 5 of whom had a positive family history. The effects of the 1246 G>A polymorphism on the hypocretin binding sites were evaluated using different computer-assisted analyses. Results., Three new polymorphisms of the HCRTR2 gene resulted significantly associated with CH. The GTAAGG haplotype resulted more frequent in cases than in controls (OR: 3.68; 95% CI: 1.85-7.67). No point mutation of the HCRTR2 gene was found. Binding analyses showed that the 1246 G>A polymorphism (substitution of valine at position 308 by isoleucine) has no effect on the hypocretin binding sites but could influence the dimerization process of the receptor. Conclusion., Our data confirm previous studies suggesting that the HCRTR2 gene or a linked locus significantly modulates the risk for CH. In addition, we suggest that the V308I substitution of the HCRTR2 may interfere with the dimerization process of the receptor, thereby influencing its functional activity. [source] Cadmium accumulation and binding characteristics in intact Sertoli/germ cell units, and associated effects on stage-specific functions in vitro: insights from a shark testis modelJOURNAL OF APPLIED TOXICOLOGY, Issue 2 2008Leon M. McClusky Abstract The increased human use of cadmium (Cd) and its increased occurrence in the environment is of concern. The testis is sensitive to Cd because of the steroid-mediated regulation of spermatogenesis, high levels of DNA synthesis and gene transcription, all of which varies in a stage-related manner. Validated techniques (acridine orange vital staining to detect apoptosis and dextran-rhodamine exclusion to assess blood,testis barrier function) were recently developed and the shark testis was proposed as an alternative model for assessing stage-specific functions in living testicular tissue and to study toxicant actions on spermatogenesis. The present paper shows that 109Cd accumulation and binding in vitro was stage-dependent (premeiotic, PrM > meiotic, M > postmeiotic, PoM), rapid and persisted in spermatocysts (intact germ cell/Sertoli cell units) 49 h after washout. In competitive binding analyses of all three spermatocyst stages, Hg, but not Zn, could replace bound 109Cd, suggesting that Cd binding was specific. These findings were associated with a biphasic apoptotic response in the PrM spermatocysts, which was maximal at 10 µm CdCl2 and 1 µm CdCl2 after 2 and 4 days in culture, respectively. Although Cd uptake in PoM cysts was more than 2-fold less than uptake in PrM cysts, the percentage dextran-rhodamine permeant PoM cysts was ,8-fold greater than in controls in the presence of both 10 µm CdCl2 and 30 µm CdCl2 after 4 days culture, indicating that blood,testis barrier function in PoM spermatocysts was compromised. These findings demonstrate that this model has utility for use in screening assays of environmental toxicants. Copyright © 2007 John Wiley & Sons, Ltd. [source] Interactions between the L1 cell adhesion molecule and ezrin support traction-force generation and can be regulated by tyrosine phosphorylationJOURNAL OF NEUROSCIENCE RESEARCH, Issue 12 2008Takeshi Sakurai Abstract An Ig superfamily cell-adhesion molecule, L1, forms an adhesion complex at the cell membrane containing both signaling molecules and cytoskeletal proteins. This complex mediates the transduction of extracellular signals and generates actin-mediated traction forces, both of which support axon outgrowth. The L1 cytoplasmic region binds ezrin, an adapter protein that interacts with the actin cytoskeleton. In this study, we analyzed L1,ezrin interactions in detail, assessed their role in generating traction forces by L1, and identified potential regulatory mechanisms controlling ezrin,L1 interactions. The FERM domain of ezrin binds to the juxtamembrane region of L1, demonstrated by yeast two-hybrid interaction traps and protein binding analyses in vitro. A lysine-to-leucine substitution in this domain of L1 (K1147L) shows reduced binding to the ezrin FERM domain. Additionally, in ND7 cells, the K1147L mutation inhibits retrograde movement of L1 on the cell surface that has been linked to the generation of the traction forces necessary for axon growth. A membrane-permeable peptide consisting of the juxtamembrane region of L1 that can disrupt endogenous L1,ezrin interactions inhibits neurite extension of cerebellar cells on L1 substrates. Moreover, the L1,ezrin interactions can be modulated by tyrosine phosphorylation of the L1 cytoplasmic region, namely, Y1151, possibly through Src-family kinases. Replacement of this tyrosine together with Y1176 with either aspartate or phenylalanine changes ezrin binding and alters colocalization with ezrin in ND7 cells. Collectively, these data suggest that L1,ezrin interactions mediated by the L1 juxtamembrane region are involved in traction-force generation and can be regulated by the phosphorylation of L1. © 2008 Wiley-Liss, Inc. [source] The soybean Dof-type transcription factor genes, GmDof4 and GmDof11, enhance lipid content in the seeds of transgenic Arabidopsis plantsTHE PLANT JOURNAL, Issue 4 2007Hui-Wen Wang Summary Soybean is one of the most important leguminous seed crops among the oil crops. Although the pathways for lipid biosynthesis have been identified, the factors that regulate the biosynthetic pathways at the transcriptional level are largely unknown. Here, we report our findings on the involvement of soybean Dof-type transcription factor genes in the regulation of the lipid content in soybean seeds. We identified 28 Dof-type transcription factor genes in soybean plants, and these genes displayed diverse patterns of expression in various organs. Seven flower/pod-specific genes and one constitutively expressed gene were further investigated. The proteins encoded by these seven genes were localized in the nucleus, and exhibited different abilities for transcriptional activation and DNA binding. Two genes, GmDof4 and GmDof11, were found to increase the content of total fatty acids and lipids in GmDof4 and GmDof11 transgenic Arabidopsis seeds. We also found that the 1000-seed weight was increased in the GmDof4 and GmDof11 transgenic plants. Using microarray and DNA binding analysis, we found that the two Dof-like proteins, GmDof4 and GmDof11, activated the acetyl CoA carboxylase gene and long-chain-acyl CoA synthetase gene, respectively, by direct binding to the cis -DNA elements in their promoter regions. In addition, both proteins downregulated the storage protein gene, CRA1, through direct binding. These results suggest that the two GmDof genes may augment the lipid content of soybean seeds by upregulating genes that are associated with the biosynthesis of fatty acids. [source] | ||||||||||