In silico Characterization of Class II Plant Defensins from Arabidopsis thaliana

Defensins comprise a polyphyletic group of multifunctional defense peptides. Cis-defensins, also known as cysteine stabilized αβ (CSαβ) defensins, are one of the most ancient defense peptide families. In plants, these peptides have been divided into two classes, according to their precursor organization. Class I defensins are composed of the signal peptide and the mature sequence, while class II defensins have an additional C-terminal prodomain, which is proteolytically cleaved. Class II defensins have been described in Solanaceae and Poaceae species, indicating this class could be spread among all flowering plants. Here, a search by regular expression (RegEx) was applied to the Arabidopsis thaliana proteome, a model plant with more than 300 predicted defensin genes. Two sequences were identified, A7REG2 and A7REG4, which have a typical plant defensin structure and an additional C-terminal prodomain. TraVA database indicated they are expressed in flower, ovules and seeds, and being duplicated genes, this indicates they could be a result of a subfunctionalization process. The presence of class II defensin sequences in Brassicaceae and Solanaceae and evolutionary distance between them suggest class II defensins may be present in other eudicots. Discovery of class II defensins in other plants could shed some light on flower, ovules and seed physiology, as this class is expressed in these locations.

A Systematic Review and Bioinformatics Study on Genes and micro-RNAs Involving the Transformation of Endometriosis into Ovarian Cancer

Background: Along with the description of tumorigenesis processes in endometriosisrelated ovarian cancer, identifying dysregulated miRNAs, the target genes of these miRNAs, and the processes abnormally affected by dysregulated miRNAs is essential, which was our goal. Methods: Two reviewers individually evaluated the articles which collected relevant information including genes and miRNAs involved in the transformation of endometriosis into ovarian cancer. To assess the mature sequence of miRNAs and also their chromosomal positions, miRPathDB software was employed. To determine the main target gene predicted for each considered miRNAs, the TargetScanS Web server was applied. The interaction of each gene with other genes associated with endometrial- related ovarian cancer was determined by GeneMANIA software. Finally, to design integrated model of miRNAs-targeted genes interaction network, the Cytoscape software was used. Results: The final number of studies available for analysis was 6 manuscripts including 22 miRNAs described as involved in the transformation of endometriosis into different subtypes of ovarian cancers (14 miRNAs up-regulated and 8 miRNAs down-regulated). Three miRNAs of miR-141 (upregulated), miR-205 (down-regulated), and miR-125b (down-regulated) were revealed as the originator for genetic interactions leading to carcinogenesis. We could show some common loops and pathways including uncontrolled cell proliferation and abnormal apoptosis (mediated by PTEN gene induced by miR-21 and miR-214), and disaggregation and epithelialization (mediated by ZEB1 and ZEB2 genes induced by miR-200). Conclusions: According to our analysis, up-regulation of miR-141 and down-regulation of miR-205 and miR-125b have a central role in transforming endometriosis to ovarian cancer.

Truncated Prosequence of Rhizopus oryzae Lipase: Key Factor for Production Improvement and Biocatalyst Stability

Recombinant Rhizopus oryzae lipase (mature sequence, rROL) was modified by adding to its N-terminal 28 additional amino acids from the C-terminal of the prosequence (proROL) to obtain a biocatalyst more suitable for the biodiesel industry. Both enzymes were expressed in Pichia pastoris and compared in terms of production bioprocess parameters, biochemical properties, and stability. Growth kinetics, production, and yields were better for proROL harboring strain than rROL one in batch cultures. When different fed-batch strategies were applied, lipase production and volumetric productivity of proROL-strain were always higher (5.4 and 4.4-fold, respectively) in the best case. rROL and proROL enzymatic activity was dependent on ionic strength and peaked in 200 mM Tris-HCl buffer. The optimum temperature and pH for rROL were influenced by ionic strength, but those for proROL were not. The presence of these amino acids altered lipase substrate specificity and increased proROL stability when different temperature, pH, and methanol/ethanol concentrations were employed. The 28 amino acids were found to be preferably removed by proteases, leading to the transformation of proROL into rROL. Nevertheless, the truncated prosequence enhanced Rhizopus oryzae lipase heterologous production and stability, making it more appropriate as industrial biocatalyst.

Dysregulated Expression of MicroRNA-16 Contributes to Abnormal Erythropoiesis in Patients with Polycythemia Vera

Discovery of JAK2V617F mutation in myeloproliferative neoplasms (MPN) raised intriguing question on how a single mutation might underlie different clinical phenotypes. Micro-RNAs are important regulators of gene expression, intervening in normal and neoplastic hematopoiesis. An abnormal expression profile of miRNAs in primary myelofibrosis granulocytes (Guglielmelli P, Exp Hematol 2007) and in polycythemia vera (PV) erythroid cells has been described (Bruchova H, Exp Hematol 2007; Haematologica 2008); therefore, dysregulation of miRNAs might contribute to MPN pathogenesis. In a global analysis of miRNA expression in CD34+ cells from PV pts we found abnormally increased levels of miR-16; of note, defects in miR-16 causing reduced expression have been implicated in the pathogenesis of chronic lymphocytic leukemia (CLL). There are two miR-16 genes, miR-16-1 (in a cluster with miR-15a on chr 13, mutated/deleted in CLL) and miR-16-2 (in a cluster with miR-15b on chr 3), which differ each to the other in their pre-miR sequence while the mature sequence is conserved. Using stem-looped primer technology and real-time PCR, we determined that expression levels of miR-16 in CD34+ cells from 15 PV pts were 10 to 30-fold higher than in CD34+ cells from controls or reactive erythrocytosis (P<.0001); the increase was specific for miR-16 since levels of mature miR-15a and miR-15b were unchanged. miR-16 was also slightly increased in PV granulocytes (about 2-fold vs controls; P=.026). Levels of miR-16 were independent of the JAK2V671F allele burden (r=0.4). Sequencing the entire pre- and mature miR-16-1/ miR-15a and miR-16-2/miR15b regions disclosed no abnormality, in contrast with CLL for miR-16-1. We also determined gene copy number by RT-PCR, but found no evidence of miR-16-1/2 copy number changes. Since the mature sequence is shared by the two miR-16, we designed a RT-PCR approach to determine the levels of their respective pre-microRNAs. We found that levels of pre-miR-16-2 were significantly increased in PV CD34+ cells compared to controls, while pre-miR-16-1 did not show appreciable changes; there was also a significant correlation between the levels of pre-miR16-2 (unlike premiR- 16-1) and those of total mature miR-16 in PV CD34+ cells (r=0.7, P<.01). The preferential contribution of miR-16-2 to abnormally high total miR-16 levels in PV CD34+ cells was supported by knock-down experiments using siRNAs specific for either miR-16-1 or miR-16-2. In fact, while in normal CD34+ cells the two siRNAs produced a comparable reduction of total miR16 levels, in PV cells only the miR-16-2 siRNA significantly reduced the abnormally increased mature miR-16. It has been previously shown that miR-16 increases in latest stages of normal erythropoiesis in vitro; we determined the kinetics of miR-16 during cultures of CD34+ cells induced to differentiate along erythroid lineage from normal subjects and PV pts. In both control and PV cells there was a prompt down-regulation of miR-16 soon after starting culture; thereafter, miR-16 levels started to increase progressively from day 6 reaching a maximum on day 12–14; at any time point considered, the miR-16 levels measured in PV cells were significantly greater than controls. To address the function of miR-16 in erythroid differentiation, we over-expressed (Amaxa technology) mature miR16 in normal CD34+ cells both at the beginning of culture and at day 6, when progenitors were switched from a proliferative (in the presence of IL-3, IL-6, SCF) to a differentiative (plus Epo) culture phase. No significant modification compared to mock controls was observed in cells transfected at day 0, while in those transfected at day 6 the percentage of CD36+/GPA+ cells in the presence of EPO at least doubled; notably, also in the absence of EPO GPA+ cells, virtually absence in mocktreated cultures, were generated. Finally, preliminary evidences indicated that knockingdown miR-16-2 in PV CD34+ cells resulted in significant reduction of erythroid colony formation. Overall, these data indicate dysregulated expression of miR-16 in CD34+ and in in-vitro generated erythroid cells from PV patients, and point to miR-16-2 as being specifically involved; we also provided evidence for a role of miR-16 in normal erythroid differentiation and for its possible involvement in the abnormal erythropoiesis of PV.

ConformationalEpitopes Recognized by Protective Anti-Neisserial Surface Protein AAntibodies

ABSTRACT NspA is a conserved membrane protein that elicits protective antibody responses in mice against Neisseria meningitidis. A recent crystallographic study showed that NspA adopts an eight-strandedβ -barrel structure when reconstituted in detergent. In order to define the segments of NspA-containing epitopes recognized by protective murine anti-NspA antibodies, we studied the binding of two bactericidal and protective anti-NspA monoclonal antibodies (MAbs), AL12 and 14C7. Neither MAb binds to overlapping synthetic peptides (10-mers, 12-mers, and cyclic 12-mers) corresponding to the entire mature sequence of NspA, or to denatured recombinant NspA (rNspA), although binding to the protein can be restored by refolding in liposomes. Based on the ability of the two MAbs to bind to Escherichia coli microvesicles prepared from a set of rNspA variants created by site-specific mutagenesis, the most important contacts between the MAbs and NspA appear to be located within the LGG segment of loop 3. The conformation of loop 2 also appears to be an important determinant, as particular combinations of residues in this segment resulted in loss of antibody binding. Thus, the two anti-NspA MAbs recognize discontinuous conformational epitopes that result from the close proximity of loops 2 and 3 in the three-dimensional structure of NspA. The data suggest that optimally immunogenic vaccines using rNspA will require formulations that permit proper folding of the protein.