Extracellular release of the ‘differentiation enhancing factor’, a HMG1 protein type, is an early step in murine erythroleukemia cell differentiation

AbstractDifferentiation enhancing factor (DEF) is a 29 kDa protein expressed in murine erythroleukemia (MEL) cells and active in promoting a significant increase in the rate of hexamethylenebisacetamide induced differentiation of these cells. The factor was recently shown to possess an amino acid sequence identical to that reported for one of the HMG1 proteins, designated as ‘amphoterin’ on the basis of its highly dipolar sequence. In the present study, we have expressed DEF cDNA in an E. coli strain and found that the recombinant protein has functional properties identical to those observed with native DEF. Furthermore, we demonstrate that, following MEL cell stimulation with the chemical inducer, DEF is secreted in large amounts in the extracellular medium. In fact, the N-terminal sequence and the partial amino acid sequence of tryptic peptides from the secreted protein correspond to those of DEF isolated from the soluble fraction of resting MEL cells. These results are indicative for an extracellular localization as the site of action of DEF and suggest a novel function for proteins belonging to the HMG1 family. Finally, the early decay of DEF mRNA, in chemical induced MEL cells, support the hypothesis that the involvement of the enhancing factor occurs and is completed in the early phases of cell differentiation

PN_SCD1, VESICLE TRAFFICKING REGULATOR IS DEMETHYLATED AND OVEREXPRESSED IN FLORETS OF APOMICTIC PASPALUM NOTATUM GENOTYPES

Apomixis (asexual reproduction through seeds) is considered a deviation of the sexual reproductive pathway leading to the formation of clonal progenies genetically identical to the mother plant. It has been suggested that apomixis might be a consequence of epigenetic alterations, such as interspecific hybridization and polyploidization, resulting in a wide deregulation of reproductive development. Studies on epigenetic are transforming our actual idea of the structural variation and diversity that prevails at key steps of plant female gametogenesis, with deep implications for understanding the evolutionary trends that model innovation in reproductive development and adaptation. Recent results have provided evidences indicating that epigenetic mechanisms are crucial to control events that distinguish sexual from apomictic development. Therefore, the epigenetic regulation of apomixis is an attractive theory as it potentially accounts for the facultative nature of apomixis as well as the ability of apomictic to revert back to sexuality. In this work we used the Methylation-Sensitive Amplification Polymorphism (MSAP) technique to characterize floral genome cytosine methylation patterns occurring in sexual and aposporous Paspalum notatum genotypes, in order to identify epigenetically-controlled genes putatively involved in apomixis development. A partial and rather divergent methylation reprogramming was detected in apomictic genotypes. From twelve polymorphic MSAP-derived sequences, one (PN_6.6, renamed PN_SCD1) was selected due to its relevant annotation and differential representation in 454 floral transcriptome libraries of sexual and apomictic P. notatum. PN_6.6 encodes the DENN domain/WD repeat-containing protein SCD1, which interacts with RAB GTPases- and/or MAPKs to promote specialized cell division, functions in clathrin-mediated membrane transport and was defined as potential substrate receptor of CUL4 E3 ubiquitin ligases. Quantitative RT-PCR and comparative RNAseq analyses of laser microdissected nucellar cells confirmed PN_SCD1 upregulation in florets of apomictic plants and revealed that overexpression takes place just before the onset of apospory initials. Moreover, we found that several SCD1 molecular partners are upregulated in florets of P. notatum apomictic plants. Our results revealed a specific vesicle trafficking molecular pathway epigenetically modulated during apomixis. Results will be presented and critically discussed

Novel Genotypes of the Subtropical Grass \u3cem\u3eEragrostis Curvula\u3c/em\u3e for the Analysis of Apomixis (Diplospory)

Eragrostis curvula (Schrad.) Nees is a variable grass native to Southern Africa. Its several forms, known as lovegrasses, were introduced to Australia, USA and Argentina as forage perennial grasses. Apomixis is a common trait in the genus Eragrostis, with diplospory being the most frequent type. Sexual reproduction also occurs in Eragrostis, although not frequently. Since most tetraploid Eragrostis lines are apomictic, the generation of a sexual tetraploid strain is a requirement for linkage analysis of the gene(s) governing the apomictic character. Furthermore, isogenic lines of the same ploidy, reproducing alternatively by sexuality or apomixes, represent an ideal system for comparative transcriptome analysis. The aim of this work was the generation and characterization of two novel genotypes of E. curvula: a dihaploid strain obtained in vitro from an apomictic cultivar and a tetraploid plant derived from the dihaploid after chromosome duplication

Discovery and Functional Categorisation of Expressed Sequence Tags from Flowers of \u3cem\u3eEragrostis Curvula\u3c/em\u3e Genotypes Showing Different Ploidy Levels and Reproductive Modes

Two novel genotypes of weeping lovegrass (Eragrostis curvula) - a dihaploid strain obtained in vitro from an apomictic cultivar and a tetraploid plant derived from the dihaploid after chromosome duplication – have recently been developed. These materials represent an excellent system for the identification, through transcriptional profiling, of genes involved in diplospory and/or ploidy level gene regulation. The aim of this work was the discovery and functional classification of expressed sequence tags (ESTs) from immature inflorescences of the apomictic E. curvula cultivar Tanganyika (2n=4x=40), a dihaploid sexual strain derived from it (2n=2x=20) and a tetraploid sexual strain (2n=4x=40) obtained by colchicine duplication of the dihaploid

Identification of ovule transcripts from the Apospory-Specific Genomic Region (ASGR)-carrier chromosome

<p>Abstract</p> <p>Background</p> <p>Apomixis, asexual seed production in plants, holds great potential for agriculture as a means to fix hybrid vigor. Apospory is a form of apomixis where the embryo develops from an unreduced egg that is derived from a somatic nucellar cell, the aposporous initial, via mitosis. Understanding the molecular mechanism regulating aposporous initial specification will be a critical step toward elucidation of apomixis and also provide insight into developmental regulation and downstream signaling that results in apomixis. To discover candidate transcripts for regulating aposporous initial specification in <it>P. squamulatum</it>, we compared two transcriptomes derived from microdissected ovules at the stage of aposporous initial formation between the apomictic donor parent, <it>P. squamulatum </it>(accession PS26), and an apomictic derived backcross 8 (BC₈) line containing only the Apospory-Specific Genomic Region (ASGR)-carrier chromosome from <it>P. squamulatum</it>. Toward this end, two transcriptomes derived from ovules of an apomictic donor parent and its apomictic backcross derivative at the stage of apospory initiation, were sequenced using 454-FLX technology.</p> <p>Results</p> <p>Using 454-FLX technology, we generated 332,567 reads with an average read length of 147 base pairs (bp) for the PS26 ovule transcriptome library and 363,637 reads with an average read length of 142 bp for the BC₈ovule transcriptome library. A total of 33,977 contigs from the PS26 ovule transcriptome library and 26,576 contigs from the BC₈ovule transcriptome library were assembled using the Multifunctional Inertial Reference Assembly program. Using stringent <it>in silico </it>parameters, 61 transcripts were predicted to map to the ASGR-carrier chromosome, of which 49 transcripts were verified as ASGR-carrier chromosome specific. One of the alien expressed genes could be assigned as tightly linked to the ASGR by screening of apomictic and sexual F₁s. Only one transcript, which did not map to the ASGR, showed expression primarily in reproductive tissue.</p> <p>Conclusions</p> <p>Our results suggest that a strategy of comparative sequencing of transcriptomes between donor parent and backcross lines containing an alien chromosome of interest can be an efficient method of identifying transcripts derived from an alien chromosome in a chromosome addition line.</p

Expression Analysis of the Ligands for the Natural Killer Cell Receptors NKp30 and NKp44

BACKGROUND: The natural cytotoxicity receptors (NCR) are important to stimulate the activity of Natural Killer (NK) cells against transformed cells. Identification of NCR ligands and their level of expression on normal and neoplastic cells has important implications for the rational design of immunotherapy strategies for cancer. METHODOLOGY/PRINCIPAL FINDINGS: Here we analyze the expression of NKp30 ligand and NKp44 ligand on 30 transformed or non-transformed cell lines of different origin. We find intracellular and surface expression of these two ligands on almost all cell lines tested. Expression of NKp30 and NKp44 ligands was variable and did not correlate with the origin of the cell line. Expression of NKp30 and NKp44 ligand correlated with NKp30 and NKp44-mediated NK cell lysis of tumor cells, respectively. The surface expression of NKp30 ligand and NKp44 ligand was sensitive to trypsin treatment and was reduced in cells arrested in G(2)/M phase. CONCLUSION/SIGNIFICANCE: These data demonstrate the ubiquitous expression of the ligands for NKp30 and NKp44 and give an important insight into the regulation of these ligands

Multi-Determinants Analysis of Molecular Alterations for Predicting Clinical Benefit to EGFR-Targeted Monoclonal Antibodies in Colorectal Cancer

KRAS mutations occur in 35-45% of metastatic colorectal cancers (mCRC) and preclude responsiveness to EGFR-targeted therapy with cetuximab or panitumumab. However, less than 20% patients displaying wild-type KRAS tumors achieve objective response. Alterations in other effectors downstream of the EGFR, such as BRAF, and deregulation of the PIK3CA/PTEN pathway have independently been found to give rise to resistance. We present a comprehensive analysis of KRAS, BRAF, PIK3CA mutations, and PTEN expression in mCRC patients treated with cetuximab or panitumumab, with the aim of clarifying the relative contribution of these molecular alterations to resistance.We retrospectively analyzed objective tumor response, progression-free (PFS) and overall survival (OS) together with the mutational status of KRAS, BRAF, PIK3CA and expression of PTEN in 132 tumors from cetuximab or panitumumab treated mCRC patients. Among the 106 non-responsive patients, 74 (70%) had tumors with at least one molecular alteration in the four markers. The probability of response was 51% (22/43) among patients with no alterations, 4% (2/47) among patients with 1 alteration, and 0% (0/24) for patients with > or =2 alterations (p<0.0001). Accordingly, PFS and OS were increasingly worse for patients with tumors harboring none, 1, or > or =2 molecular alteration(s) (p<0.001).When expression of PTEN and mutations of KRAS, BRAF and PIK3CA are concomitantly ascertained, up to 70% of mCRC patients unlikely to respond to anti-EGFR therapies can be identified. We propose to define as 'quadruple negative', the CRCs lacking alterations in KRAS, BRAF, PTEN and PIK3CA. Comprehensive molecular dissection of the EGFR signaling pathways should be considered to select mCRC patients for cetuximab- or panitumumab-based therapies