ISRN Molecular Biology The latest articles from Hindawi Publishing Corporation © 2014 , Hindawi Publishing Corporation . All rights reserved. Identification of QTLs for Resistance to Sclerotinia sclerotiorum in Carioca Common Bean by the Moving Away Method Thu, 06 Feb 2014 16:09:42 +0000 The aim of this study was to use multiple DNA markers for detection of QTLs related to resistance to white mold in an F2 population of common bean evaluated by the straw test method. The DNA from 186 F2 plants and from the parents was extracted for genotypic evaluation using SSR, AFLP, and SRAP markers. For phenotypic analysis, 186 F2:4 progenies and ten lines were evaluated, in a 14 × 14 triple lattice experimental design. The adjusted mean values of the F2:4 progenies were used for identification of QTLs by Bayesian shrinkage analysis. Significant differences were observed among the progenies for reaction to white mold. In identification of QTLs, 17 markers identified QTLs for resistance—13 SSRs and 4 AFLPs. The moving away method under the Bayesian approach proved to be efficient in the identification of QTLs when a genetic map is not used due to the low density of markers. The ME1 and BM211 markers are near the QTLs, with the effect of increasing resistance to white mold, and they have high heritability. They are thus promising for marker-assisted selection. Letícia A. de C. Lara, João B. dos Santos, Juliana S. Veloso, Marcio Balestre, Filipe C. Alves, and Monik E. Leite Copyright © 2014 Letícia A. de C. Lara et al. All rights reserved. Expression of a Mutant kcnj2 Gene Transcript in Zebrafish Sun, 12 Jan 2014 06:54:04 +0000 Long QT 7 syndrome (LQT7, also known as Andersen-Tawil syndrome) is a rare autosomal-dominant disorder that causes cardiac arrhythmias, periodic paralysis, and dysmorphic features. Mutations in the human KCNJ2 gene, which encodes for the subunit of the potassium inwardly-rectifying channel (), have been associated with the disorder. The majority of mutations are considered to be dominant-negative as mutant proteins interact to limit the function of wild type KCNJ2 proteins. Several LQT7 syndrome mouse models have been created that vary in the physiological similarity to the human disease. To complement the LQT7 mouse models, we investigated the usefulness of the zebrafish as an alternative model via a transient approach. Initial bioinformatic analysis identified the zebrafish orthologue of the human KCNJ2 gene, together with a spatial expression profile that was similar to that of human. The expression of a kcnj2-12 transcript carrying an in-frame deletion of critical amino acids identified in human studies resulted in embryos that exhibited defects in muscle development, thereby affecting movement, a decrease in jaw size, pupil-pupil distance, and signs of scoliosis. These defects correspond to some phenotypes expressed by human LQT7 patients. Ivone U. S. Leong, Jonathan R. Skinner, Andrew N. Shelling, and Donald R. Love Copyright © 2014 Ivone U. S. Leong et al. All rights reserved. Quantification of the Spatial Organization of the Nuclear Lamina as a Tool for Cell Classification Thu, 07 Nov 2013 08:24:19 +0000 The nuclear lamina is the structural scaffold of the nuclear envelope that plays multiple regulatory roles in chromatin organization and gene expression as well as a structural role in nuclear stability. The lamina proteins, also referred to as lamins, determine nuclear lamina organization and define the nuclear shape and the structural integrity of the cell nucleus. In addition, lamins are connected with both nuclear and cytoplasmic structures forming a dynamic cellular structure whose shape changes upon external and internal signals. When bound to the nuclear lamina, the lamins are mobile, have an impact on the nuclear envelop structure, and may induce changes in their regulatory functions. Changes in the nuclear lamina shape cause changes in cellular functions. A quantitative description of these structural changes could provide an unbiased description of changes in cellular function. In this review, we describe how changes in the nuclear lamina can be measured from three-dimensional images of lamins at the nuclear envelope, and we discuss how structural changes of the nuclear lamina can be used for cell classification. Christiaan H. Righolt, Diana A. Zatreanu, and Vered Raz Copyright © 2013 Christiaan H. Righolt et al. All rights reserved. Genetic Analysis and Molecular Identification of Virulence in Xanthomonas oryzae pv. oryzae Isolates Sun, 08 Sep 2013 15:44:19 +0000 Bacterial leaf blight (BLB) of rice is a very destructive disease worldwide and is caused by Xanthomonas oryzae pv. oryzae (Xoo). The aim of the present study was to examine if the Xoo virulence pathotypes obtained using phenotypic pathotyping could be confirmed using molecular approach. After screening of 60 Operon primers with genomic DNA of two Xoo isolates (virulent pathotype, Vr, and mildly virulent pathotype, MVr), 12 Operon primers that gave reproducible and useful genetic information were selected and used to analyze 50 Xoo isolates from 7 West African countries. Genetic analysis revealed two major Xoo virulence genotypes (Mta and Mtb) with Mta having two subgroups (Mta1 and Mta2). Mta1 (Vr1) subgroup genotype has occurrence in six countries and Mta2 (Vr2) in three countries while Mtb genotype characterized mildly virulence (MVr) Xoo isolates present in five countries. The study revealed possible linkage and correlation between phenotypic pathotyping and molecular typing of Xoo virulence. Xoo virulence genotypes were known to exist within country and there was evidence of Xoo pathogen migration between countries. Durable resistance rice cultivars would need to overcome both Mta and Mtb Xoo virulence genotypes in order to survive after their deployment into different rice ecologies in West Africa. Amos Onasanya, R. O. Onasanya, Abiodun A. Ojo, and B. O. Adewale Copyright © 2013 Amos Onasanya et al. All rights reserved. Characterization of Histone H2A Derived Antimicrobial Peptides, Harriottins, from Sicklefin Chimaera Neoharriotta pinnata (Schnakenbeck, 1931) and Its Evolutionary Divergence with respect to CO1 and Histone H2A Mon, 03 Jun 2013 08:07:00 +0000 Antimicrobial peptides (AMPs) are humoral innate immune components of fishes that provide protection against pathogenic infections. Histone derived antimicrobial peptides are reported to actively participate in the immune defenses of fishes. Present study deals with identification of putative antimicrobial sequences from the histone H2A of sicklefin chimaera, Neoharriotta pinnata. A 52 amino acid residue termed Harriottin-1, a 40 amino acid Harriottin-2, and a 21 mer Harriottin-3 were identified to possess antimicrobial sequence motif. Physicochemical properties and molecular structure of Harriottins are in agreement with the characteristic features of antimicrobial peptides, indicating its potential role in innate immunity of sicklefin chimaera. The histone H2A sequence of sicklefin chimera was found to differ from previously reported histone H2A sequences. Phylogenetic analysis based on histone H2A and cytochrome oxidase subunit-1 (CO1) gene revealed N. pinnata to occupy an intermediate position with respect to invertebrates and vertebrates. Naveen Sathyan, Rosamma Philip, E. R. Chaithanya, P. R. Anil Kumar, V. N. Sanjeevan, and I. S. Bright Singh Copyright © 2013 Naveen Sathyan et al. All rights reserved. Efficient IDUA Gene Mutation Detection with Combined Use of dHPLC and Dried Blood Samples Mon, 22 Apr 2013 10:21:50 +0000 Objectives. Development of a simple mutation directed method in order to allow lowering the cost of mutation testing using an easily obtainable biological material. Assessment of the feasibility of such method was tested using a GC-rich amplicon. Design and Methods. A method of denaturing high-performance liquid chromatography (dHPLC) was improved and implemented as a technique for the detection of variants in exon 9 of the IDUA gene. The optimized method was tested in 500 genomic DNA samples obtained from dried blood spots (DBS). Results. With this dHPLC approach it was possible to detect different variants, including the common p.Trp402Ter mutation in the IDUA gene. The high GC content did not interfere with the resolution and reliability of this technique, and discrimination of G-C transversions was also achieved. Conclusion. This PCR-based dHPLC method is proved to be a rapid, a sensitive, and an excellent option for screening numerous samples obtained from DBS. Furthermore, it resulted in the consistent detection of clearly distinguishable profiles of the common p.Trp402Ter IDUA mutation with an advantageous balance of cost and technical requirements. Diogo Ribeiro, Ana Cardoso, Ana Joana Duarte, Luis Vieira, and Olga Amaral Copyright © 2013 Diogo Ribeiro et al. All rights reserved. Molecular Characterization and Phylogenetic Analysis of a Histone-Derived Antimicrobial Peptide Teleostin from the Marine Teleost Fishes, Tachysurus jella and Cynoglossus semifasciatus Sun, 03 Mar 2013 13:58:18 +0000 Antimicrobial peptides (AMPs) are host defense peptides that are well conserved throughout the course of evolution. Histones are classical DNA-binding proteins, rich in cationic amino acids, and recently appreciated as precursors for various histone-derived AMPs. The present study deals with identification of the potential antimicrobial peptide sequence of teleostin from the histone H2A of marine teleost fishes, Cynoglossus semifasciatus and Tachysurus jella. A 245 bp amplicon coding for 81 amino acids was obtained from the cDNA transcripts of these fishes. The first 52 amino acids from the N terminal of the peptide were identical to previously characterized histone-derived antimicrobial peptides. Molecular and physicochemical characterizations of the sequence were found to be in agreement with previously reported histone H2A-derived AMPs, suggesting the possible role of histone H2A in innate defense mechanism in fishes. E. R. Chaithanya, Rosamma Philip, Naveen Sathyan, and P. R. Anil Kumar Copyright © 2013 E. R. Chaithanya et al. All rights reserved. The Not4 RING E3 Ligase: A Relevant Player in Cotranslational Quality Control Mon, 21 Jan 2013 08:50:48 +0000 The Not4 RING E3 ligase is a subunit of the evolutionarily conserved Ccr4-Not complex. Originally identified in yeast by mutations that increase transcription, it was subsequently defined as an ubiquitin ligase. Substrates for this ligase were characterized in yeast and in metazoans. Interestingly, some substrates for this ligase are targeted for polyubiquitination and degradation, while others instead are stable monoubiquitinated proteins. The former are mostly involved in transcription, while the latter are a ribosomal protein and a ribosome-associated chaperone. Consistently, Not4 and all other subunits of the Ccr4-Not complex are present in translating ribosomes. An important function for Not4 in cotranslational quality control has emerged. In the absence of Not4, the total level of polysomes is reduced. In addition, translationally arrested polypeptides, aggregated proteins, and polyubiquitinated proteins accumulate. Its role in quality control is likely to be related on one hand to its importance for the functional assembly of the proteasome and on the other hand to its association with the RNA degradation machines. Not4 is in an ideal position to signal to degradation mRNAs whose translation has been aborted, and this defines Not4 as a key player in the quality control of newly synthesized proteins. Martine A. Collart Copyright © 2013 Martine A. Collart. All rights reserved. Autophagy: New Questions from Recent Answers Sun, 30 Dec 2012 16:31:04 +0000 Macroautophagy (hereafter autophagy) is currently one of the areas of medical life sciences attracting a great interest because of its pathological implications and therapy potentials. The discovery of the autophagy-related genes (ATGs) has been the key event in this research field because their study has led to the acquisition of new knowledge about the mechanism of this transport pathway. In addition, the investigation of these genes in numerous model systems has revealed the central role that autophagy plays in maintaining the cell homeostasis. This process carries out numerous physiological functions, some of which were unpredicted and thus surprising. Here, we will review some of the questions about the mechanism and function of autophagy that still remain unanswered, and new ones that have emerged from the recent discoveries. Fulvio Reggiori Copyright © 2012 Fulvio Reggiori. All rights reserved. The Dual Role of TGF in Human Cancer: From Tumor Suppression to Cancer Metastasis Mon, 24 Dec 2012 18:47:10 +0000 The transforming growth factor-beta (TGFβ) superfamily encompasses widespread and evolutionarily conserved polypeptide growth factors that regulate and orchestrate growth and differentiation in all cell types and tissues. While they regulate asymmetric cell division and cell fate determination during early development and embryogenesis, TGFβ family members play a major regulatory role in hormonal and immune responses, cell growth, cell death and cell immortalization, bone formation, tissue remodeling and repair, and erythropoiesis throughout adult life. The biological and physiological functions of TGFβ, the founding member of this family, and its receptors are of central importance to human diseases, particularly cancer. By regulating cell growth, death, and immortalization, TGFβ signaling pathways exert tumor suppressor effects in normal cells and early carcinomas. Thus, it is not surprising that a high number of human tumors arise due to mutations or deletions in the genes coding for the various TGFβ signaling components. As tumors develop and progress, these protective and cytostatic effects of TGFβ are often lost. TGFβ signaling then switches to promote cancer progression, invasion, and tumor metastasis. The molecular mechanisms underlying this dual role of TGFβ in human cancer will be discussed in depth in this paper, and it will highlight the challenge and importance of developing novel therapeutic strategies specifically aimed at blocking the prometastatic arm of the TGFβ signaling pathway without affecting its tumor suppressive effects. Jean-Jacques Lebrun Copyright © 2012 Jean-Jacques Lebrun. All rights reserved. Processing of Damaged DNA Ends for Double-Strand Break Repair in Mammalian Cells Tue, 04 Dec 2012 13:53:57 +0000 Most DNA double-strand breaks (DSBs) formed in a natural environment have chemical modifications at or near the ends that preclude direct religation and require removal or other processing so that rejoining can proceed. Free radical-mediated DSBs typically bear unligatable 3′-phosphate or 3′-phosphoglycolate termini and often have oxidized bases and/or abasic sites near the break. Topoisomerase-mediated DSBs are blocked by covalently bound peptide fragments of the topoisomerase. Enzymes capable of resolving damaged ends include polynucleotide kinase/phosphatase, which restores missing 5′-phosphates and removes 3′-phosphates; tyrosyl-DNA phosphodiesterases I and II (TDP1 and TDP2), which remove peptide fragments of topoisomerases I and II, respectively; and the Artemis and Metnase endonucleases, which can trim damaged overhangs of diverse structure. TDP1 as well as APE1 can remove 3′-phosphoglycolates and other 3′ blocks, while CtIP appears to provide an alternative pathway for topoisomerase II fragment removal. Ku, a core DSB joining protein, can cleave abasic sites near DNA ends. The downstream processes of patching and ligation are tolerant of residual damage and can sometimes proceed without complete damage removal. Despite these redundant pathways for resolution, damaged ends appear to be a significant barrier to rejoining, and their resolution may be a rate-limiting step in repair of some DSBs. Lawrence F. Povirk Copyright © 2012 Lawrence F. Povirk. All rights reserved. Assembly of the β-Barrel Outer Membrane Proteins in Gram-Negative Bacteria, Mitochondria, and Chloroplasts Tue, 20 Nov 2012 11:39:32 +0000 In the last decade, there has been an explosion of publications on the assembly of β-barrel outer membrane proteins (OMPs), which carry out diverse cellular functions, including solute transport, protein secretion, and assembly of protein and lipid components of the outer membrane. Of the three outer membrane model systems—Gram-negative bacteria, mitochondria and chloroplasts—research on bacterial and mitochondrial systems has so far led the way in dissecting the β-barrel OMP assembly pathways. Many exciting discoveries have been made, including the identification of β-barrel OMP assembly machineries in bacteria and mitochondria, and potentially the core assembly component in chloroplasts. The atomic structures of all five components of the bacterial β-barrel assembly machinery (BAM) complex, except the β-barrel domain of the core BamA protein, have been solved. Structures reveal that these proteins contain domains/motifs known to facilitate protein-protein interactions, which are at the heart of the assembly pathways. While structural information has been valuable, most of our current understanding of the β-barrel OMP assembly pathways has come from genetic, molecular biology, and biochemical analyses. This paper provides a comparative account of the β-barrel OMP assembly pathways in Gram-negative bacteria, mitochondria, and chloroplasts. Rajeev Misra Copyright © 2012 Rajeev Misra. All rights reserved. DNA Extraction Protocol for Plants with High Levels of Secondary Metabolites and Polysaccharides without Using Liquid Nitrogen and Phenol Wed, 14 Nov 2012 09:29:26 +0000 Mangroves and salt marsh species are known to synthesize a wide spectrum of polysaccharides and polyphenols including flavonoids and other secondary metabolites which interfere with the extraction of pure genomic DNA. Although a plethora of plant DNA isolation protocols exist, extracting DNA from mangroves and salt marsh species is a challenging task. This study describes a rapid and reliable cetyl trimethylammonium bromide (CTAB) protocol suited specifically for extracting DNA from plants which are rich in polysaccharides and secondary metabolites, and the protocol also excludes the use of expensive liquid nitrogen and toxic phenols. Purity of extracted DNA was excellent as evident by A260/A280 ratio ranging from 1.78 to 1.84 and A260/A230 ratio was >2, which also suggested that the preparations were sufficiently free of proteins and polyphenolics/polysaccharide compounds. DNA concentration ranged from 8.8 to 9.9 μg μL−1. The extracted DNA was amenable to RAPD, restriction digestion, and PCR amplification of plant barcode genes (matK and rbcl). The optimized method is suitable for both dry and fresh leaves. The success of this method in obtaining high-quality genomic DNA demonstrated the broad applicability of this method. Sunil Kumar Sahu, Muthusamy Thangaraj, and Kandasamy Kathiresan Copyright © 2012 Sunil Kumar Sahu et al. All rights reserved. Molecular Determinants of Cav1.2 Calcium Channel Inactivation Wed, 17 Oct 2012 14:27:31 +0000 Voltage-gated L-type Cav1.2 calcium channels couple membrane depolarization to transient increase in cytoplasmic free Ca2+ concentration that initiates a number of essential cellular functions including cardiac and vascular muscle contraction, gene expression, neuronal plasticity, and exocytosis. Inactivation or spontaneous termination of the calcium current through Cav1.2 is a critical step in regulation of these processes. The pathophysiological significance of this process is manifested in hypertension, heart failure, arrhythmia, and a number of other diseases where acceleration of the calcium current decay should present a benefit function. The central issue of this paper is the inactivation of the Cav1.2 calcium channel mediated by multiple determinants. Nikolai M. Soldatov Copyright © 2012 Nikolai M. Soldatov. All rights reserved. Regulation of GLUT4 and Insulin-Dependent Glucose Flux Wed, 17 Oct 2012 11:59:37 +0000 GLUT4 has long been known to be an insulin responsive glucose transporter. Regulation of GLUT4 has been a major focus of research on the cause and prevention of type 2 diabetes. Understanding how insulin signaling alters the intracellular trafficking of GLUT4 as well as understanding the fate of glucose transported into the cell by GLUT4 will be critically important for seeking solutions to the current rise in diabetes and metabolic disease. Ann Louise Olson Copyright © 2012 Ann Louise Olson. All rights reserved. Nucleosome Positioning Mon, 15 Oct 2012 10:42:36 +0000 Nucleosome positioning is not only related to genomic DNA compaction but also to other biological functions. After the chromatin is digested by micrococcal nuclease, nucleosomal (nucleosome-bound) DNA fragments can be sequenced and mapped on the genomic DNA sequence. Due to the development of modern DNA sequencing technology, genome-wide nucleosome mapping has been performed in a wide range of eukaryotic species. Comparative analyses of the nucleosome positions have revealed that the nucleosome is more frequently formed in exonic than intronic regions, and that most of transcription start and translation (or transcription) end sites are located in nucleosome linker DNA regions, indicating that nucleosome positioning influences transcription initiation, transcription termination, and gene splicing. In addition, nucleosomal DNA contains guanine and cytosine (G + C)-rich sequences and a high level of cytosine methylation. Thus, the nucleosome positioning system has been conserved during eukaryotic evolution. Hiromi Nishida Copyright © 2012 Hiromi Nishida. All rights reserved. Identification and Molecular Characterization of Molluskin, a Histone-H2A-Derived Antimicrobial Peptide from Molluscs Mon, 15 Oct 2012 08:19:10 +0000 Antimicrobial peptides are humoral innate immune components of molluscs that provide protection against pathogenic microorganisms. Among these, histone-H2A-derived antimicrobial peptides are known to actively participate in host defense responses of molluscs. Present study deals with identification of putative antimicrobial sequences from the histone-H2A of back-water oyster Crassostrea madrasensis, rock oyster Saccostrea cucullata, grey clam Meretrix casta, fig shell Ficus gracilis, and ribbon bullia Bullia vittata. A 75 bp fragment encoding 25 amino acid residues was amplified from cDNA of these five bivalves and was named “Molluskin.” The 25 amino acid peptide exhibited high similarity to previously reported histone-H2A-derived AMPs from invertebrates indicating the presence of an antimicrobial sequence motif. Physicochemical properties of the peptides are in agreement with the characteristic features of antimicrobial peptides, indicating their potential role in innate immunity of molluscs. Naveen Sathyan, Rosamma Philip, E. R. Chaithanya, and P. R. Anil Kumar Copyright © 2012 Naveen Sathyan et al. All rights reserved. Models, Regulations, and Functions of Microtubule Severing by Katanin Thu, 27 Sep 2012 11:36:38 +0000 Regulation of microtubule dynamics depends on stochastic balance between polymerization and severing process which lead to differential spatiotemporal abundance and distribution of microtubules during cell development, differentiation, and morphogenesis. Microtubule severing by a conserved AAA family protein Katanin has emerged as an important microtubule architecture modulating process in cellular functions like division, migration, shaping and so on. Regulated by several factors, Katanin manifests connective crosstalks in network motifs in regulation of anisotropic severing pattern of microtubule protofilaments in cell type and stage dependent way. Mechanisms of structural disintegration of microtubules by Katanin involve heterogeneous mechanochemical processes and sensitivity of microtubules to Katanin plays significant roles in mitosis/meiosis, neurogenesis, cilia/flagella formation, cell wall development and so on. Deregulated and uncoordinated expression of Katanin has been shown to have implications in pathophysiological conditions. In this paper, we highlight mechanistic models and regulations of microtubule severing by Katanin in context of structure and various functions of Katanin in different organisms. Debasish Kumar Ghosh, Debdeep Dasgupta, and Abhishek Guha Copyright © 2012 Debasish Kumar Ghosh et al. All rights reserved. Inner Membrane Protein YhcB Interacts with RodZ Involved in Cell Shape Maintenance in Escherichia coli Mon, 03 Sep 2012 15:29:25 +0000 Depletion of YhcB, an inner membrane protein of Escherichia coli, inhibited the growth of rodZ deletion mutant showing that the loss of both YhcB and RodZ is synthetically lethal. Furthermore, YhcB was demonstrated to interact with RodZ as well as several other proteins involved in cell shape maintenance and an inner membrane protein YciS of unknown function, using bacterial two-hybrid system. These observations seem to indicate that YhcB is involved in the biogenesis of cell envelope and the maintenance of cell shape together with RodZ. Gaochi Li, Kentaro Hamamoto, and Madoka Kitakawa Copyright © 2012 Gaochi Li et al. All rights reserved. Isolation of Alcohol Dehydrogenase cDNA and Basal Regulatory Region from Metroxylon sagu Sun, 26 Aug 2012 08:09:11 +0000 Alcohol dehydrogenase (Adh) is a versatile enzyme involved in many biochemical pathways in plants such as in germination and stress tolerance. Sago palm is plant with much importance to the state of Sarawak as one of the most important crops that bring revenue with the advantage of being able to withstand various biotic and abiotic stresses such as heat, pathogens, and water logging. Here we report the isolation of sago palm Adh cDNA and its putative promoter region via the use of rapid amplification of cDNA ends (RACE) and genomic walking. The isolated cDNA was characterized and determined to be 1464 bp long encoding for 380 amino acids. BLAST analysis showed that the Adh is similar to the Adh1 group with 91% and 85% homology with Elaeis guineensis and Washingtonia robusta, respectively. The putative basal msAdh1 regulatory region was further determined to contain promoter signals of TATA and AGGA boxes and predicted amino acids analyses showed several Adh-specific motifs such as the two zinc-binding domains that bind to the adenosine ribose of the coenzyme and binding to alcohol substrate. A phylogenetic tree was also constructed using the predicted amino acid showed clear separation of Adh from bacteria and clustered within the plant Adh group. Ching Ching Wee and Hairul Azman Roslan Copyright © 2012 Ching Ching Wee and Hairul Azman Roslan. All rights reserved. Real-Time PCR-Coupled CE-SELEX for DNA Aptamer Selection Wed, 08 Aug 2012 13:40:52 +0000 Aptamers are short nucleic acid or peptide sequences capable of binding to a target molecule with high specificity and affinity. Also known as “artificial antibodies,” aptamers provide many advantages over antibodies. One of the major hurdles to aptamer isolation is the initial time and effort needed for selection. The systematic evolution of ligands by exponential enrichment (SELEX) is the traditional procedure for generating aptamers, but this process is lengthy and requires a large quantity of target and starting aptamer library. A relatively new procedure for generating aptamers using capillary electrophoresis (CE), known as CE-SELEX, is faster and more efficient than SELEX but requires laser-induced fluorescence (LIF) to detect the aptamer-target complexes. Here, we implemented an alternative system without LIF using real-time- (RT-) PCR to indirectly measure aptamer-target complexes. In three rounds of selection, as opposed to ten or more rounds common in SELEX protocols, a specific aptamer for bovine serum albumin (BSA) was obtained. The specificity of the aptamer to BSA was confirmed by electrophoretic mobility shift assays (EMSAs), an unlabeled competitor assay, and by a supershift assay. The system used here provides a cost effective and a highly efficient means of generating aptamers. Patrick Ruff, Rekha B. Pai, and Francesca Storici Copyright © 2012 Patrick Ruff et al. All rights reserved. Receptor Binding by Cholera Toxin B-Subunit and Amino Acid Modification Improves Minimal Peptide Immunogenicity Sun, 15 Jul 2012 08:02:54 +0000 We increase our understanding of augmenting a cellular immune response, by using an HIV-1 protease-derived epitope (PR75–84), and variants thereof, coupled to the C-terminal, of the B subunit of cholera toxin (CTB). Fusion proteins were used for immunizations of HLA-A0201 transgenic C57BL/6 mice. We observed different capacities to elicit a cellular immune response by peptides with additions of five to ten amino acids to the PR epitope. There was a positive correlation between the magnitude of the elicited cellular immune response and the capacity of the fusion protein to bind GM-1. This binding capacity is affected by its ability to form natural pentamers of CTB. Our results suggest that functional CTB pentamers containing a foreign amino acid-modified epitope is a novel way to overcome the limited cellular immunogenicity of minimal peptide antigens. This way of using a functional assay as readout for improved cellular immunogenicity might become highly valuable for difficult immunogens such as short peptides (epitopes). Andreas Boberg, Alexandra Stålnacke, Andreas Bråve, Jorma Hinkula, Britta Wahren, and Nils Carlin Copyright © 2012 Andreas Boberg et al. All rights reserved. Ion Transporters and Abiotic Stress Tolerance in Plants Sun, 03 Jun 2012 15:21:42 +0000 Adaptation of plants to salt stress requires cellular ion homeostasis involving net intracellular Na+ and Cl− uptake and subsequent vacuolar compartmentalization without toxic ion accumulation in the cytosol. Sodium ions can enter the cell through several low- and high-affinity K+ carriers. Some members of the HKT family function as sodium transporter and contribute to Na+ removal from the ascending xylem sap and recirculation from the leaves to the roots via the phloem vasculature. Na+ sequestration into the vacuole depends on expression and activity of Na+/H+ antiporter that is driven by electrochemical gradient of protons generated by the vacuolar H+-ATPase and the H+-pyrophosphatase. Sodium extrusion at the root-soil interface is presumed to be of critical importance for the salt tolerance. Thus, a very rapid efflux of Na+ from roots must occur to control net rates of influx. The Na+/H+ antiporter SOS1 localized to the plasma membrane is the only Na+ efflux protein from plants characterized so far. In this paper, we analyze available data related to ion transporters and plant abiotic stress responses in order to enhance our understanding about how salinity and other abiotic stresses affect the most fundamental processes of cellular function which have a substantial impact on plant growth development. Faïçal Brini and Khaled Masmoudi Copyright © 2012 Faïçal Brini and Khaled Masmoudi. All rights reserved. The Tousled-Like Kinases as Guardians of Genome Integrity Sun, 20 May 2012 17:24:56 +0000 The Tousled-like kinases (TLKs) function in processes of chromatin assembly, including replication, transcription, repair, and chromosome segregation. TLKs interact specifically (and phosphorylate) with the chromatin assembly factor Asf1, a histone H3-H4 chaperone, histone H3 itself at Ser10, and also Rad9, a key protein involved in DNA repair and cell cycle signaling following DNA damage. These interactions are believed to be responsible for the action of TLKs in double-stranded break repair and radioprotection and also in the propagation of the DNA damage response. Hence, I propose that TLKs play key roles in maintenance of genome integrity in many organisms of both kingdoms. In this paper, I highlight key issues of the known roles of these proteins, particularly in the context of DNA repair (IR and UV), their possible relevance to genome integrity and cancer development, and as possible targets for intervention in cancer management. Arrigo De Benedetti Copyright © 2012 Arrigo De Benedetti. All rights reserved. K-Ras Mutations in Non-Small-Cell Lung Cancer: Prognostic and Predictive Value Mon, 14 May 2012 09:03:39 +0000 Non-small-cell lung cancer (NSCLC) is a heterogeneous disease due to the presence of different clinically relevant molecular subtypes. Until today, several biological events have been identified in lung adenocarcinoma, including epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) translocations, offering new hopes to patients with metastatic disease. Unfortunately, in approximately 50% of adenocarcinoma and for those harbouring K-RAS mutations, the most frequent mutation in Caucasian lung adenocarcinoma, so far no specific drug demonstrated efficacy. The rat sarcoma (RAS) genes, including H-RAS, K-RAS, and N-RAS, encode a family of proteins regulating cell growth, differentiation, and apoptosis. K-RAS mutations are present in 20–30% of NSCLC and occur most commonly, but not exclusively, in adenocarcinoma histology and life-long smokers. Although in colorectal cancer patients K-RAS mutations represent a validated negative predictive biomarker for treatment with anti-EGFR monoclonal antibodies, their role in selecting specific treatment for NSCLC patients remains undefined. Aim of the present paper is to critically analyze the prognostic and predictive value of K-RAS mutations in NSCLC. Manolo D’Arcangelo and Federico Cappuzzo Copyright © 2012 Manolo D’Arcangelo and Federico Cappuzzo. All rights reserved. Nucleotide Sequencing and SNP Detection of Toll-Like Receptor-4 Gene in Murrah Buffalo (Bubalus bubalis) Thu, 29 Mar 2012 08:58:39 +0000 Toll-like receptor-4 (TLR-4) has an important pattern recognition receptor that recognizes endotoxins associated with gram negative bacterial infections. The present investigation was carried out to study nucleotide sequencing and SNP detection by PCR-RFLP analysis of the TLR-4 gene in Murrah buffalo. Genomic DNA was isolated from 102 lactating Murrah buffalo from NDRI herd. The amplified PCR fragments of TLR-4 comprised of exon 1, exon 2, exon 3.1, and exon 3.2 were examined to RFLP. PCR products were obtained with sizes of 165, 300, 478, and 409 bp. TLR-4 gene of investigated Murrah buffaloes was highly polymorphic with AA, AB, and BB genotypes as revealed by PCR-RFLP analysis using Dra I, Hae III, and Hinf I REs. Nucleotide sequencing of the amplified fragment of TLR-4 gene of Murrah buffalo was done. Twelve SNPs were identified. Six SNPs were nonsynonymous resulting in change in amino acids. Murrah is an indigenous Buffalo breed and the presence of the nonsynonymous SNP is indicative of its unique genomic architecture. Sequence alignment and homology across species using BLAST analysis revealed 97%, 97%, 99%, 98%, and 80% sequence homology with Bos taurus, Bos indicus, Ovis aries, Capra hircus, and Homo sapiens, respectively. M. Mitra, S. Taraphder, G. S. Sonawane, and A. Verma Copyright © 2012 M. Mitra et al. All rights reserved. Structural Modeling and Analysis of Pregnancy-Associated Glycoprotein-1 of Buffalo (Bubalus bubalis) Mon, 12 Mar 2012 09:37:33 +0000 The present study was conducted to design and analyze the structural model of buffalo pregnancy-associated glycoprotein-1 (PAG-1) using bioinformatics. Structural modeling of the deduced buffalo PAG-1 protein was done using PHYRE, CONSURF servers and its structure was subsequently constructed using MODELLER 9.9 and PyMOL softwares Buffalo PAG-1 structural conformity was analyzed using PROSA, WHATIF, and 3D-PSSM servers. Designed buffalo PAG-1 protein structure on BLAST analysis retrieved protein structures belonging to aspartic proteinase family. Moreover in silico analysis revealed buffalo PAG-1 protein retained bilobed structure with pepstatin-binding clefts near the active sites by docking studies with pepstatin A using PatchDock server. Structural studies revealed that the amino and carboxy terminal containing aspartic residues are highly conserved and buried within the protein structure. Structural conformity studies showed that more than 90% of the residues lie inside favored and allowed regions. It was also deduced that buffalo PAG-1 possesses low and high energy zones with a very low threshold for proteolysis ascertaining the stableness of the buffalo PAG-1 protein structure. This study depicts the structural conformity and stability of buffalo PAG-1 protein. Jerome Andonissamy, S. K. Singh, and S. K. Agarwal Copyright © 2012 Jerome Andonissamy et al. All rights reserved. Central Role of Ubiquitination in Genome Maintenance: DNA Replication and Damage Repair Wed, 08 Feb 2012 11:10:07 +0000 Faithful transmission of genetic information through generations ensures genomic stability and integrity. However, genetic alterations occur every now and then during the course of genome duplication. In order to repair these genetic defects and lesions, nature has devised several repair pathways which function promptly to prevent the cell from accumulating permanent mutations. These repair mechanisms seem to be significantly impacted by posttranslational modifications of proteins like phosphorylation and ubiquitination. Protein ubiquitination is emerging as a critical regulatory mechanism of DNA damage response. Non-proteolytic, proteasome-independent functions of ubiquitin involving monoubiquitination and polyubiquitination of DNA repair proteins contribute significantly to the signaling of DNA repair pathways. In this paper, we will particularly highlight the work on ubiquitin-mediated signaling in the repair processes involving the Fanconi anemia pathway, translesional synthesis, nucleotide excision repair, and repair of double-strand breaks. We will also discuss the role of ubiquitin ligases in regulating checkpoint mechanisms, the role of deubiquitinating enzymes, and the growing possibilities of therapeutic intervention in this ubiquitin-conjugation system. Soma Ghosh and Tapas Saha Copyright © 2012 Soma Ghosh and Tapas Saha. All rights reserved. Increased Phospho-Keratin 8 Isoforms in Colorectal Tumors Associated with EGFR Pathway Activation and Reduced Apoptosis Tue, 31 Jan 2012 09:01:35 +0000 Hyperphosphorylated keratin (K) 8 acts as a phosphate “sponge” for stress-activated protein kinases thereby inhibiting pro-apoptotic molecules and thus apoptosis. MAP kinase/ERK1 has increased activity in colorectal cancer (CRC) and is known to phosphorylate K8. The aims were to identify the K8 isoforms abundantly present in colon tumors, using 2D difference gel electrophoresis (DIGE), to identify the modifications using mass spectrometry, and to validate the differential abundance of these isoforms in tumors relative to matched normal mucosae. 2D DIGE showed 3 isoforms of K8 significantly increased in tumor ≥2-fold in 6/8 pairs. Metal oxide affinity chromatography mass spectrometry and bioinformatics were used to identify phosphorylated serine residues. Levels of PS24, PS432, and PS74 by western blotting were found to be significantly increased in tumor versus matched normal. Blocking of EGFR signaling in Caco2 cells showed a significant decrease (𝑃<0.0001) in K8 PS74 and PS432 levels by 59% and 66%, respectively, resulting in increased apoptosis. Georgia Arentz, Tim Chataway, Mark R. Condina, Timothy J. Price, Peter Hoffmann, and Jennifer E. Hardingham Copyright © 2012 Georgia Arentz et al. All rights reserved. Functional Characterization of a Small-Molecule Inhibitor of the DKK1-LRP6 Interaction Mon, 30 Jan 2012 14:59:42 +0000 Background. DKK1 antagonizes canonical Wnt signalling through high-affinity binding to LRP5/6, an essential component of the Wnt receptor complex responsible for mediating downstream canonical Wnt signalling. DKK1 overexpression is known for its pathological implications in osteoporosis, cancer, and neurodegeneration, suggesting the interaction with LRP5/6 as a potential therapeutic target. Results. We show that the small-molecule NCI8642 can efficiently displace DKK1 from LRP6 and block DKK1 inhibitory activity on canonical Wnt signalling, as shown in binding and cellular assays, respectively. We further characterize NCI8642 binding activity on LRP6 by Surface Plasmon Resonance (SPR) technology. Conclusions. This study demonstrates that the DKK1-LRP6 interaction can be the target of small molecules and unlocks the possibility of new therapeutic tools for diseases associated with DKK1 dysregulation. Sara Iozzi, Rosaria Remelli, Barbara Lelli, Daniela Diamanti, Silvia Pileri, Luisa Bracci, Renza Roncarati, Andrea Caricasole, and Simonetta Bernocco Copyright © 2012 Sara Iozzi et al. All rights reserved.