Localization of eukaryotic initiation factor 2 in neuron primary cultures and established cell lines

Eukaryotic initiation factor 2 (eIF-2) is a heterotrimeric protein with subunits α, β and γ that forms a ternary complex with Met-tRNA and GTP. It promotes the binding of Met-tRNA to ribosomes and controls translational rates via phosphorylation/dephosphorylation mechanisms. By means of immunofluorescence and post-embedding immunocytochemistry of intact cells and quantitative immunoblotting of cell extracts, the cellular distribution of the initiation factor has been examined in primary neuronal cultures as well as in two established cell lines: PC12 phaeochromocytoma cells and rat pituitary GH4C1 cells. Our results indicated that the initiation factor is located not only in the cytoplasm but also in the nuclei of the cultured neurons and cell lines. In the cytoplasm, immunocytochemical studies reveal that the factor is present mainly in those areas that are rich in ribosomes. In the nucleus, the immunolabelling of eukaryotic initiation factor 2 verified the presence of gold particles in both nucleolar and extranucleolar areas. The specific distribution of this factor on both sides of the nuclear envelope suggests that it might have some nuclear-related function(s) besides its already known role in the control of translation     

Cadaverine: a common polyamine in zygotic embryos and somatic embryos of the species Capsicum chinense Jacq.

The behavior of endogenous polyamines was studied in somatic embryos and zygotic embryos of Habanero pepper (Capsicum chinense). In the first part of the work, the polyamine content was evaluated in both types of embryos (somatic and zygotic). As a result, in addition to the common polyamines (putrescine, spermidine and spermine), it was also possible to detect cadaverine, a polyamine rarely found in plants. In general, all the polyamines were found to be more abundant in somatic embryos than in zygotic embryos, with significantly higher contents of putrescine and cadaverine. Subsequently, the content of putrescine, spermidine, spermine and cadaverine, in their different forms (free, bound and conjugated) was determined in somatic embryos which were cultured in non-ventilated and ventilated containers. Detection of polyamines was carried out at 28 and 42 days of culture by the HPLC method. The ethylene content was monitored during the process in both culture conditions (ventilated and non-ventilated). As a result of the analysis, cadaverine was always found present, indicating that it is a common polyamine in the species. Ethylene was detected in containers without ventilation throughout the culture, except during replenishment of the culture medium (R1, R2 and R3). The behavior pattern of each polyamine, analyzed under different culture conditions (ventilated and non-ventilated) and at different moments of culture (28 and 42 days of culture), show that the polyamines are not only involved in morphogenic processes in plants; polyamines are also significantly affected by the surrounding environment. However, the most novel result, presented for the first time in this paper, is that cadaverine is found to be a common polyamine in C. chinense since it is present in both zygotic embryos and somatic embryos.

     

Relative Contribution of P5 and Hap Surface Proteins to Nontypable Haemophilus influenzae Interplay with the Host Upper and Lower Airways

Nontypable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract disease, and initiates infection by colonizing the nasopharynx. Bacterial surface proteins play determining roles in the NTHi-airways interplay, but their specific and relative contribution to colonization and infection of the respiratory tract has not been addressed comprehensively. In this study, we focused on the ompP5 and hap genes, present in all H. influenzae genome sequenced isolates, and encoding the P5 and Hap surface proteins, respectively. We employed isogenic single and double mutants of the ompP5 and hap genes generated in the pathogenic strain NTHi375 to evaluate P5 and Hap contribution to biofilm growth under continuous flow, to NTHi adhesion, and invasion/phagocytosis on nasal, pharyngeal, bronchial, alveolar cultured epithelial cells and alveolar macrophages, and to NTHi murine pulmonary infection. We show that P5 is not required for bacterial biofilm growth, but it is involved in NTHi interplay with respiratory cells and in mouse lung infection. Mechanistically, P5_NTHi375 is not a ligand for CEACAM1 or α5 integrin receptors. Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20. We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant. Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.     

The Impact of Extra-Domain Structures and Post-Translational Modifications in the Folding/Misfolding Behaviour of the Third PDZ Domain of MAGUK Neuronal Protein PSD-95

The modulation of binding affinities and specificities by post-translational modifications located out from the binding pocket of the third PDZ domain of PSD-95 (PDZ3) has been reported recently. It is achieved through an intra-domain electrostatic network involving some charged residues in the β2–β3 loop (were a succinimide modification occurs), the α3 helix (an extra-structural element that links the PDZ3 domain with the following SH3 domain in PSD-95, and contains the phosphorylation target Tyr397), and the ligand peptide. Here, we have investigated the main structural and thermodynamic aspects that these structural elements and their related post-translational modifications display in the folding/misfolding pathway of PDZ3 by means of site-directed mutagenesis combined with calorimetry and spectroscopy. We have found that, although all the assayed mutations generate proteins more prone to aggregation than the wild-type PDZ3, those directly affecting the α3 helix, like the E401R substitution or the truncation of the whole α3 helix, increase the population of the DSC-detected intermediate state and the misfolding kinetics, by organizing the supramacromolecular structures at the expense of the two β-sheets present in the PDZ3 fold. However, those mutations affecting the β2–β3 loop, included into the prone-to-aggregation region composed by a single β-sheet comprising β2 to β4 chains, stabilize the trimeric intermediate previously shown in the wild-type PDZ3 and slow-down aggregation, also making it partly reversible. These results strongly suggest that the α3 helix protects to some extent the PDZ3 domain core from misfolding. This might well constitute the first example where an extra-element, intended to link the PDZ3 domain to the following SH3 in PSD-95 and in other members of the MAGUK family, not only regulates the binding abilities of this domain but it also protects PDZ3 from misfolding and aggregation. The influence of the post-translational modifications in this regulatory mechanism is also discussed.     

Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing

High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.     

Islands within islands: a molecular phylogenetic study of the Leucocroton alliance (Euphorbiaceae) across the Caribbean Islands and within the serpentinite archipelago of Cuba

Aim Our aim was to investigate the historical biogeography of the three genera of the Leucocroton alliance (i.e. Garciadelia Jestrow & Jiménez Rodr., Lasiocroton Griseb., and Leucocroton Griseb., Euphorbiaceae). Location  The alliance is restricted to the Bahamas, Cuba, Hispaniola and Jamaica. Methods Members of the Leucocroton alliance, along with representatives from tribe Adelieae (Adelia L. and Philyra Klotzsch.), were included in a molecular phylogenetic analysis based upon nucleotide sequences of the internal transcribed spacer region of the nuclear ribosomal DNA and the non‐coding chloroplast regions psbM–trnD and ycf6–pcbM. The program s‐diva was used to calculate ancestral areas based on the phylogenetic trees and present species distributions. Results Phylogenetic analyses support the monophyly of the three genera. The ancestral area of the Leucocroton alliance is eastern Cuba and Hispaniola. Ancestral forms of Leucocroton arose on eastern Cuba and underwent two migrations across the island. The ancestor of Lasiocroton also originated on eastern Cuba followed by later dispersal to and speciation events on the other islands. Our study also suggests that ancestral forms of the Leucocroton alliance probably occurred on limestone soils. Main conclusions Our study concurs with previous hypotheses suggesting that the flora of serpentinite regions of the Caribbean derives from other types of soils. The serpentine endemics of the Leucocroton alliance have a single origin and represent one of the most extraordinary examples of speciation in this unique environment of the New World. The high colonization success achieved by the members of Leucocroton on serpentine soils was not attained by the other genera of the alliance, which occur on limestone areas.