Human CD5 protects circulating tumor antigen-specific CTL from tumor-mediated activation-induced cell death

We previously characterized several tumor-specific T cell clones from PBL and tumor-infiltrating lymphocytes of a lung cancer patient with identical TCR rearrangements and similar lytic potential, but with different antitumor response. A role of the TCR inhibitory molecule CD5 to impair reactivity of peripheral T cells against the tumor was found to be involved in this process. In this report, we demonstrate that CD5 also controls the susceptibility of specific T cells to activation-induced cell death (AICD) triggered by the tumor. Using a panel of tumor-infiltrating lymphocytes and PBL-derived clones expressing different levels of CD5, our results indicate that T lymphocyte AICD in response to the cognate tumor is inversely proportional to the surface expression level of CD5. They also suggest a direct involvement of CD5 in this process, as revealed by an increase in tumor-mediated T lymphocyte AICD following neutralization of the molecule with specific mAb. Mechanistically, our data indicate that down-regulation of FasL expression and subsequent inhibition of caspase-8 activation are involved in CD5-induced T cell survival. These results provide evidence for a role of CD5 in the fate of peripheral tumor-specific T cells and further suggest its contribution to regulate the extension of CTL response against tumor.     

Localisation of caveolin in mammary tissue depends on cell type

Caveolins, components of caveolae, are expressed in mammary tissue. In order to determine whether caveolins are present in different mammary cell types and whether their localisation depends on the physiological stage or species, cav-1 and cav-2 were characterised by immunoblotting in mammary tissues from the mouse, ewe and rabbit and localised, by immunofluorescence and electron microscopy, in mammary tissues from the mouse and ewe. At all the physiological stages studied, cav-1 and cav-2 were present in endothelial and myoepithelial cells in which flask-shaped caveolae were abundant. However, labelling of cav-1 and cav-2 associated with small vesiculo-tubular structures (including those close to lipid droplets) was low in epithelial cells. To study the possible association of cav-1 with lipid droplets, lactating ewe mammary fragments were treated in vitro with brefeldin A. This treatment did not modify the association of cav-1-labelled structures with lipid droplets. Finally, HC11 and MCF-10A mammary cell lines were treated with oleic acid. The total quantity of cav-1 was little affected by the treatment, although the lipid droplet labelling of cav-1 was amplified in MCF-10A cells. Thus, the synthesis and localisation of caveolins are mostly dependent upon the cell types of mammary tissue and upon their state of differentiation.     

Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors

We describe a chemical proteomics approach to profile the interaction of small molecules with hundreds of endogenously expressed protein kinases and purine-binding proteins. This subproteome is captured by immobilized nonselective kinase inhibitors (kinobeads), and the bound proteins are quantified in parallel by mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ). By measuring the competition with the affinity matrix, we assess the binding of drugs to their targets in cell lysates and in cells. By mapping drug-induced changes in the phosphorylation state of the captured proteome, we also analyze signaling pathways downstream of target kinases. Quantitative profiling of the drugs imatinib (Gleevec), dasatinib (Sprycel) and bosutinib in K562 cells confirms known targets including ABL and SRC family kinases and identifies the receptor tyrosine kinase DDR1 and the oxidoreductase NQO2 as novel targets of imatinib. The data suggest that our approach is a valuable tool for drug discovery.     

Fucosylated pentaerythrityl phosphodiester oligomers (PePOs): automated synthesis of DNA-based glycoclusters and binding to Pseudomonas aeruginosa lectin (PA-IIL)

The synthesis of propargylated pentaerythrityl phosphodiester oligomers (PePOs) was achieved using a DNA synthesizer with a bis-propargylated pentaerythritol-based phosphoramidite. An azido fucose derivative was reacted under "click" chemistry conditions activated by microwaves to construct a series of glycosylated PePOs bearing 4, 6, 8, and 10 L-fucose residues. Binding to the fucose-specific bacterial lectin (PA-IIL) was determined for the fucosylated PePOs through an enzyme-linked lectin amplification competition assay. The IC50 values measured are 10-20 times better than for monovalent l-fucose and denotate for a "macromolecular" effect rather than a "cluster" effect.     

Induced parthenogenesis in mandarin for haploid production: induction procedures and genetic analysis of plantlets

This study focused on haploid induction in mandarin through in situ gynogenesis by pollination with irradiated pollen of ‘Meyer’ lemon. Pollination was carried out for three genotypes of mandarin with four levels of gamma-ray-irradiated pollen (150, 300, 600, and 900 Gy). The resulting seeds were characterised by a small size. Embryos were rescued in vitro and the ploidy level of the plantlets was determined by flow cytometry analysis. Haploid, diploid, triploid plantlets were obtained. The haploid parthenogenetic origin was confirmed using microsatellite marker analysis and chromosome count. Diploid and triploid plants were the result of crosses between mandarin and lemon. The induction of gynogenetic haploids of ‘Fortune’ (Citrus clementina Hort ex Tan. × Citrus tangerina Hort ex Tan.) and ‘Ellendale’ (Citrus reticulata Blanco × Citrus sinensis L. Osb) is reported here for the first time.     

A new generation of pPRIG-based retroviral vectors

Background  

Retroviral vectors are valuable tools for gene transfer. Particularly convenient are IRES-containing retroviral vectors expressing both the protein of interest and a marker protein from a single bicistronic mRNA. This coupled expression increases the relevance of tracking and/or selection of transduced cells based on the detection of a marker protein. pAP2 is a retroviral vector containing eGFP downstream of a modified IRES element of EMCV origin, and a CMV enhancer-promoter instead of the U3 region of the 5'LTR, which increases its efficiency in transient transfection. However, pAP2 contains a limited multicloning site (MCS) and shows weak eGFP expression, which previously led us to engineer an improved version, termed pPRIG, harboring: i) the wild-type ECMV IRES sequence, thereby restoring its full activity; ii) an optimized MCS flanked by T7 and SP6 sequences; and iii) a HA tag encoding sequence 5' of the MCS (pPRIG HAa/b/c).     

Altered retinoic acid signalling underpins dentition evolution

Small variations in signalling pathways have been linked to phenotypic diversity and speciation. In vertebrates, teeth represent a reservoir of adaptive morphological structures that are prone to evolutionary change. Cyprinid fish display an impressive diversity in tooth number, but the signals that generate such diversity are unknown. Here, we show that retinoic acid (RA) availability influences tooth number size in Cyprinids. Heterozygous adult zebrafish heterozygous for the cyp26b1 mutant that encodes an enzyme able to degrade RA possess an extra tooth in the ventral row. Expression analysis of pharyngeal mesenchyme markers such as dlx2a and lhx6 shows lateral, anterior and dorsal expansion of these markers in RA-treated embryos, whereas the expression of the dental epithelium markers dlx2b and dlx3b is unchanged. Our analysis suggests that changes in RA signalling play an important role in the diversification of teeth in Cyprinids. Our work illustrates that through subtle changes in the expression of rate-limiting enzymes, the RA pathway is an active player of tooth evolution in fish.     

Revisiting the link between breeding effort and oxidative balance through field evaluation of two sympatric sibling insect species

The idea that oxidative stress could be a major force governing evolutionary trade‐offs has recently been challenged by experimental approaches in laboratory conditions, triggering extensive debates centered on theoretical and methodological issues. Here, we revisited the link between oxidative stress and reproduction by measuring multiple antioxidant and oxidative damages in wild‐caught females of two sibling weevil species (Curculio elephas, C. glandium). The strength of our study arised from (1) studied species that were sympatric and exploited similar resource, but displayed contrasting reproductive strategies and (2) individuals were sampled throughout adult life so as to relate oxidative status to breeding effort. We found that the short‐lived C. elephas sacrifices red‐ox homeostasis for immediate reproduction upon emergence as characterized by low antioxidant defenses and elevated oxidative damage. Comparatively, C. glandium massively invests in antioxidant and maintains low oxidative damage, which may contribute to their extended prereproductive period. Intriguingly, we also reveal, for the first time in a field study, an unexpected reactivation of antioxidant defenses with the onset of reproduction. Our results thus support the existence of a strong, but complex relationship between oxidative stress and life‐history evolution and highlight the need for a finer‐scale picture of antioxidant strategies.     

Endemic predators, invasive prey and native diversity

Interactions between native diversity and invasive species can be more complex than is currently understood. Invasive ant species often substantially reduce diversity in the native ants diversity that act as natural control agents for pest insects. In Indonesia (on the island of Sulawesi), the third largest cacao producer worldwide, we show that a predatory endemic toad (Ingerophrynus celebensis) controls invasive ant (Anoplolepis gracilipes) abundance, and positively affects native ant diversity. We call this the invasive-naivety effect (an opposite of enemy release), whereby alien species may not harbour anti-predatory defences against a novel native predator. A positive effect of the toads on native ants may facilitate their predation on insect vectors of cacao diseases. Hence, toads may increase crop yield, but further research is needed on this aspect. Ironically, amphibians are globally the most threatened vertebrate class and are strongly impacted by the conversion of rainforest to cacao plantations in Sulawesi. It is, therefore, crucial to manage cacao plantations to maintain these endemic toads, as they may provide critical ecosystem services, such as invasion resistance and preservation of native insect diversity.