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Lactococcus lactis IBB477 presenting adhesive and muco-adhesive properties as a candidate carrier strain for oral vaccination against influenza virus
Get PDFIn the gastrointestinal tract (GIT), adhesion is a prerequisite for bacterial colonization. Lactococci can be used in functional food (probiotics) and health-related applications (mucosal vaccines, therapeutic drug delivery), both potentially involving adhesive properties. A candidate lactic acid bacterium for influenza antigen delivery through the GIT should display the ability to adhere. The present work probes the interactions between Lactococcus lactis and mucins using pig gastric mucin (PGM) as a model. Two strains were used for the optimization of the screening method for adhesion: L. lactis subsp. cremoris IBB477 persistent in the GIT of germ-free rats, and the low-adhering control strain MG1820. High adhesion to bare and mucin-coated polystyrene of IBB477 in comparison with MG1820 was observed. We searched for genetic determinants potentially involved in the adhesion/muco-adhesion of IBB477, identifying two such genes: prtP and a gene coding for a protein with MUB and MucBP domains. Based on its persistence in the GIT and adhesive properties, L. lactis IBB477 is a candidate carrier strain for expression of influenza haemagglutinin (HA) protein for induction of mucosal immune response
Immobilization of His-tagged kinase JAK2 onto the surface of a plasmon resonance gold disc modified with different copper (II) complexes.
Get PDFNew surface plasmon resonance (SPR) sensing platforms which consists of copper (II) complexes of a pentetic acid thiol ligand (DPTA-Cu(II)) and of a thiol derivative of dipyrromethene (DPM-Cu(II) created on the surface of gold SPR disc were applied to oriented immobilization of His-tagged Janus kinase 2 (GST-His6-JAK2). This method is based on the covalent bond formation between histidine from a His-tag chain of a protein and Cu(II) centres from the complexes. The kinetic and thermodynamic parameters of the oriented immobilization of GST-His6-JAK2 protein to DPTA-Cu(II) and DPM-Cu(II) complexes attached to the Au surface of a SPR disc were discussed
Transcriptional profiling of ParA and ParB mutants in actively dividing cells of an opportunistic human pathogen Pseudomonas aeruginosa.
Get PDFAccurate chromosome segregation to progeny cells is a fundamental process ensuring proper inheritance of genetic material. In bacteria with simple cell cycle, chromosome segregation follows replication initiation since duplicated oriC domains start segregating to opposite halves of the cell soon after they are made. ParA and ParB proteins together with specific DNA sequences are parts of the segregation machinery. ParA and ParB proteins in Pseudomonas aeruginosa are important for optimal growth, nucleoid segregation, cell division and motility. Comparative transcriptome analysis of parA null and parB null mutants versus parental P. aeruginosa PAO1161 strain demonstrated global changes in gene expression pattern in logarithmically growing planktonic cultures. The set of genes similarly affected in both mutant strains is designated Par regulon and comprises 536 genes. The Par regulon includes genes controlled by two sigma factors (RpoN and PvdS) as well as known and putative transcriptional regulators. In the absence of Par proteins, a large number of genes from RpoS regulon is induced, reflecting the need for slowing down the cell growth rate and decelerating the metabolic processes. Changes in the expression profiles of genes involved in c-di-GMP turnover point out the role of this effector in such signal transmission. Microarray data for chosen genes were confirmed by RT-qPCR analysis. The promoter regions of selected genes were cloned upstream of the promoter-less lacZ gene and analyzed in the heterologous host E. coliΔlac. Regulation by ParA and ParB of P. aeruginosa was confirmed for some of the tested promoters. Our data demonstrate that ParA and ParB besides their role in accurate chromosome segregation may act as modulators of genes expression. Directly or indirectly, Par proteins are part of the wider regulatory network in P. aeruginosa linking the process of chromosome segregation with the cell growth, division and motility
Contribution of RIT1 mutations to the pathogenesis of Noonan syndrome: four new cases and further evidence of heterogeneity.
No full textNoonan syndrome (NS) is a common developmental disorder presenting with dysmorphic craniofacial features, heart defects, and short stature. It belongs to the group of RASopathies caused by germline mutations in genes encoding proteins involved in the RAS/MAPK signaling pathway. Although mutations in nine genes are known to cause NS, approximately 30% of the cases still have unexplained etiology. To identify the new causative genes, 42 patients with a clinical diagnosis of NS, who had negative results on Sanger sequencing of PTPN11, SOS1, and RAF1 (the most common NS genes), were selected for whole exome sequencing. In two patients, mutations in recently described new NS gene—RIT1 were found (c.244T>G [p.Phe82Val] and c.270G>C [p.Met90Ile]). Further analysis of a larger cohort (n = 64) of NS patients with classic Sanger sequencing revealed the presence of RIT1 mutation c.284G>C (p.Gly95Ala) in two additional patients. All the detected mutations were localized in switch II domain responsible for GTPase activity. The modeling of RIT1 protein structure revealed that the mutated amino acids and their interacting residues are evolutionary conserved and any residue replacement might change the structural stability and/or protein internal dynamics influencing catalytic activity of the protein. It seems that the identified mutations might alter protein function and therefore, the activity of ERK and P38 MAPK pathways, thus underlying the specific phenotype observed in NS patients. Our study independently confirms the role of RIT1 in the pathogenesis of Noonan syndrome
Assessing the importance of human activities for the establishment of the invasive Poa annua in Antarctica
Get PDFBecause of its harsh environmental conditions and remoteness, Antarctica is
often considered to be at low risk of plant invasion. However, an increasing
number of reports have shown the presence and spread of non-native plants in
Antarctica; it is therefore important to study which factors control the invasion
process in this ecosystem. Here, we assessed the role of different human
activities on the presence and abundance of the invasive Poa annua. In
addition, we performed a reciprocal transplant experiment in the field, and a
manipulative experiment of germination with P. annua and the natives
Colobanthus quitensis and Deschampsia antarctica, in order to unravel the effects
of physical soil disturbance on the establishment and survival of P. annua. We
found a positive correlation between abundance of P. annua and level of soil
disturbance, and that survival of P. annua was 33% higher in sites with
disturbed soil than non-disturbed. Finally, we found that disturbance conditions
increased germination for P. annua, whereas for native species germination
in experimentally disturbed soil was either unchanged or reduced
compared to undisturbed soil. Our results indicate that human activities that
modify abiotic soil characteristics could play an important role in the
abundance of this invasive species. If the current patterns of human activities
are maintained in Antarctica, the establishment success and spread of P. annua
could increase, negatively affecting native flora
Proteins involved in the degradation of cytoplasmic mRNA in the major eukaryotic model systems.
Get PDFThe process of mRNA decay and surveillance is considered to be one of the main posttranscriptional gene expression regulation platforms in eukaryotes. The degradation of stable, protein-coding transcripts is normally initiated by removal of the poly(A) tail followed by 5'-cap hydrolysis and degradation of the remaining mRNA body by Xrn1. Alternatively, the exosome complex degrades mRNA in the 3'>5'direction. The newly discovered uridinylation-dependent pathway, which is present in many different organisms, also seems to play a role in bulk mRNA degradation. Simultaneously, to avoid the synthesis of incorrect proteins, special cellular machinery is responsible for the removal of faulty transcripts via nonsense-mediated, no-go, non-stop or non-functional 18S rRNA decay. This review is focused on the major eukaryotic cytoplasmic mRNA degradation pathways showing many similarities and pointing out main differences between the main model-species: yeast, Drosophila, plants and mammals
Proper functioning of the GINS complex is important for the fidelity of DNA replication in yeast
Get PDFThe role of replicative DNA polymerases in ensuring genome stability is intensively studied, but the role of other components of the replisome is still not fully understood. One of such component is the GINS complex (comprising the Psf1, Psf2, Psf3 and Sld5 subunits), which participates in both initiation and elongation of DNA replication. Until now, the understanding of the physiological role of GINS mostly originated from biochemical studies. In this article, we present genetic evidence for an essential role of GINS in the maintenance of replication fidelity in S. cerevisiae. In our studies we employed the psf1-1 allele (Takayama et al., 2003) and a novel psf1-100 allele isolated in our laboratory. Analysis of the levels and specificity of mutations in the psf1 strains indicates that the destabilization of the GINS complex or its impaired interaction with DNA polymerase epsilon increases the level of spontaneous mutagenesis and the participation of the error-prone DNA polymerase zeta. Additionally, a synergistic mutator effect was found for the defects in Psf1p and in the proofreading activity of Pol epsilon, suggesting that proper functioning of GINS is crucial for facilitating error-free processing of terminal mismatches created by Pol epsilon
NMR structural studies of the first catalytic half-domain of ubiquitin activating enzyme
Get PDFWe report a high resolution NMR structure and 15N relaxation studies of the first catalytic cysteine half-domain (FCCH) of the mouse ubiquitin-activating enzyme E1, together with interaction studies of FCCH and the other catalytic E1 subdomain - SCCH (second catalytic cysteine half-domain). In solution, mouse FCCH forms a well-defined six-stranded antiparallel β-barrel structure, a common fold for many proteins with a variety of cellular functions. 15N relaxation data reveal FCCH complex backbone dynamics and indicate which residues experience slow intramolecular motions. Some of these residues make contacts with the polar face of ubiquitin in the co-crystal structure of yeast E1 and ubiquitin. However, the titration of FCCH with ubiquitin does not show any visible chemical shift changes in the 2D 1H/15N HSQC spectra of the FCCH. The 2D 1H/15N HSQC experiments performed both for each catalytic half-domain individually and for their equimolar mixture in the milimolar concentration range display no detectable chemical shift perturbation, suggesting a lack of interaction between the two subdomains unless they are covalently linked via the adenylation domain
Structural basis of the methylation specificity of R.DpnI
Get PDFR.DpnI consists of N-terminal catalytic and C-terminal winged helix domains that are separately specific for the Gm6ATC sequences in Dam-methylated DNA. Here we present a crystal structure of R.DpnI with oligoduplexes bound to the catalytic and winged helix domains and identify the catalytic domain residues that are involved in interactions with the substrate methyl groups. We show that these methyl groups in the Gm6ATC target sequence are positioned very close to each other. We further show that the presence of the two methyl groups requires a deviation from B-DNA conformation to avoid steric conflict. The methylation compatible DNA conformation is complementary with binding sites of both R.DpnI domains. This indirect readout of methylation adds to the specificity mediated by direct favorable interactions with the methyl groups and solvation/desolvation effects. We also present hydrogen/deuterium exchange data that support ‘crosstalk’ between the two domains in the identification of methylated DNA, which should further enhance R.DpnI methylation specificity
Genetic diversity of hemagglutinin gene of A(H1N1)pdm09 influenza strains isolated in Taiwan and its potential impact on HA-neutralizing epitope interaction.
Get PDFPandemic influenza A(H1N1)pdm09 virus is a global health threat and between 2009-2011 it became the predominant influenza virus subtype circulating in the world. The research describes the MSSCP (Multitemperature Single Strand Conformation Polymorphism) analysis of the hemagglutinin (HA) region encompassing major neutralizing epitope in pandemic influenza isolates from Taiwan. Several genetically distinct changes appeared in isolates obtained in 2010 and 2011. The majority of changes in HA protein did not result in significant modifications, however three modifications were localized in epitope E of H1 and one was part of the interface binding antibodies BH151 and HC45 possibly making the current vaccine less effective.-Taking into account the possibility of the emergence of influenza A with antibody evading potential, the MSSCP method provides an alternative approach for detection of minor variants which escape detection by conventional Sanger sequencing