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BACKGROUND: The α1,3-galactosyl epitope (α1-3Gal epitope), a major xenotransplant antigen, is synthesized by α1,3-galactosyltransferase (α1-3Gal transferase), which is evolutionarily related to the histo-blood group A/B transferases. STUDY DESIGN AND METHODS: We constructed structural chimeras between the human type A and murine α1-3Gal transferases and examined their activity and specificity. RESULTS: In many instances, a total loss of transferase activity was observed. Certain areas could be exchanged, with a potential diminishing of activity. With a few constructs, changes in acceptor substrate specificity were suspected. Unexpectedly, a functional conversion from A to B transferase activity was observed after replacing the short sequence of human A transferase with the corresponding sequence from murine α1-3Gal transferase. CONCLUSION: Because these two paralogous enzymes differ in 16 positions of the 38 amino acid residues in the replaced region, our finding may suggest that despite separate evolution and diversified acceptors, these glycosyltransferases still share the three-dimensional domain structure that is responsible for their sugar specificity, arguing against the functional requirement of a strong purifying selection playing a role in the evolution of the ABO family of genes. © 2009 American Association of Blood Banks. Source

Buj R.,Institute Dinvestigacions Biomediques Of Barcelona Iibb | Buj R.,Institute Of Medicina Predictiva I Personalitzada Del Cancer Imppc | Iglesias N.,Institute Dinvestigacions Biomediques Of Barcelona Iibb | Planas A.M.,Institute Dinvestigacions Biomediques Of Barcelona Iibb | And 3 more authors.
BMC Molecular Biology

Background: Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous.Results: We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit's component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells.Conclusions: We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior modifications. Using this technology, any existing Gateway destination expression vector with its model-specific properties could be easily adapted for expressing fusion proteins. © 2013 Buj et al.; licensee BioMed Central Ltd. Source

Institute Of Medicina Predictiva I Personalitzada Del Cancer Imppc and Sanford Burnham Institute for Medical Research | Date: 2013-02-08

Disclosed herein are systems and methods for using demethylation of genomic DNA for diagnosing, predicting, and/or monitoring the status or outcome of a neoplasm or a cancer in a subject.

Jorda M.,Institute Of Medicina Predictiva I Personalitzada Del Cancer Imppc | Peinado M.A.,Institute Of Medicina Predictiva I Personalitzada Del Cancer Imppc
Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis

It is well established that epigenetic events, in an intimate cooperation with genetic events, are involved in every step of tumorigenesis. DNA methylation, which in mammals takes place in the cytosines that precede a guanine (CpG dinucleotide), is the most well-characterized epigenetic mark. The study of aberrant DNA methylation patterns, such as hypermethylation of CpG islands and global genomic hypomethylation, are common issues in the studies on all types of cancer, and as in other areas of molecular oncology, colorectal cancer has become a privileged target. Besides the great variety of technologies available for the analysis of DNA methylation, most methods are based on three principles: methylation-sensitive enzymes, bisulphite conversion of unmethylated cytosines and immunoprecipitation of 5-methylcytosines. By combining each one of these principles with other genomic methodologies, a large range of approaches aimed at the analysis of methylation from one specific CpG site to a large number of sequences on the genome scale and suitable for different research needs have been developed. The goal of this review is to describe the most widely used methylation methods in the study of cancer, as well as the potential clinical applications of DNA methylation biomarkers in colorectal cancer. © 2010 Elsevier B.V. Source

Gomez-Diaz E.,Institute Of Biologia Evolutiva Ibe | Jorda M.,Institute Of Medicina Predictiva I Personalitzada Del Cancer Imppc | Peinado M.A.,Institute Of Medicina Predictiva I Personalitzada Del Cancer Imppc | Rivero A.,IRD Montpellier
PLoS Pathogens

A growing body of evidence points towards epigenetic mechanisms being responsible for a wide range of biological phenomena, from the plasticity of plant growth and development to the nutritional control of caste determination in honeybees and the etiology of human disease (e.g., cancer). With the (partial) elucidation of the molecular basis of epigenetic variation and the heritability of certain of these changes, the field of evolutionary epigenetics is flourishing. Despite this, the role of epigenetics in shaping host-pathogen interactions has received comparatively little attention. Yet there is plenty of evidence supporting the implication of epigenetic mechanisms in the modulation of the biological interaction between hosts and pathogens. The phenotypic plasticity of many key parasite life-history traits appears to be under epigenetic control. Moreover, pathogen-induced effects in host phenotype may have transgenerational consequences, and the bases of these changes and their heritability probably have an epigenetic component. The significance of epigenetic modifications may, however, go beyond providing a mechanistic basis for host and pathogen plasticity. Epigenetic epidemiology has recently emerged as a promising area for future research on infectious diseases. In addition, the incorporation of epigenetic inheritance and epigenetic plasticity mechanisms to evolutionary models and empirical studies of host-pathogen interactions will provide new insights into the evolution and coevolution of these associations. Here, we review the evidence available for the role epigenetics on host-pathogen interactions, and the utility and versatility of the epigenetic technologies available that can be cross-applied to host-pathogen studies. We conclude with recommendations and directions for future research on the burgeoning field of epigenetics as applied to host-pathogen interactions. © 2012 Gómez-Díaz et al. Source

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